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J. Biol. Chem., Vol. 276, Issue 29, 27657-27662, July 20, 2001
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From the Department of Molecular and Cellular Biochemistry,
Graduate School of Medical Sciences, Kyushu University,
Fukuoka 812-8582, Japan
Received for publication, April 17, 2001, and in revised form, May 9, 2001
The transcription factor nuclear factor- The nuclear factor- Although the activation of NF- Lipopolysaccharide (endotoxin or LPS), a major cell wall component of
Gram-negative bacteria, is one of the strongest stimulators to activate
NF- In this study, to better understand the mechanisms regulating
inflammation or NF- Cells and Reagents--
Peritoneal macrophages were collected by
peritoneal lavage with Hank's balanced salt solution (Life
Technologies) at 4 or 5 days after intraperitoneal injection of 2 ml of
3% sterile thioglycollate (Difco) into 8-10-week-old mice and
maintained in RPMI 1640 medium containing 10% heat-inactivated fetal
calf serum supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin. The other cells were cultured in Dulbecco's
modified Eagle's medium with the heat-inactivated fetal calf serum,
penicillin, and streptomycin. LPS from Escherichia coli
0111:B4 was purchased from List Biological Laboratories, Inc. Tumor
necrosis factor (TNF)- cDNA Cloning of I Plasmids--
The expression plasmids for transfection were
constructed by subcloning the DNA fragments obtained by polymerase
chain reaction into pcDNA3 (Invitrogen Corp.), pCI (Promega Corp.),
or pEGFP-N1 (CLONTECH Laboratories, Inc.) with or
without NH2-terminal Myc- or Flag-tag. pRK-IKK- Electrophoretic Mobility Shift Assay (EMSA)--
EMSA was
performed essentially as described in Ref. 18 with annealed
oligonucleotides (5'-TTAACAGAGGGGACTTTCCGAG-3' and 5'-GGCTCGGAAAGTCCCCTCTGTTAA-3') as a probe.
Immunofluorescent Microscopy--
HeLa and COS-7 cells grown on
a coverslip were transfected with the indicated expression vector with
FuGENETM 6. Twenty-four hours after transfection, the cells were fixed
with 4% paraformaldehyde in phosphate-buffered saline at room
temperature for 1 h. The cells were permeablized with 0.1% Triton
X-100 and then incubated with anti-p65 (Santa Cruz Biotechnology),
anti-Myc (9E10, Roche Molecular Biochemicals), or anti-Flag (M2, Sigma)
in phosphate-buffered saline containing 2% fetal calf serum. After
washing, the cells were further incubated with Alexa 488-conjugated
goat anti-mouse IgG F(ab')2 fragment (Molecular Probes)
and/or CyTM 3-labeled goat anti-rabbit IgG (Amersham Pharmacia
BiotechAB). Cells were stained with 4',6-diamidino-2-phenylindole
before analyses under a fluorescent microscopy.
Immunoprecipitation and Immunoblotting--
HEK293T cells were
transfected with the indicated expression plasmids by the calcium
phosphate method (17). Cells were lysed with phosphate-buffered saline
containing 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.01% SDS, and
10 µg/ml aprotinin. Immunoprecipitate with anti-Flag (M2) or anti-Myc
(9E10) antibody was resolved by 10% SDS-polyacrylamide gel
electrophoresis and probed with the indicated antibody.
Apoptosis Analysis--
Cells were transfected with the
indicated plasmid with LipofectAMINETM 2000 (Life Technologies).
Twenty-four hours after transfection, cells were treated with 50 ng/ml
TNF- To identify genes that were up-regulated after LPS stimulation in
macrophages, subtractive hybridization was performed with cDNAs
from LPS-treated and untreated murine macrophage-like cell line
RAW264.7. Among the clones specifically induced by LPS treatment of the cells, one clone was identified to encode a protein containing the ankyrin repeats as found in I No or little mRNA for I In order to determine the specificity for the induction, the expression
of I
A Novel I
B Protein, IB-
, Induced by Proinflammatory
Stimuli, Negatively Regulates Nuclear Factor-B in the Nuclei*
, and
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(NF-B) plays crucial roles in a wide variety of cellular functions
and its activity is strictly regulated by cytosolic inhibitors known as
IBs. We here report a new member of the IB protein family,
IB-
, harboring six ankyrin repeats at its carboxyl terminus.
