IKKi/IKK
Plays a Key Role in Integrating Signals Induced by Pro-inflammatory Stimuli*
Vladimir V. Kravchenko,
John C. Mathison,
Klaus Schwamborn
,
Frank Mercurio
and
Richard J. Ulevitch
From the
Scripps Research Institute, La Jolla, California 92037 and
Celgene Research San Diego, California
92121
Received for publication, March 24, 2003
, and in revised form, May 2, 2003.
 |
ABSTRACT
|
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We report that the product of the inducible gene encoding the kinase known
as IKKi/IKK
(IKKi) is required for expression of a group of genes
up-regulated by pro-inflammatory stimuli such as bacterial endotoxin
(lipopolysaccharide (LPS)). Here, using murine embryonic fibroblasts obtained
from mice bearing deletions in IKK2, p65, and IKKi genes, we provide evidence
to support a link between signaling through the NF-
B and
CCAAA/enhancer-binding protein (C/EBP) pathways. This link includes an
NF-
B-dependent regulation of C/EBP
and C/EBP
gene
transcription and IKKi-mediated activation of C/EBP. Disruption of the
NF-
B pathway results in the blockade of the inducible up-regulation of
C/EBP
, C/EBP
, and IKKi genes. Cells lacking IKKi are normal in
activation of the canonical NF-
B pathway but fail to induce
C/EBP
activity and transcription of C/EBP and C/EBP-NF-
B target
genes in response to LPS. In addition we show that, in response to LPS or
tumor necrosis factor
, both
and
subunits of C/EBP
interact with IKKi promoter, suggesting a feedback mechanism in the regulation
of IKKi-dependent cellular processes. These data are among the first to
provide insights into the biological function of IKKi.
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INTRODUCTION
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Gene expression during innate and adaptive immune responses involves the
combined effects of multiple transcription factors. Among these are members of
the activating protein 1, NF-
B, signal transducers and activators of
transcription, and CCAAA/enhancer-binding proteins
(C/EBP)1 families
(13).
Prototypic activators of innate immunity such as bacterial endotoxin
(lipopolysaccharide (LPS)) are known to regulate both the NF-
B and the
C/EBP pathways (4,
5). The C/EBP family is
comprised of at least six proteins containing basic leucine zipper (bZIP)
motifs (6,
7). The NF-
B family is
made up of fewer members including p50, p52, p65/RelA, c-Rel, and RelB
(8). These transcription
factors regulate expression of distinct and overlapping subsets of genes
encoding immune and pro-inflammatory modulators
(3,
9). Whether or not NF-
B
and C/EBP family members influence each other is not well understood at this
time. Current paradigms suggest that the rapidly and transiently activated
NF-
B pathway is central in a primary wave of gene induction followed by
a second wave of gene transcription some hours later mediated by other
transcription factors including members of the C/EBP family
(3,
5).
Despite participating in regulation of overlapping sets of genes, the
NF-
B and the C/EBP pathways are activated by distinct intracellular
signaling mechanisms. Activation of the canonical NF-
B pathway depends
on stability of the inhibitor known as I
B
. It stabilizes
NF-
B complexes so that after its degradation the remaining subunits
translocate from the cytoplasm to the nucleus. NF-
B-dependent
transcription of I
B
gene also provides a potent feedback
mechanism maintaining the balance between cytoplasmic and nuclear localization
of the NF-
B subunits
(8). Treatment of cells with
specific inducers, such as TNF, IL-1, or LPS, results in the phosphorylation
of I
B
at two serines (Ser-32 and Ser-36). This is a signal for
its rapid ubiquitin-dependent proteolysis and translocation of free
NF-
B to the nucleus. I
B
phosphorylation is catalyzed by
the I
B kinase (IKK), a complex composed of three subunits,
IKK
/IKK1, IKK
/IKK2, and NEMO/IKK
/IKKAP1/FIP3. IKK1 and
IKK2 are the catalytic subunits, whereas NEMO serves a non-enzymatic,
regulatory function. Biochemical and genetic analyses demonstrate that IKK2 is
essential for NF-
B activation in response to TNF, IL-1, and LPS,
whereas IKK1 is not required for such responses
(10). Targeted deletion of p65
or IKK2 gene affects TNF-induced transcription of the I
B
gene
(11,
12). Recently two additional
IKK2-related kinases, IKKi/IKK
(13,
14) and TANK-binding kinase
1/NF-
B-activating kinase/T2K
(1517)
have been identified. Whether these latter proteins play a role as enzymatic
or non-enzymatic regulatory factors is not fully understood
(4).
Regulatory mechanisms for the C/EBP pathways differ markedly from those of
NF-
B and include transcriptional and/or post-translational mechanisms
as well as protein-protein interactions via dimerization through
leucine-zipper domains (6,
7,
18,
19). Phosphorylation also
regulates the C/EBP family by directing nuclear localization and
transcription-activating potential
(2023).
Some members of this family, specifically C/EBP
and C/EBP
, have
been linked to gene expression in the acute phase response and during
inflammation
(2426).
Furthermore, up-regulation of C/EBP
and C/EBP
gene expression
occurs after exposure to pro-inflammatory stimuli such as TNF, IL-1, IL-6, or
LPS (3,
5).
