(Received for publication, May 9, 1995; and in revised form, September 12, 1995)
From the
In view of the observations that the calcium ionophores, A23187
and ionomycin, enhance the processing and secretion of interleukin-1
(IL-1) and IL-1
from macrophages, and IL-1
processing is
mediated by calpain, a calcium-dependent protease, we evaluated the
possibility that calpain might also play a role in the processing of
IL-1
. Whereas calpain-containing P388D1 macrophage lysates and
purified calpain processed precursor IL-1
to its mature 17-kDa
form, precursor IL-1
was degraded by both sources of calpain.
However, the activation of calpain in P388D1 cells that were
transiently transfected with a cDNA expression vector encoding the
precursor form of IL-1
did not result in the degradation of
precursor IL-1
, but did result in the processing and secretion of
IL-1
, implying that precursor IL-1
is protected from calpain
degradation in vivo. Furthermore, calpain did not enhance the
processing of the IL-1
precursor by the IL-1
-converting
enzyme. These results indicate that calpain is not involved in the
processing of precursor IL-1
in vitro or in
vivo. The IL-1
precursor may be protected from calpain
degradation by a sequestering mechanism that involves a cytoplasmic
factor(s) that reduces the sensitivity of IL-1
to attack by
calpain or localizes IL-1
to a site that precludes any interaction
with the protease.
Although MDL 28,170, a calpain inhibitor,
prevented the ionomycin-induced processing of precursor IL-1 to
the mature protein in P388D1 cells, it did not inhibit the
ionomycin-induced secretion of the mature IL-1
and -
proteins
expressed in these cells. These results indicate that a
calcium-dependent factor other than calpain is involved in the
secretion of the mature IL-1 proteins.
Interleukin-1 (IL-1) ()is an important inflammatory
cytokine produced primarily by monocytes, macrophages, and
polymorphonuclear leukocytes. By mediating the production of potent
molecules such as prostaglandins, leukotrienes, platelet-activating
factor, and nitric oxide, and by up-regulating endothelial
cell-adhesion molecules, IL-1 affects processes as diverse as immune
cell recruitment, blood pressure, vascular smooth muscle contraction,
kidney function, cell proliferation, bone resorption, and central
nervous system function(1) . The two forms of IL-1, IL-1
and -
, are produced as precursor proteins of 31-33 kDa and
are subsequently processed to mature proteins of 15-17
kDa(2, 3, 4) . The precursor form of
IL-1
is processed in vitro by calpain, a
calcium-dependent protein; human IL-1
is cleaved between
Phe
and Leu
, whereas murine IL-1
is
cleaved between Arg
and
Ser
(5, 6) . The precursor form of
IL-1
is processed by the IL-1
-converting enzyme (ICE) between
Asp
and
Ala
(7, 8, 9) . Although
precursor IL-1
is biologically active, precursor IL-1
is
biologically inactive due to weak receptor binding(10) . Even
though the amino acid sequence identity between the mature forms of
IL-1
and IL-1
is low (26%), they bind to the same receptor
and invoke the same set of biological
responses(11, 12) .
IL-1 and -
, basic
fibroblast growth factor, hisactophilin, and soy bean trypsin inhibitor
belong to a family of proteins whose structure is comprised of 12
antiparallel
-strands connected by several loops and turns and
arranged around a 3-fold axis of symmetry (13, 14, 15, 16, 17) .
These proteins are also characterized by the lack of an
NH
-terminal secretory signal sequence. Examples of other
proteins with a defined extracellular function but devoid of a
secretory signal sequence include thioredoxin, transglutaminase,
thymosin, parathymosin, and the soluble lectins L-14, L-29, and CBP30 (18, 19, 20, 21) . Drugs such as
brefeldin A and monensin, which block the trafficking of proteins
through the endoplasmic reticulum-Golgi pathway do not affect the
secretion of several of these proteins, suggesting an alternative or
``nonclassical'' route to the cell
exterior(20, 21, 22, 23, 24) .
The secretion of this group of proteins is enhanced by heat shock or
calcium ionophores, suggesting that these proteins may share a common
secretory pathway(20, 21, 23, 24) .