IB- mRNA is strongly induced after stimulation by
lipopolysaccharide. The induction of IB- is also observed by
stimulation with interleukin-1
but not by tumor necrosis factor-
.
In contrast to cytosolic IB-, -, and -
, the induced
IB- localizes in the nucleus via its amino-terminal region, which
shows no homology with other proteins. Transiently expressed IB-
inhibits the NF-B activity without affecting the nuclear
translocation of NF-B upon stimulation. The expressed IB-
preferentially associates with the NF-B subunit p50 rather than p65
and recombinant IB- proteins inhibit the DNA binding of the
p65/p50 heterodimer and the p50/p50 homodimer. Thus, IB-
negatively regulates NF-B activity in the nucleus, possibly in order
to prevent excessive inflammation. Moreover, transfection of IB-
renders cells more susceptible to apoptosis induced by tumor necrosis
factor-. The proapoptotic activity of IB- further suggests
that it might be one of key regulators for inflammation and other
biologically relevant processes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(NF-B)1 is an
evolutionarily conserved transcription factor that controls the
expression of a large number of genes involved in a wide variety of
biological functions, such as inflammation, stress and immune
responses, embryonic development, and apoptosis (1, 2). The NF-B
activity is attributed to the homo- and heterodimers of Rel/NF-B
family proteins. In resting cells, NF-B is present in the cytosol as
an inactive complex with its inhibitor proteins, IBs. Exposure of
cells to various proinflammatory stimuli including microbial cell-wall
components, viral products, cytokines, or cellular stresses induces
proteasome-mediated degradation of IB following its phosphorylation
and ubiquitination. Thus liberated NF-B translocates into the
nucleus where it activates the transcription of target genes, such as
cytokines/chemokines, cell adhesion molecules, the nitric-oxide
synthase, or co-stimulatory molecules (1, 2).
B is essential for the initiation of
inflammation to eliminate pathogens, its activity must be tightly
regulated: too strong or prolonged activation of NF-B leads to
overproduction of cytokines, which culminates in the induction of fever
or life-threatening shock, and thus is seriously detrimental to the
host. In order to regulate the activity, NF-B activates genes, such
as IB- (3-6) and A20 (7, 8), whose products inhibit the activity
or activation of NF-B itself, thus comprising negative-feedback loop.
B for monocytes and macrophages, which play critical roles in
innate immunity (9). The cellular activation triggered by LPS and some
of the other microbial cell-wall components has recently been shown to
be mediated by members of the toll-like receptor (TLR) family (10-13).
TLRs share a cytoplasmic domain (TIR domain) with homology to that of
the interleukin (IL)-1 receptor (IL-1R) or the IL-1 receptor accessory
protein (IL-1RAcP), and it is suggested that the intracellular pathways
for the LPS and IL-1 signaling are shared.
B activity, we screened for genes that are induced by proinflammatory stimuli on these cells. During the screening, we identified a protein with partial similarity with the
IB protein family. The protein, induced by the proinflammatory stimuli, negatively regulates the NF-B activity in the nucleus, whereas typical IB proteins, represented by IB-, -, and
-, are constitutively expressed and present in the cytosol to
prevent nuclear translocation of NF-B. The distinct characteristics
of this protein from the cytosolic IB proteins would expand our understanding of mechanisms for the regulation of NF-B and
inflammatory reactions.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and IL-1 were from Genzyme Corp.
Cambridge, MA.
