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INTRODUCTION |
Lipopolysaccharide
(LPS)1 is a surface component
of Gram-negative bacteria that is released following host infection and
causes tissue injury and shock (1). LPS mediates such adverse effects by inducing the production of pro-inflammatory cytokines. One of the
most important of these LPS-induced pro-inflammatory cytokine mediators
is tumor necrosis factor-
(TNF-
). TNF-
production in monocytes
and macrophages constitutes between 1 and 2% of secreted proteins in
response to LPS (2). Purified TNF-
induces many of the deleterious
effects of LPS in vivo (3); passive immunization against
TNF-
protects animals from the lethal effects of LPS (4).
The LPS signaling cascade leading to TNF-
production bifurcates to
control both transcription of the TNF-
gene and translation of
TNF-
mRNA (5). Translational regulation of TNF-
mRNA is mediated by a short AU-rich sequence that is conserved among various cytokines and oncogenes and is present within the 3'-untranslated regions of such genes (6). This element confers a repression of
translation that must be overcome in order for translation to proceed
(7-11). Recent studies also suggest a role for this element in
destabilization of TNF-
mRNA (12). Signaling molecules shown to
play a role in translational regulation of TNF-
mRNA include p38
(13) and Jun-N-terminal kinase/stress-activated protein kinase
(JNK/SAPK) (14), members of the mitogen-activated protein kinase
family, as well as the more proximal signaling molecules Raf and Ras
(15). Transcriptional regulation of the TNF-
gene is quite complex;
differences exist across species as well as among cell types.
Regulation of TNF-
transcription is conferred by transcription
factor binding sites present within the TNF-
promoter. The
cis-acting elements within the TNF-
promoter that are conserved
among species include a Y box motif, an SP-1 binding site, as well as
multiple nuclear factor-
B (NF-
B) sites (16, 17). In addition, the
human TNF-
promoter contains an AP-1 site (18). LPS induction of
both murine and human TNF-
promoter activity is dependent on NF-
B
binding sites. Mutation or deletion of such sites results in a loss of
LPS responsiveness, whereas multiple copies of the NF-
B sites
inserted in front of a reporter gene are LPS-responsive (16, 19, 20).
Furthermore, compounds that inhibit NF-
B block TNF-
transcription
and TNF-
production in human monocytes (21). Evidence arguing
against a role for NF-
B in regulation of human TNF-
gene
transcription exists (22, 23). However, the discrepancy may be due to
the stimulus used to induce TNF-
promoter activity, as well as the cell type.
LPS treatment of macrophages stimulates the nuclear mobilization of
NF-
B (24, 25). Under normal conditions, NF-
B is found in a
cytoplasmic complex with an inhibitory protein, inhibitor of NF-
B
(I
B) (26). Many signals that lead to the nuclear translocation of
NF-
B result in the phosphorylation and subsequent degradation of
I
B. Phosphorylation of Ser-32 and Ser-36 of I
B-
targets I
B-
for ubiquitination and degradation by the proteosome,
allowing NF-
B to translocate to the nucleus (27-29). Recently, two
kinases have been identified that inducibly phosphorylate I
B: I
B
kinase-1 (IKK-1), also called IKK-
(30-33), and I
B kinase-2
(IKK-2), also called IKK-
(31, 33, 34). These kinases are activated
by TNF-
and interleukin-1
(30, 33, 34) and are required for cytokine-induced activation of NF-
B (30, 32, 33). IKK-1 and IKK-2
show 52% identity at the amino acid level and can exist in a
heterodimer that is able to interact with another kinase called
NF-
B-inducing kinase (NIK). NIK was originally identified based on
its ability to bind to TNF-receptor-associated factor 2 and is a member
of the mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase kinase (MEKK) family (35). Co-expression of NIK with
IKK-1 or IKK-2 enhances IKK activity, whereas overexpression of a
kinase mutant NIK blocks cytokine-induced NF-
B activation (30, 34).
Another kinase implicated in the regulation of NF-
B activation is
MEKK1. MEKK1 activates an I
B-
kinase complex (36, 37) and is also
implicated in TNF-
-induced NF-
B activation (38). Furthermore,
MEKK1 is required for Tax-induced NF-
B activation (39) and is
involved in Fc
RI-induced activation of the human TNF-
promoter
(40).
