From the Departments of Pharmacology and
§ Biochemistry, Graduate School of Pharmaceutical Sciences,
Hokkaido University, Sapporo 060-0812, Japan
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ABSTRACT |
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We previously reported that several stresses can
induce cytokine-induced neutrophil chemoattractant expression in a
nuclear factor Nuclear factor The ubiquitin-dependent degradation of regulatory
short-lived proteins plays an important role in cellular processes,
including the cell cycle, immune system functions, inflammatory
responses, and tissue differentiation. A key element in the regulation
process is E3, a member of the ubiquitin-substrate ligase family of
enzymes. After binding of the substrate through a specific structural
motif, E3 transfers activated ubiquitin moieties from a
ubiquitin-conjugating enzyme, E2, to a Lys residue in the target
protein to generate a polyubiquitin chain. The tagged substrate is
proteolyzed by a 26 S proteasome complex with the release of free and
reutilizable ubiquitin (11-13). The activation of the enzymatic
component of the ubiquitin system can render the substrates susceptible
to conjugation and subsequent degradation. The proteasome is considered to be a crucial component in the ubiquitin-dependent
proteolytic system (14). In this system, the proteasome functions in an ATP-dependent manner as a 26 S complex, which is assembled
from a 20 S complex and other several regulatory subunits in the
presence of ATP (15-17). These proteins are involved in physiological
homeostasis processes such as the cell cycle, DNA replication, and
stress response. However, regulation of these activities is unclear.
In this study, we attempted to elucidate how NF- Materials--
C6 glioma cells were obtained from the American
Type Culture Collection. Restriction endonucleases, dNTP mixture, and
RNase inhibitor were purchased from Takara (Kyoto, Japan).
Oligo(dT)12-18 primer, 5× first strand buffer, and
reverse transcriptase were from Life Technologies, Inc. The Expand High
Fidelity PCR system and rat IL-8 (CINC/gro) ELISA kits were obtained
from Roche Molecular Biochemicals and Amersham International
(Buckinghamshire, United Kingdom), respectively. Herbimycin A was
obtained from Wako (Osaka, Japan). MG132 and PSI were purchased from
Calbiochem. Anti-I Cell Culture--
C6 glioma cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum, 50 µg/ml penicillin, and 100 µg/ml streptomycin in a humidified
incubator containing 5% CO2.
RNA Isolation and PCR Analysis--
Total RNA was prepared from
1.2 × 107 cells using guanidium/cesium chloride as
described previously (18, 19). PCR was performed on total RNA extracted
from cultures. Total RNA (2 µg) was incubated at 37 °C for 60 min
with a mixture of 100 units of reverse transcriptase, 1× first strand
buffer, 10 mM DTT, a 0.5 mM concentration of
each dNTP, and 50 units of RNase inhibitor to a final volume of 20 µl. The reaction mixture was then incubated for 10 min at 70 °C to
inactive the reverse transcriptase. An aliquot (2 µl) of reverse transcriptase product was mixed with 1 milliunit of DNA polymerase and
a 200 nM concentration of each of the sense and antisense primers in a buffer containing 1× PCR buffer and a 0.2 mM
concentration of each dNTP in a final volume of 20 µl. The mixture
was overlaid with 30 µl of liquid paraffin to prevent evaporation and
then amplified by 20 cycles of PCR as described (19). The number of
cycles that produced a linear relationship between the amount of input
RNA and the resulting PCR products was used for each primer pair. The
PCR products were resolved by electrophoresis on a 6% polyacrylamide
gel in 0.5× Tris borate/EDTA. The gel was stained with ethidium
bromide and photographed. The primers used are as follows: rat CINC-1,
5'-ATG GTC TCA GCC ACC CGC TCG-3' (positions 37-57; upstream) and
5'-GAC ACC CTT TAG CAT CTT TTG-3' (positions 298-318; downstream); rat
I ELISA--
ELISA for rat IL-8 (CINC/gro) antigen expressed in
the culture supernatant was performed using a kit from Amersham
International. The limit of detection was <0.08 ng/ml.
Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extracts were prepared using previously described methods (6, 19).
Briefly, 5 × 106 cells were harvested; washed once
with 2 ml of ice-cold phosphate-buffered saline and resuspended in 400 µl of buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride,
and 20 units/ml aprotinin. After incubation for 15 min on ice, Nonidet
P-40 was added to a final concentration of 0.6%, and the mixture was
vortexed vigorously for 10 s. The nuclei were precipitated;
resuspended in 50 µl of buffer containing 20 mM HEPES (pH
7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM
EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl
fluoride, and 20 units/ml aprotinin; and vortexed vigorously for 15 min
at 4 °C. The lysate were centrifuged at 15,000 rpm for 20 min at
4 °C, and the supernatants containing the nuclear proteins were
transferred into new vials. The protein concentration of each extract
was measured using a Bio-Rad protein assay kit.