B---
Subtractive hybridization was
performed using polymerase chain reaction-select cDNA subtraction
kit (CLONTECH Laboratories, Inc.) with cDNAs
from LPS-treated (1 µg/ml, 1 h) and untreated RAW264.7 cells,
according to the manufacturer's instructions. A partial cDNA
fragment obtained by the subtractive hybridization was used as a probe
for Northern blotting to confirm the induction by LPS and for the
screening of a cDNA library constructed from LPS-stimulated
RAW264.7 cells. The 5' non-coding region was isolated by 5'-rapid
amplification of cDNA ends to confirm the initiation codon.
-Flag
(14) and pcDNA3-Flag-NIK (15) were described previously. The entire
coding region or the COOH-terminal region (amino acids 315-629) of
IB- was subcloned into pMALg (provided by Dr. T. Ito) or pGEX-2T
(Amersham Pharmacia Biotech AB), respectively, to prepare recombinant proteins.
B-Luciferase Reporter Assay--
RAW264.7 or HEK293
cells were transfected with the indicated expression plasmid together
with pELAM1-Luc (16) and pRL-TK (Promega Corp.) by the FuGENETM 6 method (Roche Molecular Biochemicals) or the calcium-phosphate method
(17), respectively. Two days after transfection, the cells were
stimulated as indicated at 37 °C for 6 h. Luciferase activities
were measured by using the Dual-luciferase Reporter System (Promega
Corp.).
for 24 h and stained with annexin V-biotin (BD
Pharmingen), followed by streptavidin-TRIcolor (Caltag Laboratories)
and analyzed by flow cytometry.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bs. The protein was named IB- and further analyzed, since it exhibited similarity to known IB family proteins both in structure and function but had distinct characteristics (see below).
B- was detected in the resting cells,
but it was strongly induced by LPS treatment in RAW264.7 cells (Fig.
1A). A similar induction was
observed with mouse peritoneal macrophages (Fig. 1B). The
induction of IB- peaked at 1 h after the stimulation. The
levels of the mRNA then gradually decreased, but significant
expression was detected even 24 and 48 h after the stimulation
(Fig. 1C). As low as 0.1 ng/ml LPS elicited IB- mRNA and the induction was abolished by polymyxin B-treatment, a
polypeptide that neutralizes lipid A, the biological center of LPS
(Fig. 1D).
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Fig. 1.
Induction of
I B- mRNA.
A-C, time course of IB- mRNA induction after LPS
stimulation. RAW264.7 cells (A and C) or
thioglycollate-elicited mouse peritoneal macrophages (B)
were treated with LPS (10 or 100 ng/ml) for the indicated periods of
time. D, LPS dose dependence of IB- mRNA
induction. RAW264.7 cells were treated with the indicated concentration
of LPS for 1 h in the presence (+) or absence (
) of polymyxin B
(Pmx, 1,000 units/ml). E, time course of IB- and
IB- mRNA induction by LPS, TNF-, or IL-1. NIH3T3 cells
were treated with LPS (100 ng/ml), TNF- (10 ng/ml), or IL-1 (10 ng/ml) for the indicated periods. Total RNA was extracted from the
cells and subjected to Northern blot analysis with a probe for
IB-, IB-, or glyceraldehyde-3-phosphate dehydrogenase
(G3PDH).
B- was examined with cells stimulated with two proinflammatory cytokines, tumor necrosis factor (TNF)- and IL-1. mRNA for IB- was also induced by IL-1 treatment in NIH3T3
cells, with a time course similar to that observed in the
LPS-stimulated cells (Fig. 1E). In contrast, its induction
by TNF- was negligible (Fig. 1E). To monitor whether the
cells were activated by these stimuli, the expression of IB- was
examined in parallel, as a control for a typical gene induced by
NF-B (3-6). The IB- mRNA was strongly induced by all
three stimulants, indicating that these stimuli induced cells to
activate NF-B. To investigate the molecular functions of IB-,
cDNAs for IB- were isolated from a cDNA library
constructed from LPS-stimulated RAW264.7 cells. The composite sequence
of the two partial cDNA clones encoded an open reading frame for
629 amino acids of IB- (Fig.