Processing of the precursor forms of IL-1 and -
is also
enhanced by calcium ionophores in monocytes and
macrophages(5, 22, 25) . When these cells are
stimulated to synthesize and secrete IL-1 proteins, the mature forms of
IL-1 are found only in the culture medium and not in association with
cells, suggesting that processing and secretion may be so closely
linked as to result in the rapid and efficient secretion of the mature
IL-1 proteins. Analysis of IL-1 secretion in cell line macrophages as
well as nonmacrophage cell lines expressing recombinant forms of the
precursor and mature forms of IL-1
and -
(following transient
transfection of an expression plasmid) revealed that the mature form is
the preferred substrate for secretion(26) ; such cells secrete
relatively low levels of precursor IL-1
and -
compared with
the mature forms in the presence of ionomycin or LPS(26) .
Whereas transiently transfected cells secrete only 2% of the precursor
IL-1 proteins following pulse labeling with
[
S]methionine and incubation with ionomycin or
LPS during a 6-h period, cells transiently transfected with an
expression plasmid encoding mature IL-1 secrete essentially all of the
radiolabeled IL-1(26) . The requirement for secondary stimuli
such as ionomycin or LPS to induce the secretion of the mature IL-1
proteins indicates that processing by itself is not a sufficient
stimulus for IL-1 secretion and that secretion, like processing, is
also modulated by an increase in the intracellular level of calcium.
Calcium ionophore-induced processing and the subsequent release of
mature IL-1 from monocytes and macrophages (5, 26) is not surprising given the role of calpain in
the in vitro processing of precursor
IL-1
(5, 6) . Calpain is found in a wide variety
of tissues, including monocytes, macrophages, lymphocytes, and
fibroblasts. It is an heterodimer composed of an 80-kDa and a 30-kDa
subunit, both of which contain calcium-binding EF-hand domains near the
COOH termini(27) . Calcium binding to these subunits induces a
conformational change and the subsequent activation of the
enzyme(27) . The activity of calpain is regulated by
calpastatin, a naturally occurring inhibitor of calpain. Thus the
ability to process the precursor form of IL-1
is restricted to
cell types in which the balance of calpain and calpastatin favors
calpain(6) . Processing of precursor IL-1
to the mature
form on the other hand appears to be mediated by ICE. The importance of
ICE in precursor IL-1
processing was demonstrated by the ability
of ICE-specific inhibitors to prevent the secretion of mature IL-1
from activated monocytes and macrophages(8, 28) . In
addition, monocytes and macrophages derived from ICE-deficient mice do
not secrete IL-1
when stimulated with LPS (29, 30) . ICE is synthesized as an inactive 45-kDa
precursor and is cleaved to 20-kDa and 10-kDa peptides, presumably by
autocatalysis(8) . Unlike calpain, ICE does not require calcium
to function. Thus it is not readily evident as to why calcium
ionophores enhance the processing and secretion of mature
IL-1
. Could calpain be involved in the processing and secretion of
IL-1
? Calpain might, under certain conditions, convert precursor
IL-1
to its mature form, thus representing a second processing
enzyme for precursor IL-1
. Alternatively, calpain may enhance the
activity of ICE or convert precursor IL-1
to a form that is more
efficiently cleaved by ICE. The current study was undertaken to
evaluate these possibilities.
For the analysis of precursor IL-1 turnover,
the labeled media was replaced with RPMI with or without 1
µM ionomycin, and culture supernatants and cells were
collected during a 2-h period.
Cleavage of precursor
IL-1 or precursor IL-1
protein was also analyzed in the
presence of P388D1 or THP.1 cell lysates. Nine fmol of each protein was
incubated with 20 µl of lysate in the presence of 5 mM EGTA or 1 mM CaCl
(total volume of 30 µl)
at 37 °C for 1 h. Following the addition of an equal amount of
sample buffer, the reactions were boiled for 2 min, cooled, and
40-45 µl was loaded on SDS-PAGE gels. For the ICE assays, 24
fmol of precursor IL-1
was added to 20 µl of THP.1 lysate
(incubated overnight at 4 °C, on a shaker to activate ICE) (32) (total reaction volume of 30 µl). Following the
addition of an equal amount of sample buffer, the reactions were boiled
for 2 min, cooled, and loaded on SDS-PAGE gels.