We set out to determine the signaling events initiated by LPS that lead
to activation of the murine TNF-
promoter. This promoter lacks
elements, such as AP-1, present in the human promoter, thereby allowing
a more straightforward analysis of the underlying signaling mechanisms
using a physiological promoter. Here, we demonstrate a requirement for
one or both IKKs and examine roles of NIK and MEKK1 in LPS-induced
TNF-
transcription.
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EXPERIMENTAL PROCEDURES |
DNA Constructs--
Expression vectors containing the
constitutively active version of MEKK-1 (MEKK-C) and dominant-negative
MEKK-1 (D1369A) were described previously (41). The FLAG-tagged
wild-type IKK-2, dominant-negative IKK-2 (S177A/S181A), FLAG-tagged
wild-type IKK-1, and dominant-negative IKK-1 (S176A/S180A) constructs
were described previously (31, 39). Wild-type and dominant-negative NIK
(K429A/K430A) were also described previously (39). The TNF-
transcriptional reporter (TNFpro-CAT) was provided by B. Beutler and
was described previously (14). The NF-
B reporter (NF-
B-luc)
consisted of a triple repeat of the human immunodeficiency virus
NF-
B site driving a luciferase cDNA and was provided by A. Thorburn. The dominant-negative I
B construct (I
B-
SS32, 36AA)
was provided by R. Gaynor.
Cells and Transfections--
RAW 264.7 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum (Life Technologies, Inc.), 50 units/ml penicillin, 50 µg/ml streptomycin, and 2-mM L-glutamine at 37 °C in
5% CO2. Transfections were performed using the Profection
DNA transfection kit (DEAE-dextran) from Promega and following the
protocol provided by the manufacturer. DNA amounts transfected were
kept constant by the addition of empty expression vector. Where
indicated, cells were stimulated with LPS (1 µg/ml) or diluent
(sterile saline). Cells were treated with LPS for 6 h for both
luciferase and chloramphenicol acetyltransferase (CAT) assays. CAT
assays were performed as described previously (14). CAT activity was
quantitated using a PhosphorImager. Data are represented as percent
conversion of chloramphenicol to acetylated chloramphenicol. Luciferase
assays were performed using the luciferase assay reagent (Promega) and
following the manufacturer's protocol.
In Vitro Kinase Assays--
Where indicated, FLAG-IKK-1 and
FLAG-IKK-2 were immunoprecipitated from equal amounts of protein (2 mg)
from lysates of transfected RAW 264.7 cells using 1 µg of the M-2
anti-FLAG monoclonal antibody (Sigma) for 4 h at 4 °C on a
rocking platform. Where indicated, endogenous IKK-1 and IKK-2 were
immunoprecipitated from equal amounts of lysates using 1 µg of
anti-IKK-1 (M-280; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or
anti-IKK-2 (H-470; Santa Cruz Biotechnology, Inc.), respectively, for
4 h at 4 °C on a rocking platform. Protein A-Sepharose was
added to each immunoprecipitate for 1.5 h. Immunoprecipitates were
washed three times with lysis buffer (50 mM HEPES (pH 7.5),
150 mM NaCl, 1.5 mM MgCl2, 10%
glycerol, 1% Triton X-100, 100 mM NaF, 1%
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin) and once with
kinase buffer (50 mM Tris (pH 7.4), 10 mM
MgCl2, 1 mM dithiothreitol). The pellets were
resuspended in kinase buffer with 50 µM ATP, 10 µCi of
[
-32P]ATP/sample, and 9 µg/sample of glutathione
S-transferase-I
B (amino acids, 1-54). The reactions were
carried out for 35 min at 30 °C. Samples were centrifuged,
supernatants were added to Laemmli buffer, and the mixtures were boiled
for 2 min. Aliquots were loaded on SDS-10% polyacrylamide gels for
electrophoresis. The gels were dried and exposed to x-ray film.