2A). An in-flame stop codon
preceding the first methionine codon in the cDNA was confirmed by
5'-rapid amplification of cDNA ends.
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Fig. 2.
Amino acid sequence and tissue distribution
of mouse I B-.
A, the amino acid sequences corresponding to the ankyrin
repeats are black boxed. B, alignment of amino
acid sequences of IB- and Bcl-3. Identical amino acids between
IB- and Bcl-3 are shown by black boxes. C,
a multiple tissue Northern blot (CLONTECH
Laboratories) probed with 32P-labeled IB-
cDNA.
IB- contained a COOH-terminal region with 6 ankyrin-repeats,
which was most similar to that of Bcl-3 (Fig. 2B). On the
other hand, the NH2-terminal region did not show any
significant homology to proteins in the data bases. When expression of
IB- in the normal tissue was examined by Northern blotting, a
4-kilobase mRNA for IB- was detected in the kidney, liver,
lung, and heart, but was hardly detected in the skeletal muscle,
spleen, and brain (Fig. 2C). A strong band with a smaller
size was detected in the testis, but its identity remains unknown.
During the course of the following functional characterization, a
protein identical to IB- was reported as MAIL-S (19).
All of the IB proteins thus far identified contain multiple repeats
of the ankyrin motifs, which interact with the Rel homology domain of
the Rel/NF-B proteins to mask their nuclear localization signal and
block their DNA binding. To explore the involvement of IB- in the
activation of NF-B, we transfected IB--expression plasmid into
cells and measured their NF-B activities in response to
proinflammatory stimuli. LPS stimulation of RAW264.7 cells induced
strong activation of NF-B, as measured with an NF-B-luciferase reporter. Expression of IB- resulted in a
dose-dependent inhibition of the NF-B activity in the
stimulated cells (Fig. 3A). To
identify the functional domains responsible for the inhibition, we
constructed truncated mutants of IB- and examined their effects.
The COOH-terminal half (IB-(C), amino acids 315-629) of
IB- containing the ankyrin-repeat region effectively inhibited
the NF-B activity. The NH2-terminal region
(IB-(N), amino acids 1-314) without the ankyrin-repeat sequence
showed weaker but significant activity (Fig. 3, A and G).
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Since LPS stimulation of RAW264.7 cells led to strong induction of
IB- (Fig. 1A), we examined the effect of endogenous
expression of IB- on the NF-B activities upon LPS stimulation,
by preventing the induction with antisense RNA for IB-. We
transfected a construct for sense- or antisense-IB- into RAW264.7
cells and stimulated them with LPS (Fig. 3B). In contrast to
the effect of the sense construct, LPS-induced NF-B activities were
augmented by expressing the antisense mRNA for IB-, possibly
by inactivation of induced IB- mRNA. Neither the sense nor
the antisense construct had any effect on basal NF-B activity. Thus,
LPS stimulation of the cells, leading to activation of NF-B,
simultaneously induces IB- expression, which is inhibitory for
the NF-B activities.
In addition to LPS-induced NF-B activity, transfected IB-
inhibited the IL-1- and TNF--induced NF-B activities (Fig. 3,
C and D). NF-B is activated by degradation of
cytosolic IB- or -, which is induced by phosphorylation
catalyzed by IKK (IB kinase)-/ (14). In order to determine the
signaling step(s) of the IB- inhibition of NF-B, we activated
NF-B by overexpressing IKK- or NIK (NF-B-inducing kinase), the
latter of which is a known activator for IKKs (15), and examined the
effects of the co-expression of IB-. Transfection of IB-
inhibited both IKK-- and NIK-mediated activation of NF-B (Fig. 3,
E and F). Furthermore, the transactivation
activity of transfected p65/p50 of the NF-B subunits was inhibited
by IB- (Fig. 3G). The IB-(N) and IB-(C) also had this inhibitory activity. These results indicate that IB- inhibits NF-B at the later step(s) than IKK activation. The stronger activity of IB-(C) might be due to higher expression levels of this mutant compared with the full-length IB- or
IB-(N) (data not shown).