Our
initial experiments focused on the effect of purified calpain on the in vitro cleavage of radiolabeled recombinant precursor
IL-1. When radiolabeled precursor IL-1
was incubated with
increasing amounts of purified calpain in the presence of calcium,
IL-1
was converted to several species with molecular masses (m) of 27, 17.4, 17.0, and 16.2 kDa (Fig. 1A, lanes
3-7). The 17-kDa band co-migrated with the mature IL-1
standard (lane 11). The appearance of these species was
inhibited by the calpain-specific inhibitor calpastatin (lane
8) and the cysteine protease inhibitors E-64 and MDL 28,170 (lanes 9 and 10). When precursor IL-1
was
incubated with calcium alone, there was no change in the molecular mass
of the protein, indicating that the reticulocyte lysate used to
translate the protein did not have any endogenous calpain activity (lane 2). To determine if any of the lower molecular mass
calpain cleavage products were actually mature IL-1
, we evaluated
their proteinase K sensitivity, since mature IL-1
is insensitive
to proteinase K. Authentic recombinant mature IL-1
and the
calpain-treated precursor IL-1
were treated with 10 µg/ml
proteinase K (22 °C, 30 min) prior to analysis on
SDS-polyacrylamide gels. Whereas proteinase K treatment of the
calpain-treated samples resulted in a complete loss of all of the lower
molecular mass species, there was no loss of the authentic mature
IL-1
control sample (data not shown). Thus it is highly unlikely
that any of these species is mature IL-1
.
Figure 1:
In
vitro processing of precursor IL-1 and precursor IL-1
by
purified calpain. Recombinant
[
S]methionine-labeled precursor IL-1
or
precursor IL-1
was incubated with increasing amounts of purified
calpain at 37 °C for 1 h. A, precursor IL-1
. B, precursor IL-1
. Lane 1, 5 mM EGTA; lane 2, 1 mM CaCl
; lanes
3-7, 1 mM CaCl
and 0.5, 1, 2.5, 5, or
10 units/ml, purified calpain; lanes 8-10, 1 mM CaCl
and 5 units/ml purified calpain; lane 8,
200 µg/ml calpastatin; lane 9, 200 µg/ml E-64; lane 10, 100 µM MDL 28,170; lane 11,
mature IL-1
standard.
The calpain-mediated
generation of multiple products with precursor IL-1 is in contrast
to the situation with precursor IL-1
in which only a single major
product, mature IL-1
, is obtained (Fig. 1B, lanes
3-7; (6) ). Incubation of precursor IL-1
with
increasing amounts of calpain in vitro resulted in a
progressive decrease in the level of radioactivity associated with the
IL-1
cleavage products (Fig. 2A) compared with the
average total input radiolabeled precursor IL-1
(Fig. 1A, lanes 1 and 2). Thus at higher
concentrations of calpain an increasing percentage of the precursor
IL-1
was converted to very low molecular weight fragments that
would be lost from the gels. The loss of IL-1
radioactivity at 5
and 10 units/ml of purified calpain exceeded the expected 50%
associated with the removal of 6 of 12 methionine residues as a result
of the conversion of precursor IL-1
to mature IL-1
. These
results are consistent with the notion that precursor IL-1
is degraded and not processed by calpain in vitro. In
contrast to the results with precursor IL-1
, calpain did not
degrade radiolabeled recombinant mature IL-1
(Fig. 2B) or precursor IL-1
(Fig. 2C) even at high concentrations, demonstrating a
selective effect of calpain on precursor IL-1
. Conversion of
murine precursor IL-1
to mature IL-1
involves a loss of 2 out
of 6 methionines and therefore a theoretical loss of 33% of the total
input radioactivity. Whereas an average of 18% of the input precursor
IL-1
radioactivity is lost in experiments, most of the input
precursor IL-1
radioactivity was recovered in the mature IL-1
protein in the experiment presented in Fig. 2C.
However, in other experiments, the loss of radioactivity approached the
expected value.
Figure 2:
Degradation of precursor IL-1 by
calpain in vitro. Equal amounts of recombinant radiolabeled
precursor IL-1
(A), mature IL-1
(B), or
precursor IL-1
(C) were incubated with increasing amounts
of purified calpain at 37 °C for 1 h in the presence of 1 mM CaCl
as described under ``Materials and
Methods.'' CI = 200 µg/ml
calpastatin.