Here we have performed experiments to identify possible regulatory links
between the NF-
B and C/EBP pathways, with an emphasis on events related
to innate immune responses. We have used murine embryonic fibroblasts (MEFs)
obtained from various strains of mice bearing targeted gene deletions in
components of the NF-
B pathway. We show that fibroblasts isolated from
p65/ or IKK2/, but not from control, mice fail to
induce C/EBP
and C/EBP
transcripts in response to cell
stimulation with LPS. Furthermore, we observed that both p65 and IKK2 are
required for induction of IKKi mRNA. In turn, in primary fibroblasts lacking
the IKKi gene LPS induces C/EBP
and C/EBP
mRNAs and activates
NF-
B, as observed in control cells, but importantly, fail to induce
C/EBP
-specific DNA binding activity. Furthermore, we report that LPS
treatment of IKKi/ MEFs reveals deficits in expressions of genes
associated with immune and pro-inflammatory responses. In totality, these data
support the contention that there is a link between the NF-
B and C/EBP
pathways and that IKKi may be a key element in integration of signals from
both pathways during inflammatory and immune responses.
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EXPERIMENTAL PROCEDURES
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Cells, Antibodies, and ReagentsRelA/p65-, IKK2-, IKKi-, or
Egr1-deficient MEFs and the corresponding immortalized MEFs (imMEFs) were
generated and maintained as described
(11,
12,
27,
28). Human umbilical vein
endothelial cells were purchased from Clonetics Corp. and maintained in EGM
media (Cambrex, Gaithersburg, MD). The IKKi, IKK2, IKK1, NEMO, p65, p50,
c-Rel, C/EBP
, and C/EBP
antibodies were purchased from Santa
Cruz. LPS (Escherichia coli 0111:B4) was purchased from List
Biological Laboratories. Total RNA was prepared by using TRIzol reagent
(Invitrogen). The NF-
B, Oct-1, and C/EBP gel shift oligonucleotides
were from Santa Cruz. RNA oligonucleotides were purchased from Dharmacon
Research. Double-stranded small interfering RNAs (siRNAs) (IKKi,
5'-GUGAAGGUCUUCAACACUACC-3'x
5'-UAGUGUUGAAGACCUUCACAG-3'; firefly luciferase as a nonspecific
control, 5'-CGUACGCGGAAUACUUCGAAA-3'x
5'-UCGAAGUAUUCCGCGUACGUG-3') were prepared and used for
transfection of human umbilical vein endothelial cells (
5 x
106 cells per transfection) by using electroporation performed as
described (29).
AssaysSamples of total RNA (10 µg) were analyzed by
Northern blot as described
(13). A blot was hybridized
with specific antisense oligonucleotide labeled by T4 polynucleotide kinase
using [
-32P]ATP. Nuclear extracts were prepared and used for
electrophoretic mobility shift assay (EMSA) as described
(30). Kinase activity of
endogenous IKK2 or IKKi were measured by immune-complex kinase assay with
glutathione S-transferase-I
B
(146)
as substrate (12,
13). The immune-complexes were
also subjected to Western blot to estimate the amount of precipitated
proteins. Chromatin immunoprecipitation (ChIP) were performed as described
(31). The antibodies specific
for C/EBP
, C/EBP
, or p65 were used for the ChIP assay. The levels
of IKKi or I
B
promoter DNA were determined by PCR using
oligonucleotides from the 5'-untranslated region of IKKi gene
(5'-TCTGTAAAGCAATGAGCAAG-3';
5'-AGGAAGCTGACACAGTGTGG-3') or I
B
gene
(5'-AGGGAAAGAAGGGTTCTTGC-3';
5'-CTGACTGTTGTGGGCTCG-3').
Metabolic Labeling106 cells were plated per
60-mm dish. On the second day, the cells were washed 3 times with
phosphate-free Dulbecco's modified Eagle's medium containing 5% of dialyzed
fetal bovine serum and then incubated in the same medium containing 400 mCi/ml
[32P]H3PO4 for 4 h. The last 2 h some cells
were incubated with 100 ng/ml LPS. The cells were then washed three times with
cold phosphate-buffered saline and used for preparation of nuclear extract
according to EMSA protocol (see above). The nuclear extracts were diluted by
the addition of 10 volumes of standard radioimmune precipitation assay buffer,
and C/EBP
or p65 proteins were recovered by immunoprecipitation with
specific antibodies as indicated in Fig.
5C. The immunoprecipitates were analyzed by SDS-PAGE and
autoradiography.

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FIG. 5. IKKi is required for post-transcriptional regulation of C/EBP
A, EMSA of nuclear extracts from IKKi/ and IKKi+/+ MEFs
before and after 4 h of treatment with LPS. The nuclear extracts were
incubated in the presence of specific antibody (Ab) to C/EBP
( ) or C/EBP ( ), as indicated on the top of each
lane. The DNA binding activity of C/EBP or Oct-1 (as a control)
transcription factor is shown. B, Western blot (WB) analysis
for C/EBP and actin (as a loading control) protein expression in
IKKi+/+ and IKKi/ MEFs before and after 4 h of treatment with
LPS (100 ng/ml). C, analysis of 32P-labeled C/EBP
and p65 (as a control) proteins in nuclear extracts from IKKi+/+ and
IKKi/ MEFs before and after 3 h of treatment with LPS. After
preincubation with [32P]orthophosphate for 2 h and LPS treatment,
the nuclear extracts were prepared, immunoprecipitated (IP) by
anti-C/EBP or anti-p65, and consequently subjected to SDS-PAGE and
autoradiography. The phosphorylated products (C/EBP and p65) are shown
on the left. The positions of size markers are shown on the
right. D, Northern blot analysis of steady-state IKKi, IL-6,
I B , or GAPDH mRNA levels in untreated or treated with LPS human
umbilical vein endothelial cells; cells were pre-transfected with siRNA as
indicated. ns, nonspecific control.