The above finding prompted us to study its effects on the nuclear
translocation of NF-B upon stimulation. HeLa cells transiently transfected with Myc-tagged IB- or IB- were treated with
TNF-, and then nuclear translocation of the p65 subunit of NF-B
was examined. p65 exclusively localized in the cytosol in unstimulated cells (Fig. 4A, left) and was
translocated into the nucleus upon TNF- stimulation (Fig.
4A, untransfected cells in upper middle and
right). Transfection of IB- resulted in inhibition of
the nuclear translocation of p65 by the stimulation (Fig. 4A,
upper middle; compare the transfected cells shown by arrowheads
with the others). In contrast to the effect of IB-, IB- did
not affect the translocation of p65 (Fig. 4A, upper
right).
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We found simultaneously that IB- was concentrated in the nucleus,
whereas IB- localized throughout the cells (Fig. 4A, lower panels). The same results were observed in COS-7 cells
(Fig. 4B, upper row). Thus, IB- inhibits the activated
NF-B in the nucleus rather than affecting its nuclear translocation.
We further determined the subcellular localization of the truncated
mutants of IB- (Fig. 4B). While the
NH2-terminal half (Fig. 4B, Flag- IB-(N))
showed nuclear staining similar to that of intact IB-
(Flag-IB-), the COOH-terminal half (Flag-IB-(C)) was
distributed throughout the entire cells, indicating that the nuclear
localization signal(s) is present in the NH2-terminal region. All the above results were also confirmed with green
fluorescence protein fusion proteins (data not shown), which had
inhibitory activities (Fig. 3A). We also found that
the cytosolic IB-(C) inhibited the nuclear translocation of
NF-B upon stimulation (data not shown), which might explain the
stronger activity of this mutant (Fig. 3, A and
G).
To further study the mechanisms underlying the inhibition of NF-B by
IB-, we examined the physical association of IB- with the
NF-B subunits. Myc-tagged IB- or IB- was co-expressed with Flag-tagged p65 or p50 in HEK293T cells. The cell lysates were
immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc antibody. IB- was found to associate with p50, but
its association with p65 was hardly detectable (Fig.
5, top). Strong interaction
was observed between IB- and both p65 and p50. (Fig. 5,
top, and data not shown). That IB- showed a preference for p50 appears to be reasonable, since the ankyrin repeat region of
Bcl-3, which is most homologous to that of IB-, have been reported to preferentially associate with p50 rather than p65 (20).
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The nuclear localization and its association with the p50 subunit
suggest that IB- might affect the DNA binding activity of
NF-B. In Fig. 6, the effect of
recombinant IB- on the DNA binding activity of NF-B was
examined by EMSA. An NF-B probe was retarded when incubated with
nuclear extract prepared from RAW264.7 cells treated with LPS (Fig.
6A). The band was competed out with the unlabeled probe and
supershifted by either anti-p65 or anti-p50 antibody, demonstrating
that it represented the p65/p50 heterodimer (data not shown). The
addition of a recombinant maltose-binding protein fusion protein with
the full-length IB- efficiently inhibited the appearance of the
band (Fig. 6A, MBP-IB). A glutathione S-transferase fusion protein with the COOH-terminal ankyrin
repeats alone (Fig. 6, GST-KB-CC) exhibited the same
activity, whereas no effect was observed with control maltose-binding
protein or glutathione S-transferase protein. Furthermore,
we prepared the p65/p50 heterodimer and the p50 homodimer by
transfecting both p65 and p50 or p50 alone into HEK293 cells. The
nuclear extracts from the cells transfected with p65 and p50 gave two
bands (Fig. 6B), each of which was confirmed to be the
p65/p50 heterodimer and the p50/p50 homodimer by supershift analyses
(data not shown). The recombinant IB- protein inhibited the
appearance of both bands (Fig. 6B). The COOH-terminal half
(IB-(C)) alone also exhibited the inhibitory activity and the
inhibition of the p50 homodimer was weaker than that of the p65/p50
heterodimer (Fig. 6C). Thus, IB- inhibits the DNA
binding of p65/p50 heterodimers as well as the p50 homodimer, through
its association with the p50 subunit.