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RESULTS |
LPS Activates the TNF-
Promoter and an NF-
B Reporter in RAW
264.7 Cells; LPS-induced TNF-
Promoter Activity Is
NF-
B-dependent--
LPS is a potent inducer of TNF-
biosynthesis, activating both transcription of the TNF-
gene and
translation of TNF-
mRNA. We previously demonstrated that
multiple kinase signaling pathways are stimulated by LPS and that the
JNK/SAPK pathway is required for translational induction of TNF-
by
LPS (14). In addition, we found no requirement for JNK/SAPK in
LPS-induced TNF-
transcription, despite the previously described
effects of JNK/SAPK on TNF-
transcription in another system (14,
40). Therefore, we have explored signaling pathways leading to
LPS-induced TNF-
promoter activity. It is well documented that LPS
activates NF-
B and that NF-
B is crucial for LPS induction of
TNF-
gene transcription (20, 21, 24, 25). As a starting point for
our experiments, we confirmed that LPS could induce NF-
B activation,
as well as TNF-
promoter activity in our system. We transiently
transfected RAW 264.7 cells with an NF-
B reporter (NF-
B-luc)
or a TNF-
transcriptional reporter consisting of the murine TNF-
promoter driving a CAT cDNA (TNFpro-CAT). After 24 h, cells
were stimulated with LPS or diluent for 6 h and were harvested for
assessment of luciferase or CAT activity. LPS enhanced NF-
B and
TNF-
promoter activity, as demonstrated by an increase in luciferase
(Fig. 1A, left) or CAT
activity (Fig. 1A, right) in LPS-treated cells, over that in
control cells. The stimulation across experiments was variable, ranging
from 2- to 20-fold. To determine the extent to which NF-
B
contributes to LPS-induced TNF-
promoter activity, we tested whether
overexpression of a dominant-negative I
B could inhibit LPS-induced
TNF-
promoter activity. As shown in Fig. 1B,
overexpression of the dominant-negative I
B completely abolished LPS-induced TNF promoter activity.

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Fig. 1.
LPS activates TNF- promoter activity and
an NF- B reporter; TNF- promoter activity is
NF- B-dependent. A, RAW 264.7 cells were
transfected with either a TNF- transcriptional reporter, TNFpro-CAT
(right), or an NF- B reporter, NF- B-luc
(left). 24 h after transfection, cells were stimulated
with diluent (No LPS) or LPS (1 µg/ml) for 6 h. Cells
were harvested for assessment of CAT activity or luciferase activity.
B, dominant-negative I B was co-transfected with the
TNF- transcriptional reporter. Cells were treated as described above
and were harvested for assessment of CAT activity. Results are shown as
-fold increase over diluent for NF- B reporter activity
(n = 3) (error bars represent S.E.) or
percent conversion of chloramphenicol to acetylated chloramphenicol for
TNF- promoter activity (n = 2, range indicated in
A; n = 3, error bars represent
S.E. in B).
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LPS Activates IKK-1 and IKK-2; IKKs Are Required for LPS-induced
TNF-
Promoter Activity--
Because LPS induction of the TNF-
promoter appears strongly linked to NF-
B (20, 21, 24, 25), we set
out to examine the signaling molecules involved in NF-
B activation
by LPS. Recently, two kinases, IKK-1 and IKK-2, were identified that
phosphorylate I
B, releasing NF-
B, leading to its nuclear
translocation (30-34). To determine whether LPS is capable of
activating IKK-1 or IKK-2, we immunoprecipitated endogenous IKK-1 or
IKK-2 from lysates of RAW 264.7 cells that had been treated with LPS
for varying amounts of time or from unstimulated cells (control). After
immunoprecipitation, IKK-1 or IKK-2 activity was assessed in
vitro in the presence of [
-32P]ATP using
bacterially expressed glutathione S-transferase-I
B (amino acid,
1-54) as a substrate. As shown in Fig.
2A, LPS activates both IKK-1
and IKK-2. Peak activity occurred 15 min poststimulation and rapidly
decreased within 30 min. Because LPS activates an NF-
B reporter and
IKK activity, we wanted to determine whether IKK-1 or IKK-2 is involved
in LPS induction of TNF-
promoter activity. RAW 264.7 cells were
transiently co-transfected with TNFpro-CAT, and either empty vector
(control) or inhibitory mutants of IKK-1 (IKK-1 SS/AA) or IKK-2 (IKK-2
SS/AA). These are phosphorylation-defective mutants previously shown to
block I
B phosphorylation (39). Cells were treated with diluent or
LPS for 6 h and harvested for assessment of CAT activity. As shown
in Fig. 2B, both dominant-negative IKK-2 and
dominant-negative IKK-1 decreased basal promoter activity and inhibited
LPS-induced TNF-
promoter activity. The inhibitory effect was
greater with IKK-2. Similar results were obtained with the NF-
B
reporter (data not shown). Thus, one or possibly both IKKs are required
for LPS-induced regulation of TNF-
transcription. These results
further emphasize the predominant role of NF-
B in the LPS-mediated
transcription of TNF-
.