Figure 3:
Cleavage of radiolabeled recombinant
precursor IL-1 protein by calpain-containing P388D1 lysates.
[
S]Methionine-labeled precursor IL-1
was
incubated with P388D1 lysate at 37 °C for 1 h as described under
``Materials and Methods.'' Lanes 1-5,
precursor IL-1
plus lysate; lane 1, 5 mM EGTA; lane 2, 1 mM CaCl
; lane 3, 1
mM CaCl
and 200 µg/ml calpastatin; lane
4, 1 mM CaCl
and 200 µg/ml E-64; lane
5, 1 mM CaCl
and 100 µM MDL
28,170; lane 6, precursor IL-1
, 1 mM CaCl
, and lysis buffer; lane 7, precursor
IL-1
, 1 mM CaCl
, and 5 units/ml purified
calpain; lane 8, mature IL-1
standard.
Since, unlike
purified calpain, the calpain-containing macrophage lysate does not
completely degrade precursor IL-1, it was possible that the
lysates might contain a much lower level of calpain. Thus it was
important to relate the active calpain content in the macrophage
lysates to the activity associated with the purified enzyme. Equal
amounts of radiolabeled precursor IL-1
was incubated with
increasing amounts of purified calpain or P388D1 lysates in the
presence of calcium at 37 °C for 1 h and the percent of unprocessed
precursor IL-1
remaining in each reaction was plotted against
calpain concentration or lysate volume. 50% of the precursor IL-1
protein was processed with approximately 1 unit/ml of the purified
calpain (Fig. 4). Since the same amount of processing was
obtained with approximately 4 µl of the P388D1 lysate (Fig. 4), we estimate that the lysate contains an equivalent
amount of enzyme activity. Thus the calpain concentration in the lysate
(as prepared from 10
cells) used in our experiments was
approximately 7.5 units/ml. Although the sensitivity of precursor
IL-1
to cleavage by purified calpain was not dramatically
different from that of precursor IL-1
(Fig. 4, top), precursor IL-1
was markedly less sensitive to
calpain cleavage in the presence of P388D1 lysate (Fig. 4, bottom). These results indicate that although P388D1 lysates
possess a relatively high level of calpain activity and process
precursor IL-1
as efficiently as purified calpain, precursor
IL-1
is partially, but significantly, protected from cleavage by
calpain in the lysate.
Figure 4:
Estimation of the calpain content in
P388D1 lysates. Equal amounts of radiolabeled recombinant precursor
IL-1 or precursor IL-1
proteins were incubated with
increasing amounts of purified calpain or P388D1 lysate as described
under ``Materials and Methods,'' and the percentage of total
input precursor IL-1
or precursor IL-1
protein remaining in
each reaction was plotted against calpain concentration or lysate
volume. Total input protein in each case was the average counts of
precursor IL-1
or precursor IL-1
obtained from two different
control reactions in which precursor protein remained
intact.
Figure 5:
Absence of ICE activity in P388D1
macrophage cell lysates. Extracts from P388D1 and THP.1 cells incubated
overnight at 4 °C (to favor the activation of ICE) were incubated
with radiolabeled recombinant precursor IL-1 protein in the
presence of 5 mM EGTA as described under ``Materials and
Methods.'' Lane 1, P388D1 lysate; lane 2, lysis
buffer; lane 3, THP.1 lysate; lane 4, mature
IL-1
standard.
To test if the 27-kDa protein is a
substrate for ICE, the 27-kDa species was initially generated by
incubating precursor IL-1 with calpain-containing P388D1 lysates,
the calpain in the lysates was inactivated using calpastatin, and THP.1
extract containing active ICE was added to the 27-kDa containing P388D1
lysate. As shown in Fig. 6, incubation of radiolabeled precursor
IL-1
with calpain-containing P388D1 lysate in the presence of
calcium at 37 °C for 1 h generated the expected 27-kDa product (lane 3), the appearance of which was inhibited by calpastatin (lane 4). Further incubation of the 27-kDa product-containing
lysate at 37 °C for 15 min with 200 µg/ml calpastatin resulted
in a complete inactivation of the calpain content in the lysate as
shown by the inability of a similarly incubated aliquot of P388D1
lysate to cleave precursor IL-1
in the presence of calcium (lane 1). Activated ICE-containing THP.1 extract which cleaves
precursor IL-1
to the mature protein in the presence of EGTA (lane 6) was added to the 27-kDa product-containing P388D1
lysate and incubated for 1 h at 37 °C. The endogenous calpain
content of the THP.1 extracts was inactivated by including leupeptin in
the buffer used to make the extract. The leupeptin-treated extracts
were unable to cleave radiolabeled precursor IL-1
protein (lane 2). Incubation of the 27-kDa product with active
ICE-containing THP.1 extract resulted in its conversion to a slightly
smaller product of 26 kDa (lane 5). This conversion was indeed
due to ICE as it was inhibited by iodoacetamide, a general inhibitor of
cysteine proteases (data not shown). These results indicate that
although the 27-kDa product is susceptible to ICE, it is not converted
to the mature IL-1
protein and very likely represents a
degradation product of calpain cleavage.