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RESULTS
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We first sought an experimental system to investigate relationships between
the C/EBP and NF-
B pathways. The promoter region of C3 gene contains
C/EBP sites (32); it is known
that C3 expression is regulated by C/EBP
(26). Despite the fact that
there are no identifiable NF-
B sites in the C3 promoter, transcription
of the C3 gene is induced by NF-
B activators including LPS
(9,
33). Thus, we reasoned that
measurements of LPS induction of C3 mRNA would be an appropriate marker for
initial studies to investigate possible relationships between the NF-
B
and C/EBP pathways. Here we have used MEFs from mice bearing targeted
deletions of genes encoding IKK2, p65, and IKKi. We first examined induction
of C3 mRNA in LPS-treated MEFs derived from IKK2/ and control
(IKK2+/+) embryos; the induction of C3 mRNA was observed in control cells but
not in the IKK2-deficient cells (Fig.
1A). In contrast, LPS-mediated induction of
c-jun mRNA was nearly identical in both types of MEFs. Moreover, as
shown here and as previously noted, IKK2/ MEFs showed
LPS-induced I
B
mRNA and NF-
B DNA binding activity that
was partially reduced when IKK2/ and IKK2+/+ cells were compared
(Ref. 12;
Fig. 1, A and
B). Similar findings were noted in experiments with
spontaneously imMEFs derived from IKK2/ and control cells
(Fig. 1C). Thus, the
absence of IKK2 revealed a deficiency in LPS-induced C3 induction that is not
likely to result from loss of LPS responsiveness. To further probe the role of
the NF-
B pathway we also examined the effects of p65 deficiency on LPS
induction of C3 and I
B
mRNA. LPS-mediated induction of both
I
B
and C3 mRNAs was completely abolished in p65/
imMEFs (Fig. 1D).
These data suggest that regulation of C3 gene expression requires an intact
NF-
B pathway. However, we hypothesize that the role of NF-
B
involves indirect mechanisms and requires additional gene expression under the
control of NF-
B. In fact it has been shown that protein synthesis is
required for LPS-induced expression of C3 mRNA
(34).

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FIG. 1. LPS responsiveness of cells derived from IKK2/ and IKK2+/+
mouse embryos. A, Northern blot analysis of steady-state mRNA
levels in IKK2/ and IKK2+/+ MEFs treated with LPS (100 ng/ml)
for the indicated times. B, EMSA of nuclear extracts from
IKK2/ and IKK2+/+ MEFs before and after1hof treatment with LPS.
The DNA binding activity of NF- B or Oct-1 (as a loading control)
transcription factor is shown. C, Northern blot analysis of
steady-state mRNA levels in IKK2/ and IKK2+/+ imMEFs treated
with LPS (100 ng/ml) for the indicated times. D, Northern blot of
steady-state mRNA levels in p65/ and p65+/+ imMEFs treated with
LPS (100 ng/ml) for the indicated times.
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The observation that LPS induces IKKi mRNA
(13) prompted us to evaluate
the effects of IKKi deletion on C3 gene expression. Thus, we next measured
LPS-induced C3, I
B
, and IKKi mRNA in IKKi/ and
control MEFs as well as in IKK2/ cells
(Fig. 2A). As shown
above, LPS-mediated induction of I
B
mRNA was reduced in cells
lacking IKK2, whereas IKKi deficiency had no effect on LPS-induced expression
of I
B
mRNA. In contrast, the induction of C3 mRNA was abolished
in the IKKi/ cells as well as in the IKK2/ cells.
Furthermore, we failed to observe induction of IKKi mRNA in the
IKK2/ cells. Nearly identical results were also obtained in
similar experiments with LPS-stimulated IKKi/ and IKKi+/+ imMEFs
derived from the corresponding primary isolates of MEFs
(Fig. 2B). As an
additional specificity control, we also examined the LPS responses in MEFs
derived from Egr-1-deficient embryos
(28). The LPS-mediated
induction of C3 or IKKi mRNA was practically identical in Egr1/
and Egr1+/+ cells (Fig.
2C), emphasizing the specificity of the effects observed
here with IKK2/, p65/, and IKKi/
MEFs. Thus, IKKi appears to be a key molecule for transcriptional induction of
C3 gene in response to LPS, and its expression requires an intact NF-
B
pathway.

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FIG. 2. IKKi is required for C3 gene induction by LPS in MEFs and
imMEFs. A, Northern blot analysis of steady-state mRNA levels in
IKK2/, IKK2+/+, IKKi/, and IKKi+/+ MEFs treated
with LPS (100 ng/ml) for the indicated times. B, Northern blot
analysis of steady-state C3, I B , or GAPDH mRNA levels
in IKKi/ and IKKi+/+ imMEFs treated with LPS (100 ng/ml) for the
indicated times. C, Northern blot analysis of steady-state Egr-1, C3,
IKKi, or GAPDH mRNA levels in Egr-1/ and Egr-1+/+ MEFs treated
with LPS (100 ng/ml) for the indicated times.
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To further define how IKKi participates in gene regulation we performed the
following experiments. Extracts from IKK2/, IKK2+/+,
IKKi/ (as negative control), or IKKi+/+ (as positive control)
were immunoprecipitated to enrich the samples for IKKi, and the resultant
immunoprecipitates were electrophoresed and then subjected to Western blot
analysis (Fig. 3A). As
was expected, expression of IKKi protein was not detected in
IKKi/ cells. In contrast, IKKi+/+ or IKK2+/+ cells showed
detectable IKKi protein expression that was increased after LPS addition.
Compared with IKK2+/+ or IKKi+/+ cells, the basal level of IKKi protein was
significantly reduced in IKK2-deficient cells. Importantly, LPS addition
failed to up-regulate IKKi expression in this cell type. These data together
with Northern blot studies support the contention that IKK2 is required for
the inducible expression of IKKi mRNA and protein. These data prompted us to
address the question of how IKKi regulates LPS-induced C3 gene
expression. Specifically. we asked whether the failure to induce C3
results from the absence of IKKi protein or whether essential signaling
including expression and/or activation of IKK2 is also affected by IKKi
deficiency. To address these issues, we used Western blot analysis to compare
the levels of IKK2 protein in extracts from IKKi/, IKKi+/+,
IKK2/ (as negative control), or IKK2+/+ (as positive control)
cells. The results showed that IKKi/, IKKi+/+, and IKK2+/+ cells
express nearly identical levels of IKK2 protein
(Fig. 3B). Expression
of IKK1 and NEMO subunits of the IKK complex was also unchanged in
IKKi/ cells (Fig.