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The inhibitory activity of IB- on NF-B, which acts as an
anti-apoptotic factor (21-24), suggests that IB- might also be involved in the regulation of apoptosis upon inflammatory stimuli or in
other physiologically relevant processes. To test this possibility, we
examined the effect of IB- expression on TNF--induced
apoptosis. HeLa cells transfected with an empty vector, a
phosphorylation-defective mutant (S32A/S36A) of IB-, or IB-
were stimulated with TNF- for 24 h and their morphological
changes were examined under a microscope. As well as the cells
expressing the mutant IB-, the IB--transfected cells
exhibited increased numbers of cells with round-shaped apoptotic
morphologies (Fig. 7A). For
more quantitative analyses, we stained apoptotic cells with annexin-V
and analyzed them by flow cytometry. When transfected 293 cells were
analyzed after TNF- stimulation, the vector-transfected cells
exhibited 17.4% of annexin V-possitive apoptotic cells (Fig.
7B). On the other hand, the IB--expressing cells
analyzed under the same conditions showed 29.3% of apoptotic cells.
The results indicate that expression of IB- promotes apoptosis.
This effect was comparable to that of the mutant IB- (22.7%
apoptotic cells). The IB- expression did not promote apoptosis
without the stimulation. Thus, IB- accelerates apoptosis,
possibly by inhibiting the anti-apoptotic NF-B activity.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We described in this paper a novel protein belonging to the IB
family. No or little expression of IB- is observed in
unstimulated macrophages and it is induced by a subset of
proinflammatory stimuli, such as LPS and IL-1, which activate
receptors containing the TIR domains, TLR4 and IL-1R, respectively.
Although both the TNF-- and IL-1/LPS signaling cascades activate
NF-B and mitogen-activated protein kinases, TNF- induces little
induction of IB-. The induction of IB- was inhibited by
proteasome inhibitors, lactacystin and MG-132, suggesting that NF-B
is involved in the induction of
IB-.2 However,
overexpression of the NF-B subunit p65 resulted in robust
up-regulation of IB- mRNA, but little induction of IB- mRNA was detected (data not shown). The results indicate that IB- is induced by a different mechanism from that of IB-, with NF-B activation being sufficient for the latter. A
transcription factor(s) that is essential for the induction of
IB- appears to be specifically activated by the signaling through
the cytoplasmic TIR domains of TLRs and IL-1R.
Transfection of IB- inhibited the NF-B activities induced by
LPS and IL-1 as well as TNF- (Fig. 3, A, B,
and D). This negative regulation of NF-B would be
important to prevent undesirable heavy inflammatory reactions, which
lead to septic shock or other detrimental effects. The introduction of
antisense RNA for IB- results in augmentation of NF-B activity
upon stimulation with LPS (Fig. 3B), suggesting
physiological significance of IB- in the regulation of
inflammatory reactions. Since IB- expression was induced with as
little as 0.1 ng/ml LPS and was sustained for at least 2 days (Fig. 1,
C and D), it could play a role in the acquisition
of LPS tolerance in LPS-sensitive cells. Cross-tolerance between LPS
and IL-1 stimulation is explained at least in part by IB-,
since both stimuli activate IB- induction (25).
The inhibitory activities of full-length IB- or IB-(N)
appeared weaker than those of IB- or -, but this finding might be attributable to the fact that IB- or IB-(N) was not as highly expressed as the other two proteins (data not shown). IB- might be subjected to degradation in the cells via its
NH2-terminal region although it does not appear to have the
consensus sequence for phosphorylation by IKK nor lysine residue(s) for
the subsequent ubiquitination.