EMSAs were performed by incubating 7.5 µg of nuclear extracts with 2 µg of poly(dI-dC) in binding buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM
DTT, 1 µg/ml bovine serum albumin, 0.05% Nonidet P-40, and 5%
glycerol) in a 20-µl final volume for 30 min at 4 °C. Then, an
end-labeled double-stranded oligonucleotide probe (50,000 cpm/0.3 ng)
was added, and the reaction mixture was incubated for 15 min at room
temperature. For the supershift assay with specific antibodies against
NF- Oligonucleotides--
The double-stranded oligonucleotides used
as competitors in the EMSA are as follows: blunt-ended competitors and
NF- Western Blot Analysis--
Cells (5 × 106)
were washed twice with ice-cold phosphate-buffered saline, and then 200 µl of lysis buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM DTT, and 1% Nonidet P-40) was
added. In particular, for the detection of ubiquitinated I Metabolic Labeling and Immunoprecipitation--
Cells (5 × 106) in 10-cm plates were washed twice with prewarmed
(37 °C) methionine- and cysteine-free Dulbecco's modified Eagle's
medium. The cells were then labeled 15 min before and 30 min after
IL-1 Assay for Chymotrypsin-like Activity in Cell Lysates--
Cells
(1 × 106) were stimulated with 5 ng/ml IL-1 Ability to Degrade Ubiquitinated I Induction of CINC mRNA and Protein in Response to
IL-1 Inhibitory Effects of Proteasome Inhibitors and Herbimycin A on
IL-1 Induction and Identification of NF-
Since proteasome inhibitors and herbimycin A suppressed IL-1 I
The phosphorylation of I
Next, we performed a pulse-chase study to examine the degradation rates
during IL-1 IL-1
On the other hand, chymotrypsin-like (Suc-LLVYase) activity in the
presence of ATP in the proteasome-containing fraction, which was
prepared by centrifugation at 100,000 × g for 20 min, was transiently enhanced and peaked 15 min after IL-1
To elucidate the causality between the enhancement of Suc-LLVYase
activity and the degradation of phosphorylated and multi-ubiquitinated I The aim of this study was to investigate the mechanism of NF- Recent studies have provided some insights into the mechanisms leading
to I Surprisingly, we found that resynthesized I We next examined Suc-LLVY cleaving activity in the cytosolic fraction.
The chymotrypsin-like (Suc-LLVYase) activity in the proteasome-containing cytosolic fraction prepared by centrifugation at
100,000 × g for 20 min in response to IL-1 Subsequently, we studied the mechanism of the transient Suc-LLVYase
activation stimulated by IL-1 It is recognized that the level of resynthesized I We demonstrated here that both NF- We propose here a novel regulation system for NF-B (NF-
B)-dependent manner. In this
study, we focused further on the regulation of NF-
B. The activation
of NF-
B and the subsequent cytokine-induced neutrophil
chemoattractant induction in response to interleukin-1
(IL-1
)
were inhibited by proteasome inhibitors, MG132 and proteasome inhibitor
I. Translocation of NF-
B into nuclei occurs by the phosphorylation,
multi-ubiquitination, and degradation of I
B
, a regulatory protein
of NF-
B. Nascent I
B
began to degrade 5 min after treatment
with IL-1
and disappeared completely after 15 min. However, I
B
returned to basal levels after 45-60 min. Interestingly, resynthesized
I
B
was already phosphorylated at Ser-32. These results suggest
that 1) the upstream signals are still activated, although the
translocation of NF-
B peaks at 15 min; and 2) the regulated
protein(s) acts downstream of I
B
phosphorylation. Western
blotting showed that the resynthesized and phosphorylated I
B
molecules were also upward-shifted by multi-ubiquitination in response
to IL-1
treatment. On the other hand, ATP-dependent Leu-Leu-Val-Tyr cleaving activity transiently increased, peaked at 15 min, and then decreased to basal levels at 60 min. Furthermore, the
cytosolic fraction that was stimulated by IL-1
for 15 min, but not
for 0 and 60 min, could degrade phosphorylated and multi-ubiquitinated I
B
. These results indicate that the transient translocation of
NF-
B in response to IL-1
may be partly dependent on transient proteasome activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(NF-
B)1 participates in
the regulation of the expression of multiple immediate-early genes
involved in immune, acute-phase, and inflammatory responses (1).
NF-
B is a heterodimer protein of the Rel family of transcription
factors. In mammalian cells, the factors include p65 (RelA), RelB,
c-Rel, p50/p105 (NF-
B1), and p52/p100 (NF-
B2). NF-
B proteins
are constitutively present in cells and are retained in the cytoplasm
associated with the inhibitory protein I
B (2, 3). Activated NF-
B
complexes, typically composed of p50 and p65, are translocated to the
nucleus in response to several cytokines (TNF-
, IL-1
, and IL-2),
bacterial endotoxin, and stresses (UV, H2O2).
(1, 4-6). The activation of NF-
B appears to require the
phosphorylation and degradation of the I
B proteins, thereby allowing
the rapid translocation of NF-
B from the cytoplasm to the nucleus
(4, 7-9). In particular, it has been shown that the phosphorylation of
Ser-32 and Ser-36 and the ubiquitination at Lys-21 and Lys-22 are
essential for targeting I
B for signal-induced degradation by the
ubiquitin/proteasome system (10).