3B). Thus, the absence of IKKi does not reduce the
protein expression of other key members of the IKK complex. Furthermore, it is
unlikely that IKKi is required for activation of IKK complex because LPS
treatment up-regulated the kinase activity of the IKK similarly in
IKKi/ and IKKi+/+ cells (Fig.
3C). Thus, the absence of IKKi does not alter signaling
that is directly related to NF-
B activation. The same extracts were
subjected in parallel to an immunoprecipitation/kinase assay for IKKi. In
addition LPS treatment did not alter the kinase activity of IKKi
(Fig. 3C), in keeping
with observations of others that pro-inflammatory mediators do not alter IKKi
kinase activity but, rather, up-regulate its expression
(13).

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FIG. 3. LPS induces expression of IKKi protein in MEFs. A, Western
blot analysis for IKKi protein expression in IKK2/, IKK2+/+,
IKKi/, and IKKi+/+ MEFs before and after 5 h of treatment with
LPS (100 ng/ml). An anti-IKKi immunoprecipitation was performed followed by
Western blot analysis with an anti-IKKi. Arrows indicate the position
of IKKi and IgG (heavy chain). B, Western blot analysis for IKK2,
IKK1, or NEMO protein expression in IKK2/, IKK2+/+,
IKKi/, and IKKi+/+ MEFs before and after 5 h of treatment with
LPS (100 ng/ml). C, IKK or IKKi kinase assay (KA) from
extracts of IKKi/ or IKKi+/+ MEFs treated with LPS for the
indicated times. An anti-NEMO or anti-IKKi immunoprecipitation (IP)
was performed followed by kinase assay with glutathione
S-transferase-I B (144) as a substrate. The
samples were also subjected to Western blot (WB) analysis for the
relevant proteins as indicated.
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To investigate whether IKKi deficiency alters the rate and extent of
LPS-induced expression of a variety of genes associated with innate immune and
inflammatory responses we measured LPS-induced expression of TNF, IL-1, IL-6,
IP-10, RANTES, and COX-2 mRNA. LPS treatment increased mRNA expression for
each of these genes in IKKi+/+ cells (Fig.
4A); we also showed that TNF, IL-1, and IL-6 protein
levels also increased (Fig. 4, B
and C). In contrast IKKi deficiency resulted in a marked
reduction of LPS-induced mRNA expression for each of this group of genes
(Fig. 4A). Parallel
decreases in protein expression for TNF, IL-1, and IL-6 were also noted
(Figs. 4, B and
C). The fact that both cell lines (IKKi/
and IKKi+/+ MEFs) remain LPS-responsive is supported by the observation that
identical levels of LPS-induced mRNA for c-jun, C/EBP
, and
C/EBP
genes were observed.
Comparative analysis of the promoters of the group of IKKimodulated genes
depicted in Fig. 4A
identified the presence of binding sites for multiple transcription factors
including NF-
B, interferon regulatory factor-3 (IRF-3), and C/EBP.
Although NF-
B is known to be involved in the regulation all of these
genes (8,
9), a number of observations
suggest that the absence of IKKi has no effect on activation of the canonical
NF-
B pathway. First, we have shown that LPS-mediated activation of IKK
complex activity (see Fig.
3C) and translocation of NF-
B were
indistinguishable when IKKi+/+ and IKKi/ MEFs were compared
(Fig. 4D). Second,
analysis of the subunit composition of the
B binding activity with
antibodies specific for NF-
B-related proteins revealed that treatment
of IKKi/and IKKi+/+ cells with LPS induces DNA binding complexes
containing essentially the same proteins including p65
(Fig. 4E).
Phosphorylation of p65 subunit increases its ability to activate transcription
of NF-
B target genes (8,
35). Although our data do not
exclude a role for IKKi in this process, we believe that to be unlikely
because LPS-induced expression of I
B
gene, a classical
NF-
B target gene (8,
11), is not affected by IKKi
deficiency (see Fig. 2, A and
B). Finally we examined LPS-induced activation of IRF-3
in IKKi/ MEFs since the promoter region of IP-10 gene contains
interferon stimulus-responsive element, a binding site for transcription
factor IRF-3 (36,
37). The results showed that
LPS treatment induces essentially the same levels of interferon
stimulus-responsive element binding activity in IKKi/ and
IKKi+/+ MEFs (Fig.
4F), suggesting that IKKi is not involved in regulation
of IRF-3.
Others show that C/EBP
is involved in IL-1-induced regulation of the
C3 gene (26).
Moreover, C/EBP
appears to synergize with C/EBP
, another member
of the C/EBP family, in transcriptional regulation of the IL-6 gene
(24,
25,
38). Consistent with previous
reports (24,
25), we also observed that LPS
induces C/EBP
and C/EBP
mRNA. Here we show that this occurs to an
identical extent in both IKKi/ and IKKi+/+ MEFs (see
Fig. 4A). As both nuclear
localization and transcriptional activating potential of C/EBP family members
may be regulated by phosphorylation
(20,
21,
23,
39), we also investigated
whether IKKi-deficiency affects the function of C/EBP
and C/EBP
proteins in response to LPS. Treatment of MEFs with LPS induces a DNA binding
complex containing C/EBP
and C/EBP
proteins in IKKi+/+ cells.