Figure 6:
Incubation of the calpain-generated 27-kDa
protein with ICE extract. Lane 1, precursor IL-1 in the
presence of 1 mM CaCl
and P388D1 lysate
preincubated with 200 µg/ml calpastatin; lane 2, precursor
IL-1
in the presence of 1 mM CaCl
and
leupeptin-treated, activated-ICE-containing THP.1 cell extract; lanes 3-6, precursor IL-1
; lane 3, 1
mM CaCl
and P388D1 lysate; lane 4, same
as lane 3 plus 200 µg/ml calpastatin; lane 5,
reaction in lane 3 incubated for a further 1 h with activated
ICE-containing THP.1 cell extract in the presence of 5 mM EGTA; lane 6, 5 mM EGTA and activated
ICE-containing THP.1 cell extract.
Figure 7:
Precursor IL-1 is not degraded in
vivo. P388D1 cells were transfected with the pRC/RSV-pIL-1
plasmid and labeled in [
S]methionine-containing
media as described under ``Materials and Methods.'' The
labeled proteins were chased in cold medium for 2 h in the presence or
absence of ionomycin, and the total precursor IL-1
counts were
quantitated on AMBIS as described.
Since the precursor form of IL-1 is processed in
vitro by calpain, a calcium-dependent protease, calcium
ionophore-induced processing and secretion of IL-1
from monocytes
and macrophages must be due, in part, to the activation of calpain and
the subsequent accumulation of mature IL-1
, the form of IL-1
that is most efficiently secreted by
cells(5, 6, 25, 26) . In this study,
we have demonstrated that calpain is indeed involved in the in vivo processing of precursor IL-1
, since MDL 28,170, a
cell-penetrating inhibitor of calpain(31) , prevented the
ionomycin-induced processing of IL-1
in the P388D1 macrophage cell
line (Table 2). Since precursor IL-1
is processed by ICE, a
protease that does not require
calcium(7, 8, 9) , it is not evident why
calcium ionophores should also enhance the processing of precursor
IL-1
. We were thus interested in determining if calpain was
involved in precursor IL-1
processing and secretion. Although both
purified calpain and calpain-containing P388D1 lysates cleave
recombinant precursor IL-1
to a single major species, the 16-kDa
mature IL-1
protein (Fig. 1B and (6) ),
the pattern of proteolysis observed when recombinant precursor
IL-1
is treated with the two calpain sources is substantially
different (compare Fig. 1A with Fig. 3). Whereas
purified calpain generated several species from the precursor IL-1
protein, calpain-containing lysates generated only two products, a
major 27-kDa species and a minor 17-kDa species. In addition, whereas
at higher concentrations of calpain, the cleavage of precursor
IL-1
was almost complete, a substantial amount of precursor
IL-1
remained intact in lysates containing equivalent amounts of
calpain. These results imply that the lysate contains a factor(s) that
not only favors the generation of the 27-kDa protein, but also protects
a significant amount of the precursor IL-1
protein from calpain
proteolysis. Recombinant precursor IL-1
expressed in P388D1 cells
was also protected from calpain-mediated proteolysis (Fig. 7),
although the same conditions facilitate the complete processing and
secretion of precursor IL-1
(26) .