In contrast to IB-, -, and -, all of which are in the
cytosol, the induced IB- localizes in the nucleus through the NH2-terminal region (Fig. 4), where it negatively regulates
the DNA binding of NF-B via association with the p50 subunit (Figs. 5 and 6). These findings are consistent with the observation that IB- does not affect the translocation of activated NF-B into the nuclei. Kitamura et al. (19) independently isolated the proteins, MAIL-L and -S, the latter of which is identical to IB-. The protein identical to MAIL-L was also reported more recently by
another group as an IL-1-inducible protein (26). The induction by LPS
stimulation and the nuclear localization of IB- described in this
paper is consistent with their results. They also showed in Swiss 3T3
cells that transfection of the cDNA resulted in the promotion of
LPS-induced IL-6 production, which is also regulated by NF-B. The
apparent discrepancy may be attributed to different assay systems or
different cell lines. Alternatively, IB- might also be involved
in some transcriptional activation as in the manner of Bcl-3, which has
been shown to act as a coactivator of several transcription factors
(27-29).
IB- exhibited the proapoptotic activity upon TNF- stimulation
(Fig. 7). TLR2, which acts as a receptor for several microbial products
to activate NF-B, has been reported to induce apoptosis upon the
stimulation (30, 31). Since TLR2 also contains a TIR domain, IB-
expression is likely induced by the activation of TLR2, and would
regulate NF-B activity and apoptosis. The proapoptotic activity of
IB- was comparable to that of IB- (Fig. 7B),
although its activity on NF-B was much weaker (Fig. 3C,
see above). The results suggest that IB- may have other activities to promote apoptosis than inhibiting NF-B.
IB- was induced by both LPS and IL-1 but not by TNF- (Fig.
1E). The specificity of the IB- induction provided
evidence that these two proinflammatory cytokines, IL-1 and TNF-,
have qualitatively different activities that would culminate in
distinct consequences. The physiological significance of the induction of IB- was exemplified by the induction of IB-, an
NF-B-regulated gene: the induction of IB- was sustained for
longer periods in TNF--stimulated cells, where little IB-
induction was observed, whereas the induction was transient in IL-1-
or LPS-treated cells (Fig. 1E). Various types of cytokines
are produced during inflammation, and a single cell is exposed to a
mixture of cytokines. The subtle balance of the cellular responses,
such as activation of NF-B and induction of IB-, may determine
the fate of the cells, including apoptosis. Since NF-B regulates not
only inflammation but also wide varieties of biologically important
processes, IB- might play critical roles in such physiological
and pathological situations. Precise analyses for the IB-
induction and its molecular function would shed more light on the
regulation of appropriate inflammatory reactions, the physiological
relevance of each inflammatory cytokine, and other biological processes
regulated by NF-B.
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ACKNOWLEDGEMENTS |
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We thank D. V. Goeddel (Tularik Inc.),
D. Wallach (The Weizmann Institute of Science), and T. Ito (Kanazawa
University) for the expression plasmids for IKK- and NIK, and pMALg,
respectively. We are grateful to T. Irie for construction of IB-
plasmids and Y. Sunakawa for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to T. M. and K. T.), and grants from the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T. M.), Sumitomo Foundation (to T. M.), and Kaibara Foundation (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.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with acession number(s) AB047549.
To whom correspondence should be addressed: Dept. of
Molecular and Cellular Biochemistry, Graduate School of Medical
Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka
812-8582, Japan. Tel./Fax: 81-92-642-6103; E-mail:
tmuta@mailserver.med.kyushu-u.ac.jp.
Published, JBC Papers in Press, May 16, 2001, DOI 10.1074/jbc.M103426200
2 S. Yamazaki, T. Muta, and K. Takeshige, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
NF-B, nuclear
factor-B;
LPS, lipopolysaccharide;
IL-1, interleukin-1;
TNF-, tumor necrosis factor-;
EMSA, electrophoretic mobility shift assay;
IKK, IB kinase;
TLR, toll-like receptor;
NIK, NF-B-inducing
kinase.
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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