B activation in
response to IL-1
is regulated. We report that CINC production through NF-
B induced by IL-1
is sensitive to proteasome
inhibitors. The phosphorylation of Ser-32 and the degradation of I
B
occurred rapidly, followed by I
B
protein resynthesis.
Interestingly, we found that the resynthesized I
B
protein was
already phosphorylated (Ser-32), suggesting that upstream kinases are
still activated during this period. Moreover, proteasome activity, but
not ubiquitination, transiently increased during IL-1
treatment
accompanying NF-
B activation. We present here initial evidence that
the chymotrypsin-like activity of the proteasome plays an important
role in the cytokine-induced transient activation of NF-
B.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
and anti-phospho-specific I
B
(Ser-32)
antibodies were from Santa Cruz Biotechnology and New England Biolabs,
Inc., respectively. Suc-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide,
benzyloxycarbonyl-leucyl-leucinal (calpain inhibitor), and E-64-d
(inhibitor of thiol protease) were from Peptide Inc. (Osaka, Japan).
All other reagents were purchased from Sigma.
B
, 5'-TCT CCA CTC CGT CCT GCA GG-3' (positions 513-532;
upstream) and 5'-TTA TAA CGT CAG ACG CTG GCC TCC-3' (positions
922-945; downstream); and rat GAPDH, 5'-AAA CCC ATC ACC ATC TTC CAG-3'
(positions 238-258; upstream) and 5'-AGG GGC CAT CCA CAG TCT TCT-3'
(positions 578-598; downstream).
B, nuclear extracts were preincubated with 1 µg of each
antibody for 2 h at 4 °C before the addition of the end-labeled
double-stranded oligonucleotide probe. Samples were separated by 5%
native polyacrylamide gel electrophoresis in low ionic strength buffer
(0.25× Tris borate/EDTA).
B-binding site (5'-AGT TGA GGG GAC
TTT CCC AGG C-3'; the core recognition sequence
of this oligonucleotide is underlined).
B
proteins, the cells were lysed with lysis buffer containing 0.1% SDS
and 5 mM N-ethylmaleimide (20). The total
lysates were centrifuged at 15,000 rpm for 30 min at 4 °C, and the
supernatants were removed as crude cytosolic fractions. The cytosol was
boiled with SDS sample buffer for 5 min. Equal amounts of each sample
were subjected to 12% SDS-polyacrylamide gel electrophoresis at 100 V
for 1 h at 4 °C, followed by transfer to a nitrocellulose
filter. The filters were then blocked with 10 mM Tris-HCl
(pH 7.5), 100 mM NaCl, and 0.1% Tween 20 containing 5%
nonfat milk for 1 h at room temperature. Anti-I
B and
anti-phospho-specific I
B antibodies were used as primary antibodies,
and horseradish peroxidase-labeled rabbit Ig was used as the secondary
antibody. The antibody-reactive bands were revealed by
chemiluminescence (ECL Western detection kit).
stimulation in 10 ml of methionine- and cysteine-free Dulbecco's modified Eagle's medium containing 0.2 mCi/ml
Tran35S-label (ICN Biomedicals, Irvine, CA). Pulse-labeled
cells were chased for various times for up to 60 and 240 min in
complete medium containing 2.5 mM methionine and cysteine,
respectively. At the end of each time point, the cells were solubilized
on ice with lysis buffer. The lysates were immunoprecipitated using
anti-I
B
antibody and analyzed on 12% SDS-polyacrylamide gel.
Dried gels on Whatman No. 3MM paper were exposed on an imaging plate
and visualized on a Fuji BAS 2000 apparatus.
for
the indicated time periods (0-60 min) and then washed twice with
ice-cold phosphate-buffered saline, scraped into 200 µl of lysis
buffer (50 mM HEPES (pH 7.5), 2 mM ATP, 0.1 mM EDTA, 0.1 mM EGTA, and 1 mM
DTT), and homogenized by 20 strokes in a Dounce homogenizer on ice. The
homogenate was centrifuged at 10,000 × g for 10 min at
4 °C, and the resultant supernatants were centrifuged at
100,000 × g for 20 min or 5 h at 4 °C. Each
supernatant was used for quantification of the chymotrypsin-like
activity as described (21, 22). The activity was assayed at 37 °C
for 15 min in 50 mM Tris-HCl (pH 7.8) containing 1 mM DTT, 2 mM ATP, 10 mM
MgCl2, and 0.1 mM
Suc-LLVY-4-methylcoumaryl-7-amide as a substrate. The reaction was
stopped by adding 1 ml of 10% SDS. The amounts of released
4-methylcoumaryl-7-amide were measured with a spectrofluorometer
(Hitachi F-2000 fluorescence spectrophotometer) with excitation at 380 nm and emission at 460 nm.
B
in Cytosolic Fractions
Stimulated by IL-1
--
Cells (5 × 106) were
stimulated with 5 ng/ml IL-1
for the indicated time periods (0, 15, and 60 min), and the supernatants were isolated as described above and
used for the assay of ubiquitinated I
B
degradation. Each
cytosolic fraction (20 µg) was incubated with the undegraded
ubiquitinated I
B
-accumulated fraction (40 µg) (see Fig.