Surprisingly, IKKi-deficiency resulted in the reduction of C/EBP DNA binding
activity induced by LPS and affected the induction of DNA complexes containing
specifically the C/EBP
but not the C/EBP
proteins
(Fig. 5A). Western blot
analysis of the C/EBP
proteins expression demonstrated that extracts
from IKKi/ and IKKi+/+ cells contains essentially the same
levels of C/EBP
(Fig.
5B). Metabolic labeling experiments using 32P revealed
that IKKi-deficiency results in a significant reduction of a phosphoprotein
precipitated by antibodies specific for C/EBP
. In contrast the absence
of IKKi did not effect p65/RelA phosphorylation
(Fig. 5C). These results
support the contention that IKKi is involved in the regulation of C/EBP
activity.
To confirm and extend the findings obtained using MEFs we also utilized an
approach relying on siRNAs targeted against IKKi. In this experiment we
evaluated LPS induction of IL-6 gene since this gene is known to be regulated
by C/EBP
and C/EBP
(25,
38). Results obtained from
siRNA-transfected human umbilical vein endothelial cells demonstrated that
specific reduction of IKKi transcripts is accompanied by significant
inhibition of IL-6 mRNA induced by LPS
(Fig. 5D). As an additional
specificity control, we also examined the effects of siRNAs on LPS induction
of NF-
B regulated gene for I
B
. As expected, LPS induction
of I
B
mRNA was unchanged by the reduction of IKKi expression
(Fig. 5D).
Our findings support the contention that IKKi acting through C/EBP
links the NF-
B and C/EBP pathways. It appears that control of IKKi gene
expression plays an important role in this process. Studies with
IKK2/ MEFs indicate that activation of NF-
B is required
for induction of IKKi and C3 mRNA. We further addressed this
possibility using p65/ and IKK2/ cells. Northern
blot analysis revealed that when compared with p65+/+ imMEFs, the
p65/ cells fail to induce IKKi, C/EBP
and C/EBP
mRNAs in response to LPS. In contrast basal levels of these transcripts were
normal in p65/ cells (Fig.
6A). Similar experiments using IKK2/ cells also
revealed a blockade in induction of C/EBP
and C/EBP
mRNA
(Fig. 6B). Thus, these data
support the contention that a functional NF-
B pathway is required for
regulation of C/EBP
and C/EBP
genes. The data also raise the
question of how IKKi is regulated- primarily through NF-
B or through
C/EBP or through combined effects of both pathways.

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FIG. 6. IKKi is a key molecule coupling NF- B and C/EBP pathways.
A, Northern blot analysis of IKKi, C/EBP , C/EBP , and
GAPDH steady-state mRNA levels in p65/ and p65+/+ imMEFs treated
with LPS (100 ng/ml) for the indicated times. B, Northern blot
analysis of C/EBP , C/EBP , and GAPDH steady-state mRNA levels in
IKK2/ and IKK2+/+ imMEFs treated with LPS (100 ng/ml) for the
indicated times. C, the 36-nucleotide sequence of mouse or human
(shown in bold) chromosome 1 identified 480 bp upstream of the
translation initiation site of IKKi gene. A sequence of C/EBP-like binding
site is underlined. D, EMSA of nuclear extract from normal imMEFs
treated with LPS for 2 h. The nuclear extracts were incubated with a
32P-labeled 36-bp DNA fragment (see a sequence of the upper strand
on the panel C) in the absence or in the presence of competitive
(Comp) unlabeled oligonucleotide (2 pmol) containing the wild type
36-bp fragment (W), the C/EBP consensus sequence (C), or the
wild type 36-bp fragment containing the mutation of a C/EBP binding motif
(M). Ab, antibody. In addition, some samples were incubated
in the presence of normal rabbit IgG (N) or in the presence of
specific antibodies (2 µg per reaction) against C/EBP , - , or
- as indicated. E, ChIP assays were carried out on chromatin
samples from IKK2/ (negative control), IKKi+/+, and
IKKi/ (positive control) imMEFs untreated or treated with LPS or
TNF for 2 h. The chromatin was immunoprecipitated with antibodies to C/EBP
( or ) or p65 as an additional control. Shown is an IKKi or an
I B (positive control) promoter fragment amplified by PCR from
the ChIP samples.
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To address questions of IKKi regulation we analyzed the
5'-untranslated regions of human and mouse DNA in the vicinity of the
IKKi gene. Interestingly our analysis did not reveal the presence of
NF-
B sites but rather indicated that both the murine and human genes
have identical sequences containing a C/EBP-like DNA binding site
(Fig. 6C). By using
electrophoretic gel shift assay carried out on a nuclear extract prepared from
LPS-treated cells we found that this sequence has the C/EBP-specific binding
activity in vitro (Fig.
6D). The interaction of C/EBP with IKKi promoter was also
demonstrated via chromatin immunoprecipitation (ChIP) assay. The results of
ChIP assay performed on chromatin samples from untreated or LPS-treated imMEFs
confirmed that both C/EBP
and C/EBP
are able to bind with the
promoter region of IKKi gene in vivo
(Fig. 6E). Similar results were
obtained on chromatin samples from TNF-treated cells
(Fig. 6E). The binding of p65
subunit of NF-
B with the I
B
promoter was used as positive
control, whereas the chromatin samples from IKK2/ imMEFs were
tested as negative control for C/EBP-specific binding with IKKi promoter.