The estimated calpain
concentration in the P388D1 lysate is approximately 7.5 units/ml. On a
per cell basis there appears to be sufficient calpain to completely
degrade precursor IL-1. The in vivo stability of the
precursor IL-1
protein in the presence of high levels of calpain
is consistent with the notion that within the cell, precursor
IL-1
, unlike precursor IL-1
, may be prevented from
interacting with calpain. Where then are the precursor IL-1 proteins
and their respective processing enzymes located within the cell? Both
forms of precursor IL-1 lack an NH
-terminal as well as
internal hydrophobic signal sequences and are believed to be
synthesized on free ribosomes in the
cytoplasm(4, 33) . Agents such as brefeldin A and
monensin do not block secretion of IL-1
from LPS-activated
monocytes, indicating that IL-1 proteins do not traverse the classical
secretory pathway (22) . Immunoelectron microscopy studies have
clearly shown that precursor IL-1
is present in the cytoplasmic
ground substance of activated monocytes and absent in all other
organelles such as the endoplasmic reticulum, the Golgi apparatus,
plasma membrane, and lysosomal vesicles(34) . The subcellular
location of precursor IL-1
is not as well studied as that of
precursor IL-1
, but it is also found mainly in the cytoplasmic
fraction of activated monocytes(22) . Although pro-ICE is found
only in the cytoplasm of cells(32) , the intracellular location
of the active form of ICE is not known(35) . Calpain, on the
other hand, is a cytosolic enzyme that has been shown to translocate to
the inner side of the plasma membrane under various stimulatory
conditions(36, 37) . Although calpain, ICE, and the
precursor IL-1 proteins may be initially located within the cytoplasm
in resting cells, the stimulation of macrophages with calcium
ionophores may result in the translocation of the processing enzymes
and precursor IL-1 proteins to the plasma membrane. Precursor
IL-1
, as well as precursor IL-1
, have been shown to be
myristoylated (38) and are theoretically capable of associating
with membranes. The results of preliminary experiments indicate,
however, that calpain is present only in the cytosolic fraction of
ionomycin-treated P388D1 cells (data not shown). Nonetheless, our
results are consistent with the hypothesis that precursor IL-1
is
spared from calpain degradation in cells. However, the available
evidence does not favor the view that IL-1
is sequestered in
vesicles. It is more likely that in vivo, precursor IL-1
is associated with a factor(s) that protects it from cleavage by
calpain. In support of this notion, Singer et al.(34) found that precursor IL-1
is present in the
cytoplasm in the form of clusters, perhaps in association with a
condensing molecule. Although our data suggest a physical separation
between the two IL-1 species and their respective enzymes during
processing, these barriers could break down under certain regulatory
conditions, facilitating the down-regulation of IL-1
levels by
calpain. Future studies regarding the intracellular location of the
IL-1 substrates and their respective enzymes during resting and
stimulatory conditions will allow us to evaluate this possibility.
The ability of ICE to process precursor IL-1 has been well
established. Treatment of human peripheral blood monocytes with potent
ICE-specific inhibitors prevents LPS and Staphylococcus
aureus-induced processing and secretion of
IL-1
(8, 28) . In addition, monocytes and
macrophages derived from mice carrying disrupted ICE alleles are unable
to process and secrete IL-1
in response to LPS
stimulation(29, 30) . Although these observations
favor ICE as the precursor IL-1
-processing enzyme, the THP.1
monocytic cells and human monocytes have been shown to contain
surprisingly low levels of the p20/p10 form of ICE that is
enzymatically active. In these cells, ICE is present predominantly as
the inactive 45-kDa protein(32) . Cytoplasmic extracts from
these cells contain no detectable precursor IL-1
cleavage
activity, even after LPS stimulation, and require overnight incubation
at 4 °C to generate enzyme activity(32) . In light of the
calcium ionophore-mediated enhancement of precursor IL-1
processing and secretion, it is possible that a calcium-dependent
co-factor is involved in promoting the efficient autocatalysis of
pro-ICE to active ICE. Alternatively, such a factor might enhance the
activity of ICE, perhaps by stabilizing the enzyme or by converting
precursor IL-1
to a form that is much more easily cleaved by ICE.
Our results showed that in vitro, the activity of ICE is not
enhanced by calpain, nor does ICE convert the calpain-generated 27-kDa
species to the mature protein ( Table 1and Fig. 6).
Although we cannot rule out the possibility that calpain may enhance
ICE activity in vivo, our inability to detect a 27-kDa species
in calpain-activated P388D1 cells argues against an indirect role of
calpain in precursor IL-1
processing.