7A, lane 6) at 37 °C for 60 min. The reaction
was terminated by adding SDS sample buffer. The samples were then boiled for 5 min and subjected to 12% SDS-polyacrylamide gel
electrophoresis, followed by transfer to a nitrocellulose filter. The
degradation of ubiquitinated I
B
was detected by Western blot
analysis using anti-phospho-specific I
B antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
Initially, we examined whether mRNA levels of CINC
and GAPDH were affected by IL-1
. A 24-h exposure to IL-1
did not
alter cell viability (data not shown). Furthermore, the housekeeping gene GAPDH mRNA was not altered at the stages examined (Fig.
1A). CINC mRNA was not
detected after treatment with IL-1
. Kinetic analysis showed that
IL-1
-induced CINC mRNA expression peaked at 30-60 min. We then
used ELISA to investigate the levels of CINC protein after treatment
with IL-1
. Treatment resulted in time-dependent
induction of CINC proteins into the culture supernatant. CINC proteins
were significantly elevated 12 h after IL-1
challenge and
continued to accumulate for up to 48 h (Fig. 1B).
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Fig. 1.
Induction of CINC mRNA and protein in
response to IL-1 in C6 glioma cells. The
mRNA levels were analyzed by RT-PCR using CINC-specific primers as
described under "Experimental Procedures." The number of cycles
selected for each primer pair produced a linear relationship between
the amounts of input RNA and the PCR products. The PCR products were
electrophoresed on 6% polyacrylamide gels and visualized by ethidium
bromide staining. These are typical results from four independent
experiments. CINC protein in the culture medium was detected by a
specific ELISA. The data represent the results of four independent
experiments. Each value is the mean ± S.E. (error
bars) of four determinations. A, time course of CINC
mRNA induction by 5 ng/ml IL-1
; B, IL-1
-induced
release of CINC protein.
, control;
, 5 ng/ml IL-1
.
-induced CINC mRNA and Protein Expression--
To elucidate
the involvement of proteasomes and herbimycin A-sensitive proteins
(possibly tyrosine kinases) in IL-1
-induced CINC expression, we
investigated the effects of several inhibitors. Both MG132 and PSI,
which are cell-permeable protease inhibitors, attenuated CINC mRNA
induction in a concentration-dependent manner (Fig.
2, A and B).
Furthermore, CINC proteins were significantly suppressed by
pretreatment with either inhibitor (Fig. 2D). Herbimycin A,
a potent tyrosine kinase inhibitor, also attenuated CINC mRNA and
protein expression (Fig. 2, C and D).
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Fig. 2.
Effects of protease inhibitors and herbimycin
A on IL-1 -stimulated CINC expression.
Cells were pretreated with the indicated concentrations of MG132, PSI,
and herbimycin A (Herb A) for 1, 2, and 12 h,
respectively, and then stimulated with 5 ng/ml IL-1
for 30 min (for
RT-PCR assay) or 6 h (for ELISA). Total cellular RNA and the
culture medium were prepared and analyzed by RT-PCR and ELISA in as
described in the legend of Fig. 1. Values represent the mean ± S.E. of triplicate cultures run in parallel. **, p < 0.01 versus effect of IL-1
alone.
B Complexes in Response to
IL-1
--
We previously demonstrated that NF-
B activation in C6
glioma cells in response to several stresses is involved in CINC
expression (19). In this study, we examined whether IL-1
can also
induce NF-
B activation. Nuclear extracts were prepared, and samples were subjected to EMSA using a DNA probe containing the NF-
B-binding element as described under "Experimental Procedures." As shown in
Fig. 3A, no binding of NF-
B
to radiolabeled probes was detected in unstimulated cells. However,
after treatment with IL-1
, there was significant transient binding,
peaking at 15 min. Binding completely disappeared when the nuclear
extracts were incubated with non-radiolabeled DNA (Fig. 3B).
Supershift assays in EMSA using specific antibodies against NF-
B p50
and p65 revealed that IL-1
-stimulated NF-
B is a heterodimer
(p50/p65) (Fig. 3C).
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Fig. 3.
IL-1 activates
NF-
B binding activity. The cells were
treated with 5 ng/ml IL-1
for the time periods indicated, and
nuclear extracts were prepared and subjected to EMSA as described under
"Experimental Procedures." For supershift assays, nuclear extracts
were incubated in the presence of 1 µg of specific antibodies against
each NF-
B component and an excess of unlabeled DNA probe (5-50-fold
with the probe). CT represents control rabbit IgG.
A, time course of NF-
B activation induced by IL-1
;
B, competition analysis of NF-
B; C,
identification of the NF-
B component.
-induced
CINC mRNA and protein expression, we examined the effects of these
inhibitors on NF-
B activation by IL-1
. Proteasome inhibitors and
herbimycin A partially and completely attenuated NF-
B activation, respectively (Fig. 4).
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Fig. 4.