Therefore our data provide support for the contention that up-regulation of
the IKKi promoter by pro-inflammatory mediators such as LPS and TNF is likely
to involve members of the C/EBP family.
 |
DISCUSSION
|
---|
The members of the C/EBP and NF-
B families are known to regulate
overlapping cellular processes, including the control of inflammation and
immunity (2,
3,
5,
8). C/EBP and NF-
B DNA
binding motifs have been identified in the promoter regions of genes encoding
immune and pro-inflammatory modulators, including IL-6
(24,
40), IL-1
(41), IP-10
(36), TNF
(42), RANTES
(43) and COX-2
(44). Previous studies have
also shown that the p50 subunit of NF-
B associates with the
C/EBP
/NF-IL6, suggesting cross-talk between NF-
B and C/EBP in
regulation of immune and acute-phase responses
(45). The nature of the
mechanisms responsible for coordination of C/EBP and NF-
B pathways has
been unclear.
Here we provide experimental data to support the contention that the
IKK2-related kinase IKKi is a key molecule for functional coordination between
these two pathways. The IKKidirected functional link between NF-
B and
C/EBP seems to be a "nonlinear" mechanism that includes a
NF-
B-dependent regulation of C/EBP
and C/EBP
gene
transcription and IKKi-mediated activation of C/EBP-specific DNA binding
activity. Consistent with this mechanism, our experiments carried out on MEFs
derived from mice with targeted deletion of IKKi gene showed that IKKi is
required for LPS induction of genes encoding IL-1
, IL-6, IP-10, TNF,
RANTES and COX-2. Further support for the role of IKKi was obtained in the
experiments showing that siRNA-directed reduction of IKKi expression in human
umbilical vein endothelial cells results in significant inhibition of
LPS-mediated up-regulation of the C/EBP-NF-
B-regulated target gene for
IL-6.
Previous work by others demonstrated that the promoter of the C3
gene contains C/EBP binding sites
(32) and is transactivated by
C/EBP
(26). Our
findings extend these observations by showing that IKKi is required for
LPS-induced activation of a C/EBP-specific DNA binding complex containing
C/EBP
and driving expression of C3. Moreover, we found that
IKKi-deficiency results in a significant reduction of LPS-induced
phosphorylation of a protein precipitated by antibodies specific for
C/EBP
. Although further work is needed to characterize the nature of
this phosphoprotein, these results are consistent with the assumption that
IKKi is involved in the regulation of C/EBP
activity induced by LPS. In
contrast to the effect on C/EBP we did not find evidence for linkage of IKKi
deficiency to other pathways initiated by LPS.
Previous studies have demonstrated that activators of the NF-
B
pathway, such as LPS, TNF and IL-1
(9), induce upregulation of
C/EBP
, C/EBP
and IKKi mRNA
(24,
25,
13). However the exact
sequence of events in this process has not been defined. Here we show that
LPS-mediated activation of the genes for both C/EBP
and C/EBP
requires intact NF-
B pathway, since neither gene is induced by LPS in
IKK2- or p65-deficient MEFs. Further examination of the C/EBP
and
C/EBP
promoters revealed the presence of
B-like sites,
suggesting that NF-
B directly involves in the LPS-mediated regulation
of C/EBP
and C/EBP
genes. However additional studies are needed
to more completely understand the role of these sites. In contrast, the
analysis of the promoter sequences in both human and mouse IKKi genes failed
to identify a
B-like site. Surprisingly we also noted that IKKi
promoter contains a C/EBP-like site with identical sequences in human and
mouse genes. Our analysis revealed that both
and
subunits of
C/EBP interact with an IKKi promoter fragment containing this sequence. This
observation thus supports the conclusion that IKKi gene expression is
regulated by C/EBP.
After the submission of the present work, Sharma et al.
(46) and Fitzgerald et
al. (47) reported on
experiments with human cell lines showing a link between TANK-binding kinase 1
and IKKi/IKK
and phosphorylation of endogenous IRF-3
(46) and reporter gene
expression regulated by interferon regulatory factor-3 (IFR-3) and IRF-7 in
response to virus infection. Here we have not examined effect of
IKKi-deficiency on virus induced responses in MEFs so it is difficult to
compare our studies with those recently published
(46,
47). However we believe each
report provides new evidence linking IKKi to innate immune responses to both
viral and bacterial infection. Here we showed that LPS-mediated activation of
IRF-3 DNA-binding activity was normal in IKKi/ cells, whereas
the activation of IRF family members target genes for IP-10 and RANTES was
blocked in IKKi-deficient cells. This result may reflect differences in
signaling via TLR4- and TLR3-dependent pathways. IKKi-deficiency also resulted
in the reduction of LPS-mediated expression of TNF, IL-6, IL-1 and COX-2
genes; in none of these cases does the promoter appear to contain IRF sites.
In contrast a common characteristic of the promoters for this group of
IKKi-affected genes, including IP-10 and RANTES, is the presence of binding
sites for C/EBP and NF-
B. Here we provide clear evidence that IKKi is
not involved in the activation of NF-
B but significantly affects the
activation of C/EBP. It has been shown that similar to IRF-3 and IRF-7, C/EBP
is also involved in the regulation of viral oncogenesis and infection
(48). Further it was shown
that VSV replication was significantly inhibited by a kinase-dead variant of
IKKi in 293 cells over-expressing IRF-3
(46). On the other hand,
interferon-mediated antiviral response against VSV is normal in
IRF-3/ MEFs
(49). Thus all of the most
recent data suggest that IKKi is involved in the innate immune responses to
viral and bacterial infection. This may well occur via its effects on multiple
transcription factors including members of NF-
B, IRF and C/EBP
families.