Calcium ionophores have
been shown to enhance the secretion of the mature IL-1 proteins as well
as several other proteins such as basic fibroblast growth factor,
thioredoxin, L-29, and CBP30 that also lack an NH-terminal
secretory signal
sequence(18, 20, 21, 22, 23, 24) .
Although the secretory mechanism for these proteins is unknown, several
models have been proposed and include 1) the fusion of secretory
vesicles containing these proteins with the plasma membrane, 2)
extrusion of the proteins localized just below the plasma membrane in
cytoplasmic blebs or podosomes which later release their contents
extracellularly, and 3) direct traversion of these proteins across the
plasma membrane assisted perhaps, by proteins belonging to the
multidrug resistance family. Whatever the secretory mechanism for IL-1,
it must be closely linked to the IL-1 processing machinery, since the
mature forms of IL-1 have not been found in association with cells.
Given the involvement of calpain in precursor IL-1
processing, we
were interested in determining if calpain was also involved in the
secretion of the mature IL-1
protein. Calpain might facilitate
secretion by promoting podosome formation or by localized disruption of
the cytoskeletal structure. Indeed, cytoskeletal proteins such as
tubulin and microtubule-associated proteins have been shown to be
susceptible to calpain digestion(27) . Inhibition of calpain by
MDL 28,170, however, did not inhibit ionomycin-induced secretion of
recombinant mature IL-1
from P388D1 cells (Table 2). This
finding implies a calcium-dependent factor other than calpain in
IL-1
secretion and underscores the independent nature of the
processing and secretory events(26) . Ionomycin-induced
secretion of mature IL-1
from the P388D1 cells was also unaffected
by MDL 28,170, indicating that calpain is also not involved in
IL-1
secretion. If calpain is not involved in the secretion of the
mature IL-1 proteins or in precursor IL-1
processing, what other
calcium-dependent factors might regulate these two processes? Other
members of the EF-hand family of calcium-binding proteins implicated in
protein secretion are calmodulin and calcyclin. Calmodulin has been
shown to be involved in the glucose-stimulated release of insulin from
pancreatic
cells(39) , catecholamine release from adrenal
medullary cells(40) , and acid secretion from
osteoclasts(41) . Calcium-dependent insulin release from
streptolysin O-permeabilized pancreatic
cells has also been shown
to be enhanced by calcyclin and inhibited by calcyclin
antibodies(42) . The calmodulin antagonists
trifluoperazine-dimaleate and W-7, however, did not inhibit
ionomycin-induced secretion of the mature IL-1
and -
proteins
from P388D1 cells in preliminary experiments. (
)Members of
the annexin family of calcium-binding proteins have also been shown to
be involved in exocytosis, perhaps by promoting membrane contact
between vesicles and the cytoplasmic face of the plasma membrane and
also in endocytosis where they may facilitate the closing off of
vesicles from the plasma membrane(43) . In view of the
observation that methylamine, an inhibitor of exocytosis and
endocytosis prevents the secretion of IL-1
as well as several
other proteins which lack an N-terminal secretory signal such as basic
fibroblast growth factor(23) , thioredoxin(24) , and
carbohydrate-binding protein 30(21) , it is possible that
annexins may function in the IL-1 secretory pathway. Processing and
secretion of IL-1
, like that of IL-1
, may involve two
different calcium-dependent factors. Secretion of both the mature IL-1
species may even involve a common calcium-dependent factor. Development
of a permeabilized cell system and the availability of compounds that
inhibit the function of calcium-binding proteins should facilitate the
analysis of the role of these proteins in IL-1 secretion.
Although
it is well accepted that processing of precursor IL-1 and
IL-1
involves two different enzymes, very little is known about
where these processes occur within the cell. The data presented in this
study indicate that the two IL-1 species and their respective enzymes
must be differentially localized, at least during a portion of the
processing and secretory pathway. Processing and secretion, which are
clearly independent events, are nonetheless closely linked as evidenced
by the absence of the mature IL-1
and -
proteins within
cells. Clearly, further studies regarding the precise intracellular
location of each of the two IL-1 species and their possible
co-localization with calpain and ICE need to be done in order to better
understand the processing and secretion of IL-1.