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Fig. 2.
LPS activates IKK activity; IKKs are required
for LPS induction of TNF- promoter activity. A, IKK-1 and
IKK-2 were immunoprecipitated from RAW 264.7 cells that had been
treated with LPS or diluent as indicated using anti-IKK-1 or anti-IKK-2
antibodies, respectively. Kinase activity was assessed in the presence
of [ -32P]ATP using glutathione
S-transferase (GST)-I B (1-54) as a substrate.
Autoradiographs represent phosphorylated I B. B, RAW 264.7 cells were co-transfected with the TNF- transcriptional reporter and
empty expression vector, a dominant-negative IKK-2, or
dominant-negative IKK-1. 24 h after transfection, cells were
stimulated with diluent or LPS for 6 h. Cells were harvested for
assessment of CAT activity as described above. Data are representative
of several independent experiments.
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LPS-induced Activation of IKK-2 and TNF-
Promoter Activity Is
Blocked by Kinase-defective Mutants of NIK and MEKK1--
NIK and
MEKK1 can activate IKK-1 or IKK-2 in HeLa cells and fibroblasts (34,
37, 39, 42). We examined the roles of NIK and MEKK1 in the regulation
of IKK-1 or IKK-2 in macrophages. We transiently co-transfected a
FLAG-tagged IKK-1 or IKK-2 with increasing amounts of either NIK or a
constitutively active version of MEKK1 (MEKKC). Overexpression of
either NIK or MEKKC activated IKK-2 by 50-fold or more, whereas neither
activated IKK-1, although equal amounts of IKKs were present (Fig.
3A and data not shown). To
determine the possible requirement of NIK or MEKK1 for LPS induction of
IKK-2 activity, we co-expressed FLAG-tagged IKK-2 with either empty
vector (control), a dominant-negative NIK (NIK KK/AA), or a
dominant-negative MEKK1 (MEKK1 D/A) in RAW 264.7 cells. After 29 h, the cells were treated with LPS or diluent for the indicated amounts
of time. As shown in Fig. 3B, dominant-negative NIK and
dominant-negative MEKK-1 were each able to block LPS-induced IKK-2
activity. These results suggest functions for both NIK and MEKK-1 in
LPS induction of IKK-2 activity. However, because each of these
proteins may bind to IKK2, it is possible that they could sequester it,
preventing activation by other kinases. Because NIK and MEKK1
stimulated IKK-2 activity (Fig. 3A), we examined their
effects on LPS-induced TNF-
promoter activity. RAW 264.7 cells
transiently transfected with the TNF-
transcriptional reporter and
either a dominant-negative NIK or a dominant-negative MEKK1 were
treated with LPS or diluent. As shown in Fig. 3C, expression of either dominant-negative NIK (right) or dominant-negative
MEKK1 (left) abolished LPS induction of TNF-
promoter
activity. Similar results were obtained with the NF-
B reporter (Fig.
3D). These results further suggest a role for NIK and MEKK-1
in LPS induction of the TNF-
promoter.

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Fig. 3.