In summary, our findings identify the function of IKKi as a signaling
molecule required for LPS-inducible transcriptional regulation of genes for
inflammatory and immune mediators in fibroblasts and endothelial cells,
suggesting an important role for IKKi in innate immune responses of cells of
non-hematopoietic origin. Studies under way with the IKKi/ mice
will provide more direct information about these pathways in vivo. It
follows from our data that modulation of IKKi function may have implications
for the development of the anti-inflammatory drugs. Interestingly, IKKi exists
as a high molecular weight complex
(14). We are currently
performing studies to identify and characterize IKKi-associated proteins that
may provide, in addition to IKKi, molecular targets for development of
anti-inflammatory therapies. The recent observation that diminished local
synthesis of C3 was beneficial for the transplanted kidney
(50) suggests that inhibition
of C3 expression by modulation of IKKi function could have
therapeutic value in tissue transplantation.
 |
FOOTNOTES
|
---|
* The costs of publication of this article were defrayed in part by the
payment of page charges. This 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: The Scripps Research Institute,
IMM-12, 10550 North Torrey Pines Rd. La Jolla, CA 92037. Tel.: 858-784-8219;
Fax: 858-784-8239; E-mail:
ulevitch{at}scripps.edu.
1 The abbreviations used are: C/EBP, CCAAA/enhancer-binding protein; RANTES,
regulated on activation normal T cell expressed and secreted; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; TNF, tumor
necrosis factor; IL-1, interleukin-1; IKK, I
B kinase; MEF, murine
embryonic fibroblast; imMEF, immortalized MEF; siRNA, small interfering RNA;
EMSA, electrophoretic mobility shift assay; ChIP, chromatin
immunoprecipitation; IRF-3, interferon regulatory factor-3; NEMO, NF-
B
essential modulator. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Alexander Hoffman and David Baltimore (California Institute of
Technology) for providing the p65/ 3T3-like fibroblasts, Qiutang
Li and Inder M. Verma (Laboratory of Genetics, The Salk Institute) for
providing the primary and immortalized fibroblasts derived from IKK2-deficient
mice, and Osamu Takeuchi and Shisuo Akira for providing the primary embryonic
fibroblasts obtained from IKKi-deficient mice before publication of an initial
characterization of the knockout mice.
 |
REFERENCES
|
---|
- Rincón, M., Flavell, R. A., and Davis, R. J.
(2001) Oncogene
20,
24902497[CrossRef][Medline]
[Order article via Infotrieve]
- Pomerantz, J. L., and Baltimore, D. (2002)
Mol. Cell 10,
693701[Medline]
[Order article via Infotrieve]
- Poli, V. (1998) J. Biol. Chem.
273,
2927929282[Free Full Text]
- Silverman, N., and Maniatis T. (2001) Genes
Dev. 15,
23212342[Free Full Text]
- Akira, S., and Kishimoto, T. (1997) Adv.
Immunol. 65,
146[Medline]
[Order article via Infotrieve]
- Lekstrom-Himes, J., and Xanthopoulos, K. G. (1998)
J. Biol. Chem. 273,
2854528548[Abstract/Free Full Text]
- McKnight, S. L. (2001) Cell
107,
259261[Medline]
[Order article via Infotrieve]
- Ghosh, S., May, M. J., and Kropp, E. B. (1998)
Annu. Rev. Immunol. 16,
225260[CrossRef][Medline]
[Order article via Infotrieve]
- Pahl, H. L. (1999) Oncogene
18,
68536866[CrossRef][Medline]
[Order article via Infotrieve]
- Karin, M., and Ben-Neriah, Y. (2000) Annu.
Rev. Immunol. 18,
621663[CrossRef][Medline]
[Order article via Infotrieve]
- Beg, A. A., Sha, W. C., Bronson, R. T., Ghosh, S., and Baltimore,
D. (1995) Nature
376,
167170[CrossRef][Medline]
[Order article via Infotrieve]
- Li, Q., Van Antwerp, D., Mercurio, F., Lee, K. F., and Verma, I. M.
(1999) Science
284,
321325[Abstract/Free Full Text]
- Shimada, T., Kawai, T., Takeda, K., Matsumoto, M., Inoue, J., and
Tatsumi, Y., Kanamaru, A., and Akira, S. (1999) Int.
Immunol. 11,
13571362[Abstract/Free Full Text]
- Peters, R. T., Liao, S. M., and Maniatis, T. (2000)
Mol. Cell 5,
513522[Medline]
[Order article via Infotrieve]
- Pomerantz, J. L., and Baltimore, D. (1999)
EMBO J. 18,
66946704[Abstract/Free Full Text]
- Tojima, Y., Fujimoto, A., Delhase, M., Chen, Y., Hatakeyama, S.,
Nakayama, K., Kaneko, Y., Nimura, Y., Motoyama, N., Ikeda, K., Karin, M., and
Nakanishi, M. (2000) Nature
404,
778782[CrossRef][Medline]
[Order article via Infotrieve]
- Bonnard, M., Mirtsos, C., Suzuki, S., Graham, K., Huang, J., Ng,
M., Itie, A., Wakeham, A., Shahinian, A., Henzel, W. J., Elia, A. J.,
Shillinglaw, W., Mak, T. W., Cao, Z., and Yeh, W. C. (2000)
EMBO J. 19,
49764985[Abstract/Free Full Text]
- Cao, Z., Umek, R. M., and McKnight, S. L. (1991)
Genes Gev. 5,
15381552
- Williams, S. C., Cantwell, C. A., and Johnson, P. F.
(1991) Genes Dev.
5,
15531567[Abstract]
- Nakajima, T., Kinoshita, S., Sasagawa, T., Sasaki, K., Naruto, M.,
Kishimoto, T., and Akira, S. (1993) Proc. Natl. Acad.
Sci. U. S. A. 90,
22072211[Abstract]
- Trautwein, C., Caelles, C., van der Geer, P., Hunter, T., Karin,
M., and Chojkier, M. (1993) Nature
364,
544547[CrossRef][Medline]
[Order article via Infotrieve]
- Chumakov, A. M., Grillier, I., Chumakova, E., Chih, D., Slater, J.,
and Koeffler, H. P. (1997) Mol. Cell.