Effects of protease inhibitors and herbimycin
A on IL-1 -stimulated
NF-
B activation. Cells were pretreated
with 10 µM MG132, 20 µM PSI, or 1 µg/ml
herbimycin A (Herb A) for 1, 2, or 12 h, respectively,
and then stimulated with 5 ng/ml IL-1
for 15 min. Nuclear extracts
were prepared and subjected to EMSA as described under "Experimental
Procedures."
B
Is Degraded and Thereafter Rapidly Resynthesized during
IL-1
Treatment--
NF-
B activation is mainly dependent on I
B
degradation. Before degradation, I
B is serine-phosphorylated by
I
B kinase and then ubiquitinated by ubiquitin ligase (23). We
analyzed I
B
levels at the indicated sampling times by Western
blotting using anti-I
B
antibody. The levels of I
B
were
decreased after 5 min in response to IL-1
and then disappeared after
15 min. Surprisingly, I
B
was re-detected at 30 min and returned
to basal levels at 45-60 min (Fig.
5A). Cycloheximide, a protein
synthesis inhibitor, completely blocked the resynthesis of I
B
.
Proteasome inhibitors and herbimycin A, which inhibited NF-
B
activation by IL-1
, blocked the degradation of I
B
. Fig.
5B shows the mRNA levels of I
B
during IL-1
treatment. I
B
mRNA was detected 5 min after stimulation and
peaked at 45-60 min.
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Fig. 5.
Resynthesis and phosphorylation of
I B
at Ser-32 in C6
glioma cells challenged with IL-1
. Cells
were pretreated with 10 µM MG132, 20 µM
PSI, 1 µg/ml herbimycin A (Herb A), or 0.1 µM cycloheximide (CHX) for 1, 2, 12, or 1 h, respectively, and then stimulated with 5 ng/ml IL-1
for the
indicated time periods. Cytosolic extracts (20 µg) and total RNA from
the cells were subjected to Western blot analysis and RT-PCR,
respectively. A, detection of I
B
during IL-1
treatment by Western blot analysis using anti-I
B
antibody;
B, detection of resynthesized I
B
mRNA by the
RT-PCR method; C, detection of the phosphorylated state of
I
B
by Western blot analysis using anti-phospho-specific (Ser-32)
I
B
antibody. Total cell extracts from HeLa cells prepared with
TNF-
(10 ng/ml, 5 min) treatment were used as positive controls
(P. C.).
B
at Ser-32 and Ser-36 stimulates
conjugation with ubiquitin and subsequent proteasome-mediated degradation, resulting in the translocation of NF-
B. Therefore, the
phosphorylation of I
B
at Ser-32 and Ser-36 is essential for the
activation of NF-
B. We investigated whether I
B
is
phosphorylated in response to IL-1
using antibody that specifically
recognizes the phosphorylated Ser-32 on I
B
. No detectable bands
were observed in the unstimulated state. Treatment with IL-1
stimulated the phosphorylation of I
B
at 5 min, and then all
phosphorylated I
B
was degraded at 15 min. Interestingly,
resynthesized I
B
was phosphorylated at Ser-32 (Fig.
5C). Although neither MG132 nor PSI influenced the
phosphorylation of I
B
, herbimycin A completely inhibited
IL-1
-induced serine phosphorylation.
stimulation in C6 glioma cells. The cells were labeled
15 min before and 30 min after IL-1
stimulation in medium containing
[35S]Met/Cys and chased in medium containing 2.5 mM Met and Cys for 60 and 240 min, respectively. As shown
in Fig. 6A, nascent I
B
degraded rapidly and almost disappeared 15 min after IL-1
stimulation, with a time course similar to the results of Western blot
analysis using anti-I
B
antibody (Fig. 5A). Thereafter,
I
B
protein was not detected. However, as shown in Fig.
6B, the resynthesized I
B
protein was not degraded, as
seen in the early step (0-15 min after stimulation). These results
indicate that the degradation rates of I
B
in the early (0-15 min
after stimulation) and late (45-240 min) steps are different.
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Fig. 6.
Degradation state of
I B
during
IL-1
treatment. Cells were metabolically
labeled for 15 min with Tran35S-label during the time
periods indicated by the closed bars and then chased for the
indicated time periods. In B, the cells were stimulated by
IL-1
for 30 min and then metabolically labeled for 15 min with
exposure to a similar concentration of IL-1
. Thereafter, the cells
were chased for up to 240 min. The reaction was stopped at each time
indicated by the arrows. I
B
proteins were
immunoprecipitated and analyzed on SDS-polyacrylamide gels as described
under "Experimental Procedures."
Transiently Enhances Chymotrypsin-like Activity, but Not
Ubiquitination--
We further examined how the activation of NF-
B
is regulated. It is believed that the phosphorylation and
multi-ubiquitination of I
B are critical for degradation by 26 S
proteasomes. We first measured the multi-ubiquitination state of
I
B
by Western blotting using anti-phospho-specific I
B
antibody because only phosphorylated I
B molecules are ubiquitinated.
We prepared cell extracts at different times after treatment of C6
cells with 5 ng/ml IL-1
in the presence or absence of MG132 and then
analyzed the samples by Western blotting. The extracts were prepared in
the presence of SDS (0.1%) and N-ethylmaleimide (5 mM) to inhibit isopeptidase activities, as described (20).