NIK and MEKK1 are involved in the regulation
of IKK and TNF- promoter activities. A, RAW 264.7 cells
were co-transfected with either FLAG-tagged IKK-1 or IKK-2 and
increasing amounts of either NIK (1, 5, 10, or 20 µg) or a
constitutively active MEKK1 (MEKKC; 1, 5, or 10 µg). IKK-1 or IKK-2
activity was assessed as described above. Autoradiographs display
phosphorylated I B. GST, glutathione S-transferase; IP,
immunoprecipitate. B, RAW 264.7 cells were co-transfected
with FLAG-tagged IKK-2 and empty expression vector, a dominant-negative
NIK (NIK KK/AA), or a dominant-negative MEKK1 (MEKK1 D/A). 29 h
after transfection, cells were stimulated with diluent or LPS for the
indicated amounts of time. IKK-2 kinase activity was assessed as
described above. Autoradiograph represents phosphorylated I B. Data
are representive of several independent experiments. C, RAW
264.7 cells were co-transfected with the TNF- transcriptional
reporter and empty expression vector, dominant-negative MEKK1
(left), or dominant-negative NIK (right). 24 h after transfection, cells were treated with diluent or LPS for 6 h and were harvested for assessment of CAT activity (n = 2; error bars indicate range). D, RAW 264.7 cells were co-transfected with the NF- B reporter (NF- B-luc) and
empty expression vector, dominant-negative NIK, or dominant-negative
MEKK1. Cells were treated with either diluent or LPS for 6 h and
were harvested for assessment of luciferase activity. Data are shown as
-fold increase over diluent-treated vector control. Data are
representive of two separate experiments.
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Overexpression of NIK or MEKK1 Activates TNF-
Promoter Activity
in the Absence of Exogenous Stimuli--
Overexpression of MEKK1
activates the human TNF-
promoter (40) and NF-
B (37, 39) in other
systems. Because kinase-dead NIK and MEKK1 block LPS-induced TNF-
promoter activity, we tested whether overexpression of NIK or MEKK1
could induce TNF-
promoter activity in the absence of exogenous
stimuli. We co-expressed the TNF-
transcriptional reporter with
either NIK or MEKK1 (MEKKC) in RAW 264.7 cells. 24 h after
transfection, the cells were treated with either diluent or LPS and
cells were harvested for assessment of CAT activity. As shown in Fig.
4, in the absence of LPS, overexpression of NIK induced TNF-
promoter activity to a level above that obtained with LPS stimulation alone. LPS did not appear to enhance the ability
of NIK to activate the TNF-
promoter. In a similar manner, in the
absence of LPS, overexpression of MEKK-1 activated the TNF-
promoter. However, the effects of LPS and MEKK1 were nearly additive in
inducing the TNF-
transcriptional reporter. Similar results were
obtained using the NF-
B reporter (data not shown). Thus, NIK and
MEKK1 are able to induce TNF-
promoter activity in the absence of
exogenous stimuli. Because LPS further enhances activation of TNF-
promoter activity by MEKK1, but not by NIK, it may be that MEKK1 is
acting by a distinct mechanism.

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Fig. 4.
NIK and MEKK1 activate TNF- promoter
activity in the absence of exogenous stimuli. RAW 264.7 cells were
co-transfected with the TNF- transcriptional reporter and either NIK
(left) or a constitutively active version of MEKK1
(right). Cells were stimulated with diluent or LPS and were
harvested for assessment of CAT activity as described above
(n = 2; range indicated).
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Overexpression of Dominant-negative IKKs Inhibit NIK- and
MEKK1-induced TNF-
Promoter Activity--
Because NIK and MEKK1 can
stimulate IKK-2, we tested whether IKK-2 was required for NIK- or
MEKK1-induced TNF-
promoter activity. We transiently transfected the
TNF-
transcriptional reporter with empty expression vector, MEKK1
(MEKKC), or NIK in the presence or absence of dominant-negative IKK-2
(IKK2 SS/AA). As shown in Fig. 5,
overexpression of dominant-negative IKK-2 inhibited NIK- and
MEKK1-induced TNF-
promoter activity. Similarly, overexpression of a
dominant-negative IKK1 (IKK-1 SS/AA) blocked NIK- and MEKK1-induced TNF-
promoter activity, despite the fact that neither NIK nor MEKK1
activated IKK1 in these cells (Fig. 5).

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Fig. 5.
IKKs mediate NIK- and MEKK1-induced TNF-
transcription. RAW 264.7 cells were transfected with the TNF-
transcriptional reporter; empty vector, NIK (A), or MEKK1
(B); and either dominant-negative IKK-2 or dominant-negative
IKK-1. Cells were treated with LPS or diluent and were harvested for
assessment of CAT activity. Data are representative of two separate
experiments. Error bars indicate range (A).