Biol. 17,
13751386[Abstract]
- Buck, M., Zhang, L., Halasz, N. A., Hunter, T., and Chojkier, M.
(2001) EMBO J.
20,
67126723[Abstract/Free Full Text]
- Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S.,
Nishio, Y., Nakajima, T., Hirano, T., and Kishimoto, T. (1990)
EMBO J. 9,
18971906[Abstract]
- Kinoshita, S., Akira, S., and Kishimoto, T. (1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
14731476[Abstract]
- Juan, T. S.-C., Wilson, D. R., Wilde, M. D., and Darlington, G. J.
(1993) Proc. Natl. Acad. Sci. U. S. A.
90,
25842588[Abstract]
- Takeda, K., Takeuchi, O., Tsujimura, T., Itami, S., Adachi, O.,
Kawai, T., Sanjo, H., Yoshikawa, K., Terada, N., and Akira, S.
(1999) Science
284,
313316[Abstract/Free Full Text]
- Yan, S. F., Fujita, T., Lu, J., Okada, K., Shan Zou, Y., Mackman,
N., Pinsky, D. J., and Stern, D. M. (2000) Nat.
Med. 6,
13551361[CrossRef][Medline]
[Order article via Infotrieve]
- Gitlin, L., Karelsky, S., and Andino, R. (2002)
Nature 418,
430434[CrossRef][Medline]
[Order article via Infotrieve]
- Kravchenko, V. V., Pan, Z., Han, J., Herbert, J.-M., Ulevitch, R.
J., and Ye, R. D. (1995) J. Biol. Chem.
270,
1492814934[Abstract/Free Full Text]
- Ivanov, V. N., Bhoumik, A., Krasilnikov, M., Raz, R., Owen-Schaub,
L. B., Levy, D., Horvath, C. M., and Ronai, Z. (2001)
Mol. Cell 7,
517528[Medline]
[Order article via Infotrieve]
- Wilson, D. R., Juan, T. S., Wilde, M. D., Fey, G. H., and
Darlington, G. J. (1990) Mol. Cell. Biol.
10,
61816191[Medline]
[Order article via Infotrieve]
- Rus, H. G., Kim, L. M., Niculesen, F. I., and Shin, M. L.
(1992) J. Immunol.
148,
928933[Abstract/Free Full Text]
- Cardinaux, J.-R., Allaman, I., and Magistretti, P. J.
(2000) Glia
29,
9197[CrossRef][Medline]
[Order article via Infotrieve]
- Zhong, H., Voll, R. E., and Ghosh, S. (1998)
Mol. Cell 1,
661671[Medline]
[Order article via Infotrieve]
- Ohmori, Y., and Hamilton, T. A. (1993) J.
Biol. Chem. 268,
66776688[Abstract/Free Full Text]
- Kawai, T., Takeuchi, O., Fujita, T., Inoue, J., Mühlradt, P.
F., Sato, S., Hoshino, K., and Akira, S. (2001) J.
Immunol. 167,
58875894[Abstract/Free Full Text]
- Hu, H.-M., Baer, M., Williams, S. C., Johnson, P. F., and Schwartz,
R. C. (1998) J. Immunol.
160,
23342343[Abstract/Free Full Text]
- Baer, M., Williams, S. C., Dillner, A., Schwartz, R. C., and
Johnson, P. F. (1998) Blood
92,
43534365[Abstract/Free Full Text]
- Matsusaka, T., Fujikawa, K., Nishito, Y., Mukaida, N., Matsushima,
K., Kishimoto, T., and Akira, S. (1993) Proc. Natl.
Acad. Sci. U. S. A. 90,
1019310197[Abstract]
- Zhang, Y., and Rom, W. N. (1993) Mol. Cell.
Biol. 13,
38313837[Abstract]
- Pope, R. M., Leutz, A., and Ness, S. A. (1994)
J. Clin. Invest. 94,
14491455[Medline]
[Order article via Infotrieve]
- Casola, A., Garofalo, R. P., Haeberle, H., Elliott, T. F., Lin, R.,
Jamalluddin, M., and Brasier, A. R. (2001) J.
Virol. 75,
64286439[Abstract/Free Full Text]
- Caivano, M., Gorgoni, B., Cohen, P., and Poli, V.
(2001) J. Biol. Chem.
276,
4869348701[Abstract/Free Full Text]
- LeClair, K. P., Blanar, M. A., and Sharp, P. A. (1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
81458149[Abstract]
- Sharma, S., tenOever, B. R., Grandvaux, N., Zhou, G.-P., Lin, R.,
and Hiscott, J. (2003) Science
300,
11481151[Abstract/Free Full Text]
- Fitzgerald, K. A., McWhirter, S. M., Faia, K. L., Rowe, D. C.,
Latz, E., Golenbock, D. T., Coyle, A. J., Liao, S.-M., and Maniatis, T.
(2003) Nat. Immunol.
4,
491496[CrossRef][Medline]
[Order article via Infotrieve]
- Hogan, T. H., Krebs, F. C., and Wigdahl, B. (2002)
J. Neurovirol. 8, Suppl.
2, 2126[CrossRef][Medline]
[Order article via Infotrieve]
- Sato, M., Suemori, H., Hata, N., Asagiri, M., Ogasawara, K., Nakao,
K., Nakaya, T., Katsuki, M., Nuguchi, S., Tanaka, N., and Taniguchi, T.
(2000) Immunity
13,
539548[Medline]
[Order article via Infotrieve]
- Pratt, J. R., Basheer, S. A., and Sacks, S. H. (2002)
Nat. Med. 8,
582587[CrossRef][Medline]
[Order article via Infotrieve]