As shown in Fig. 7A, a ladder of high molecular mass proteins appeared following stimulation with
IL-1
. The molecular mass increments of this ladder were ~8.5 kDa,
which is the size of ubiquitin. The ubiquitination of I
B
peaked
at 5 min following stimulation and then decreased at 15 min.
Resynthesized and phosphorylated I
B
was upward-shifted again by
30-60 min. We next examined the effect of MG132 on IL-1
-induced ladder formation. Treatment of C6 cells with MG132 alone (60 min) led
to a slight accumulation of phosphorylated and ubiquitinated I
B
.
Although MG132 inhibited the degradation of I
B
, but not its
phosphorylation (Fig. 5C), phosphorylated and ubiquitinated I
B
proteins were clearly detected under these conditions (Fig. 7B). The ubiquitination of I
B
peaked at 15 min after
stimulation and then decreased by 30-60 min, possibly because of
residual activities of proteasomes and isopeptidases.
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Fig. 7.
Multi-ubiquitination of
I B
and activation of
proteasomes in response to IL-1
. Cells
were pretreated with (B) or without (A) 10 µM MG132 for 1 h and then stimulated with 5 ng/ml
IL-1
for the indicated time periods. The cell extraction buffer
contained 0.1% SDS and 5 mM N-ethylmaleimide.
The extracts (40 µg) were then subjected to Western blot analysis
using anti-phospho-specific (Ser-32) I
B
antibody. The time course
of proteasome activities stimulated by IL-1
is shown (C).
Cells were stimulated with 5 ng/ml IL-1
for the indicated time
periods (0-60 min) and washed twice with ice-cold phosphate-buffered
saline. The cytosolic fraction was prepared as described under
"Experimental Procedures." The activity toward
Suc-LLVY-4-methylcoumaryl-7-amide was assayed in the presence of 2 mM ATP. p-I
B
, phosphorylated I
B
;
[Ub]n-p-I
B
, multi-ubiquinated
p-I
B
.
stimulation (Fig. 7C). In contrast, the activity in the cytosolic
fraction prepared by centrifugation at 100,000 × g for
5 h was reduced to ~75% of the sample prepared by
centrifugation at 100,000 × g for 20 min because the
high molecular mass proteasomes (700-1000 kDa) are precipitated by the
prolonged centrifugation.2 In
addition, we measured the activity in the TNF-
-treated cytosolic fractions. TNF-
, which can induce NF-
B activation, also increased Suc-LLVYase activity in a similar manner to IL-1
-treated
cells.3 Next, we investigated
the effects of several inhibitors on IL-1
-enhanced proteasome
activation. The cells were pretreated with MG132 or PSI for a specified
time period; the cytosolic fractions were isolated; and the activity
was measured. The proteasome inhibitors (MG132 and PSI) completely
blocked the activity in a quiescent state, and enhancement of the
activity was induced by IL-1
(data not shown). Treatment with
E-64-d, a thiol proteinase inhibitor, did not affect the activation in
response to IL-1
. In addition, herbimycin A (a potent tyrosine
kinase inhibitor), benzyloxycarbonyl-leucyl-leucinal (a calpain
inhibitor), and Ro 31-8220 (a nonspecific serine/threonine kinase
inhibitor) did not alter enhancement by IL-1
.
B
, we finally analyzed the ability to degrade ubiquitinated I
B
in IL-1
-stimulated cytosolic fractions. As shown in Fig. 8, only the cytosol stimulated by IL-1
for 15 min, but not for 0 or 60 min, could degrade ubiquitinated
I
B
in the cytosol 60 min after IL-1
stimulation. Treatment
with 10 µM MG132 suppressed the degradation of I
B
(Fig. 8, lane 4).
View larger version (13K):
[in a new window]
Fig. 8.
Degradation of ubiquitinated
I B
in
IL-1
-stimulated cytosolic fractions.
Cells were stimulated with 5 ng/ml IL-1
for 60 min, and then the
cytosolic fraction containing undegraded ubiquitinated I
B
was
prepared as described under "Experimental Procedures." The
proteasome-containing cytosolic fractions were prepared from the cells
stimulated with 5 ng/ml IL-1
for the indicated time periods (0, 15, and 60 min). Each cytosolic fraction was incubated with the undegraded
ubiquitinated I
B
-accumulated fraction at 37 °C for 60 min. The
reaction was terminated by adding SDS sample buffer. The degradation of
ubiquitinated I
B
was detected by Western blot analysis using
anti-phospho-specific I
B antibody. p-I
B
,
phosphorylated I
B
; [Ub]n-p-I
B
,
multi-ubiquitinated p-I
B
.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
regulation, especially via I
B
, in C6 glioma cells. We showed here
that IL-1
stimulates NF-
B activation and subsequent chemokine (CINC) production. Activation was sensitive to proteasome inhibitors (MG132 and PSI) and herbimycin A, indicating that both the proteasome, which degrades I
B
, and a tyrosine kinase are involved in this pathway. Furthermore, IL-1
treatment resulted in the rapid
resynthesis of I
B
. Surprisingly, we found that Ser-32 in
resynthesized I
B
was already phosphorylated. Although the
phosphorylation of this residue is important for ubiquitination and
degradation, no significant translocation of NF-
B was observed.