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DISCUSSION |
In this study, we investigated the signaling pathways initiated by
LPS that are involved in transcriptional regulation of the TNF-
gene
in macrophages. In confirmation of earlier findings concerning
regulation of the TNF-
promoter, our work indicates that most or all
of the signaling leading to TNF-
transcription is mediated by
NF-
B. This conclusion is based, in part, on the finding that
dominant-negative I
B completely inhibits LPS-induced TNF-
promoter activity. Dominant-negative IKKs fully block LPS induction of
the TNF-
promoter, further implicating NF-
B as the primary
mediator of LPS-induced TNF-
promoter activity. In a previous study
examining transcriptional regulation of the human TNF-
promoter, it
was determined that a dominant-negative JNK/SAPK interfered with
Fc
RI induction of transcription in mast cells (40). We showed that
dominant-negative JNK/SAPK had no effect on LPS induction of the murine
TNF-
promoter in macrophages (14). Therefore, regulation of TNF-
transcription differs among species and/or cell types. Indeed, the
human promoter contains elements, such as an AP-1 site, that are not
present in the murine promoter (18).
The mechanism by which NF-
B becomes activated by LPS has not been
examined. We demonstrate that LPS activates the recently identified
I
B kinases, IKK-1 and IKK-2. LPS stimulation of RAW 264.7 cells
resulted in similar kinetics of activation for both IKK-1 and IKK-2. We
established roles for IKK-2 and IKK-1 in LPS induction of TNF-
transcription by demonstrating that overexpression of dominant-negative
versions of either IKK blocked LPS induction of the murine TNF-
promoter. Because IKK-1 and IKK-2 form heterodimers (3, 33, 34), it is
not possible to determine whether one or the other is more important
using these approaches.
Because LPS induces TNF-
production in macrophages, one could argue
that the effects on the IKKs are caused by an autocrine mechanism.
This, however, is not the case because of the rapid kinetics of IKK
activation. We determined that LPS induced activation of other
signaling molecules, including extracellular signal-regulated kinases,
p38, JNK/SAPK, MEKs 1, 3, 4, and 6, 30-60 min poststimulation in
macrophages (14). In contrast, activation of IKKs peaks within 15 min
of LPS stimulation. Therefore, IKK activation is among the earliest
signaling events known to be induced by LPS in macrophages.
To test possible links between LPS and the IKKs, we examined two IKK
kinases, NIK and MEKK1. Both NIK and MEKK1 stimulate IKK-2 activity in
RAW 264.7 cells, and both NIK and MEKK1 are sufficient to activate not
only an NF-
B reporter but also the TNF-
promoter in the absence
of ligand. Additional findings suggesting that NIK and MEKK1 may both
be involved in LPS-mediated transcriptional regulation of the TNF-
gene come from studies with kinase-defective mutants. Either
kinase-dead NIK or MEKK1 blocks the induction of IKK-2 activity and
induction of the TNF-
promoter by LPS. These results may indicate
that both enzymes are required. Alternatively, they may block NF-
B
by sequestering IKK-2 or NF-
B activation complexes.
Our results suggest roles for IKK-1, IKK-2, or both, in LPS-induced
signaling to the TNF-
promoter. However, we propose that IKK-2 is
the more significant mediator of LPS-induced signaling to the TNF-
promoter, since known IKK kinases lead to activation of IKK-2, not
IKK-1. LPS-induced IKK activity observed in the IKK-1
immunoprecipitates could be due to the presence of IKK-2. Dominant-negative IKK-1 inhibited LPS-, MEKK1-, and NIK-induced TNF-
promoter, and NF-
B activity could also be attributed to the fact
that the IKKs form heterodimers (3, 33, 34). Thus, overexpression of
dominant-negative IKK-1 could interfere with IKK-2 function; more
extensive studies are necessary to determine such matters definitively.
It is clear that regulation of NF-
B is complex, as multiple kinases
and other molecules, such as NF-
B essential modulator (43), appear
to be involved. Here, we establish a role for IKKs in LPS-induced
NF-
B activation and, more importantly, in LPS induction of TNF-
transcription. Most studies about IKKs merely demonstrate a role for
IKKs in the regulation of NF-
B, using NF-
B reporters consisting
of multiple copies of a particular site driving the expression of a
reporter gene. Our studies establish a potential mechanism for
initiation of TNF-
promoter activity, thus providing a better
understanding of the regulation of such an important inflammatory mediator.