These results suggest that some proteins downstream from the serine
phosphorylation of I
B
inhibit degradation signaling. Therefore,
we further examined why the activation of NF-
B stimulated by IL-1
is transient.
B
degradation (7, 20, 24-30). The signal-induced degradation
of I
B
is dependent on the presence of an intact COOH-terminal
PEST (Pro, Glu, Ser, Thr) region as well as the phosphorylation of
Ser-32 and Ser-36. These phosphorylated residues probably target the
molecule for ubiquitination at Lys-21 and Lys-22 (29, 30). In turn,
this targets the molecule for degradation by 26 S proteasomes (20),
which occurs when the inhibitor and NF-
B are dissociated. The
I
B
molecule is resynthesized rapidly following degradation,
partly because the promoter of the I
B
gene is positively
regulated by NF-
B. We confirmed that IL-1
stimulates the
phosphorylation and degradation of I
B and its de novo
resynthesis (Fig. 5).
B
is already
phosphorylated at Ser-32, indicating that the receptor-mediated signal
for phosphorylation is active in IL-1
-challenged C6 glioma cells for
at least up to 60 min. Why does phosphorylated I
B
not cause
further NF-
B translocation? To address this problem, we examined the
activities of ubiquitin ligase and proteasomes. We first measured the
state of ubiquitination of the I
B
molecule by Western blotting.
The multi-ubiquitination of I
B
was detected by
anti-phospho-specific I
B
antibody. Multi-ubiquitinated I
B
proteins were detected as upward-shifted proteins (20). Treatment with
IL-1
stimulated ubiquitination (Fig. 7A). However,
multi-ubiquitinated proteins disappeared 15 min after IL-1
challenge, and resynthesized I
B
was phosphorylated and
ubiquitinated. In addition, pretreatment with MG132, a proteasome
inhibitor, caused the accumulation of multi-ubiquitinated I
B
,
which peaked 15 min after IL-1
treatment. These results suggest that
multi-ubiquitination in response to IL-1
is present for at least up
to 60 min after treatment. In addition, phosphorylated and
resynthesized I
B
proteins and the accumulated phosphorylated
I
B
protein in MG132-pretreated cytosolic fractions were barely
dephosphorylated (Fig. 5C). These results suggest that
inactivated enzyme such as some phosphatases is not mainly involved, or
the activity has a very low level in this system, although the
modification of I
B
(serine phosphorylation) that is essential for
NF-
B activation rapidly occurs.
was
transiently activated with a time course similar to that of NF-
B
translocation (Fig. 7C). Since it is well known that LLVY is
a substrate for chymotrypsin (which is slight in glial cells), calpain,
and 20 S and 26 S proteasomes, the activity in the cytosolic fraction may be derived from these proteins. We first investigated the possibility that the chymotrypsin-like (LLVYase) activity is partly derived from proteasome activity. The activity in the cytosolic fraction prepared by centrifugation at 100,000 × g for
5 h was reduced markedly compared with that in the sample prepared
by centrifugation at 100,000 × g for 20 min because
the high molecular mass proteasomes (700-1000 kDa) are precipitated by
prolonged centrifugation. The resulting activity is considered to be
derived from another protein such as calpain, and there was no
enhancement of the activity seen during IL-1
treatment. Moreover,
the calpain inhibitor did not block IL-1
-stimulated I
B
degradation in this system, suggesting that at least the proteasome is
partly activated by treatment with IL-1
. These results indicate that
a transient increase in Suc-LLVY cleaving activity is partly derived
from the proteasome activity. However, the time courses of Suc-LLVYase and NF-
B translocation are merely coincidental; we cannot completely rule out the possibility that the other proteinase is involved in
Suc-LLVY cleavage and I
B degradation.
using several inhibitors: E-64-d (a
thiol protease inhibitor), Ro 31-8220 (a nonspecific serine/threonine
kinase inhibitor), and herbimycin A (a potent tyrosine kinase
inhibitor). Pretreatment with these inhibitors did not affect
IL-1
-stimulated Suc-LLVYase activation. There have been few reports
on the transient activation of proteasomes in response to several
cytokines that can stimulate NF-
B translocation. However, Kawahara
et al. (21) reported that the 26 S proteasome is activated
in prophase and metaphase during the mitotic cell cycle of
synchronously dividing ascidium embryos. Furthermore, proteasomes are
activated during in vivo Xenopus egg activation induced by treatment with the calcium ionophore A23187 (22). The 26 S
proteasome consists of at least two subunits: one is a 700-kDa
proteolytic core complex called the 20 S proteasome with 28 subunits,
and the other is a 700-1000-kDa regulatory subunit complex made up of
~20 subunits (31-38). Although there are many reports that several
proteasome subunits can be phosphorylated, there is little or no direct
effect on proteasome activity from these modifications (39-42). Thus,
it is believed that phosphorylation is involved in assembly, targeting,
or turnover of proteasome subunits. In contrast, Kenneth et
al. (43) demonstrated that the phosphorylation of PA28, referred
to as the 11 S regulator, is required for stimulation of peptidase
activity, although the relevant mechanism remains to be defined.
Therefore, in our system, it may be possible that some modifications
such as phosphorylation are involved in the modulation of Suc-LLVYase
activity, possibly 26 S proteasome activity; however, we could not
directly demonstrate how the 26 S proteasome is regulated.
B
proteins is
very important for the down-regulation of NF-
B activity. We have
found here that the resynthesized proteins are phosphorylated and
multi-ubiquitinated. If the proteasome is involved in the degradation
of I
B
as reported previously, it was clearly expected that
resynthesized (phosphorylated and ubiquitinated) I
B
would also be
degraded, and then the activation of NF-
B would occur again.
However, these events did not occur. Alternatively, if ubiquitinated
I
B
is degraded by other proteinase in this system, the rates of
degradation in the early (0-15 min after stimulation) and late
(45-240 min after stimulation) steps could be the same. However,
pulse-chase analysis showed that the rates are quite different (Fig.
6). We suspect that the enzyme(s) involved in the degradation of
I
B
are down-regulated in the late stage (45-240 min after
IL-1
stimulation). Hence, we investigated the ability to degrade
ubiquitinated I
B
in each cytosolic fraction (0, 15, and 60 min
after IL-1
stimulation). As shown in Fig. 8, only the cytosol
stimulated by IL-1
for 15 min, but not for 0 or 60 min, could
degrade the accumulated ubiquitinated I
B
protein in the cytosol
60 min after stimulation. This reaction was suppressed by proteasome
inhibitor treatment. Therefore, these findings indicate that the
down-regulation of proteasome activity is partly involved in the
NF-
B system. However, we cannot completely deny the possibility that
the transient increase in proteasome activity is a secondary effect of
IL-1
stimulation, although the two processes (proteasome activation
and NF-
B activation) are merely coincidental. As indicated above,
another alternative is that other proteinases may be implicated in this
process. For example, it has been reported that calpain, another
possible regulator of the I
B/NF-
B system, can degrade I
B. Chen
et al. (44) reported that calpain contributes to I
B
degradation stimulated by silica, but not by lipopolysaccharide. However, Traenckner et al. (28) reported that calpain is not primarily involved in TNF-
-induced I
B
degradation. Therefore, we investigated whether or not calpain is involved in I
B
degradation using a calpain inhibitor. The m-calpain inhibitor
benzyloxycarbonyl-leucyl-leucinal was not effective, suggesting that
calpain is not primarily involved in this system.
B activation and CINC expression
in response to IL-1
are completely inhibited by treatment with
herbimycin A in C6 glioma cells. Since herbimycin A also blocked
phosphorylation of I
B
at Ser-32 by IL-1
, an intermediate protein between the receptor and I
B kinase may exist. However, a
herbimycin A-sensitive protein involved in IL-1
-stimulated signaling
has not been found. We have previously reported that NF-
B activation
is essential for inducible nitric-oxide synthase and CINC expression in
response to lipopolysaccharide, TNF-
, and
H2O2 in a herbimycin A-dependent
manner (6, 19). Taken together, our results suggest that there is a
common pathway mediating the stress-stimulated signaling through a
herbimycin A-sensitive protein (possibly tyrosine kinase).
B activation. In
glial cells, IL-1
induces the rapidly sequential phosphorylation, ubiquitination, and degradation of I
B
and the subsequent NF-
B translocation into the nuclei that peaks 15 min after IL-1
treatment. Receptor-mediated signals seem to be activated at least 60 min after stimulation. We demonstrated that the transient enhancement of Suc-LLVYase (possibly proteasome) activity has a time course similar
to that of the translocation of NF-
B. It is obvious that the
regulation of proteasome activity partly contributes to the transient
NF-
B activation. Furthermore, the cytosolic fraction stimulated by
IL-1
for 15 min, but not for 0 and 60 min, had an ability to degrade
ubiquitinated I
B
. In view of these results, it is suggested that
the transient increase in proteasome activity is partly involved in the
NF-
B regulation. Therefore, delineating the precise mechanisms of
proteasome activation in response to IL-1
is needed.
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FOOTNOTES |
---|
* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.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.
¶ To whom correspondence should be addressed. Tel.: 81-11-706-3246; Fax: 81-11-706-4987; E-mail: nomura{at}pharm.hokudai.ac.jp.
2 T. Uehara, M. Kaneko, and Y. Nomura, unpublished data.
3 T. Uehara and Y. Nomura, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
NF-B, nuclear
factor
B;
TNF-
, tumor necrosis factor
;
IL, interleukin;
E2, ubiquitin carrier protein;
E3, ubiquitin-protein isopeptide ligase;
CINC, cytokine-induced neutrophil chemoattractant;
PCR, polymerase
chain reaction;
ELISA, enzyme-linked immunosorbent assay;
PSI, proteasome inhibitor I;
Suc-, succinyl-;
DTT, dithiothreitol;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
EMSA, electrophoretic
mobility shift assay;
LLVY, Leu-Leu-Val-Tyr;
RT, reverse
transcription.
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REFERENCES |
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