1 Department of Medicine and Center for Gastrointestinal Biology and Disease and 3 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7080; and 2 Signal Pharmaceuticals, Inc., San Diego, California 92121
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we
examined the role of the nuclear factor-B (NF-
B)-inducing kinase
(NIK) in distinct signaling pathways leading to NF-
B activation. We
show that a dominant-negative form of NIK (dnNIK) delivered by
adenoviral (Ad5dnNIK) vector inhibits Fas-induced I
B
phosphorylation and NF-
B-dependent gene expression in HT-29 and HeLa
cells. Interleukin (IL)-1
- and tumor necrosis factor-
(TNF-
)-induced NF-
B activation and
B-dependent gene expression
are inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells.
Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF-
-induced
apoptosis at an early time point. However, cytokine- and
Fas-induced signals to NF-
B are finally integrated by the I
B
kinase (IKK) complex, since I
B
phosphorylation, NF-
B DNA
binding activity, and IL-8 gene expression were strongly inhibited in
HT-29 and HeLa cells overexpressing dominant-negative IKK
(Ad5dnIKK
). Our findings support the concept that cytokine signaling
to NF-
B is redundant at the level of NIK. In addition, this study
demonstrates for the first time the critical role of NIK and IKK
in
Fas-induced NF-
B signaling cascade.
interleukin-8; inflammation; intestinal epithelial cells; signal transduction
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IT HAS BEEN REPORTED
that the inhibitor of B (I
B)/nuclear factor-
B (NF-
B)
transcriptional system is implicated in multiple aspects of cell
physiology, such as immune and inflammatory processes, cell growth,
development, proliferation, and survival (6, 9). Activation of NF-
B is preceded by rapid serine-specific
phosphorylation and degradation of its cytoplasmic inhibitory proteins
of the I
B family (23). Cytokine-induced serine
phosphorylation of I
B
has been shown to require the participation
of a 700-kDa multisubunit protein complex, known as the I
B kinase
(IKK signalsome) (23). This complex contains at least two
catalytic subunits, IKK
and IKK
, and the regulatory molecule
IKK
/NEMO (56, 63). Overexpression of IKK
or IKK
triggers I
B
serine phosphorylation and NF-
B activation;
conversely, dominant-negative forms of both molecules impair
interleukin (IL)-1
- and tumor necrosis factor-
(TNF-
)-induced
NF-
B-dependent reporter gene expression (10, 35, 48, 60,
64). However, gene deletion studies reveal that cytokine-induced
I
B
phosphorylation and NF-
B activation are predominantly
accomplished by the catalytic subunit IKK
, and not by IKK
, and
that the master regulator of both kinases is the IKK
-subunit
(31, 53). Although these data established the IKK complex
as the I
B kinase, the events leading to its activation remain
largely unknown.
The mitogen-activated protein 3-kinase (MAPKKK) NF-B-inducing kinase
(NIK) has been proposed to act as proximal inducer of the IKK catalytic
subunit (9, 24), based on evidence that ectopic expression
of the protein induced IKK kinase activity that triggers I
B
serine phosphorylation and NF-
B-dependent gene transcription, a
cascade of events blocked by dominant-negative NIK (dnNIK) (28,
29, 32, 38, 40, 46, 60).
Fas ligand, IL-1, and TNF-
utilize various and distinct adapter
proteins and kinases to signal to downstream effector targets (5,
36, 42). It has been shown that Fas-, IL-1
-, and
TNF-
-induced
B-dependent transcription of a reporter gene is
blocked by transient transfection of a dnNIK molecule (4, 28, 32,
41, 44, 51). These data positioned NIK at the intersection point
of cytokines signaling to the NF-
B pathway.
The autosomal recessive mutation aly (alymphoplasia) causes
lack of lymph nodes and Peyer's patches as well as disorganized splenic and thymic structures in aly/aly mice (33,
37). Interestingly, this phenotype is caused by a point mutation
in the COOH-terminal region of NIK (54). Surprisingly,
lymphotoxin (LT)- receptor (LT
R)-mediated, but not
TNF-
-mediated, NF-
B activation is impaired in cells isolated from
aly/aly mice (13, 34, 54). An analysis of CD40
signaling to NF-
B in the same mice (13) demonstrated that NIK is a critical mediator of NF-
B activation by CD40 in B
cells, but not in dendritic cells. Moreover, embryonic fibroblasts isolated from NIK-deficient mice display a functional TNF-
-, IL-1
-, and LT
R-induced NF-
B DNA binding activity
(61). Overall, these data argue against a universal role
for NIK in NF-
B signaling pathways and suggest eventually a
cell-type and signal-specific relevance of this molecule.
Biochemical, pharmacological, and genetic data suggest that the control
of NF-B activation constitutes a relevant target for the treatment
of inflammatory diseases, including inflammatory bowel disease
(22). Therefore, the identification and functional characterization of the various kinases involved in the regulatory mechanisms of NF-
B activation are likely to help design new
therapeutic targets. In this study, we evaluated the role of NIK and
IKK
in Fas-, IL-1
-, and TNF-
-induced NF-
B activation in
HT-29 cells, an intestinal epithelial cell (IEC) line, and HeLa cells
using adenoviral vectors encoding dominant-negative forms of these
molecules. We show a strict requirement of NIK in Fas, but not in
IL-1
and TNF-
, signaling to NF-
B in IEC. However, cytokines
and Fas signaling to NF-
B are inhibited in cells infected with an
adenoviral vector encoding for dominant-negative IKK
(Ad5dnIKK
).
In this report, we have demonstrated for the first time the critical
role of NIK and IKK
in Fas signaling to the NF-
B pathway.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture.
The human HT-29 colonic epithelial cells (HTB 38, American Type Culture
Collection) were grown as described previously (18). HeLa
cells were grown in Eagle's minimum essential medium with 10% FBS,
1× MEM nonessential amino acids, and antibiotics. The human HT-29 cell
line was used, because IL-1, TNF-
, and Fas ligation stimulate
NF-
B activation and IL-8 gene expression in these cells (2, 3,
19, 50). Cells were stimulated with human recombinant IL-1
(5 ng/ml), TNF-
(5 ng/ml; both from Intergen, Purchase, NY),
LT-
1
2 (100 ng/ml; R & D Systems), or a
Fas agonistic antibody (CH-11, 100 ng/ml; USB, Lake Placid, NY).
Ad5dnNIK and Ad5dnIKK construction.
The dnNIK consists of a truncated protein where the kinase domain and
TNF receptor-associated factor (TRAF)-2-interacting domain (aa
1-623) were deleted (39). The adenovirus dnNIK
(Ad5dnNIK) was constructed using the Cre-lox recombination method, as
described previously (16). The subcloned gene contained an
extra 27 bp of DNA nucleotides coding for a peptide derived from the
hemagglutinin (HA) gene (YPYDVPDYA). The dnIKK
construct cloned in
adenoviral vector carried a point mutation in the kinase domain (K44A),
as described previously (35), and contained an extra 24 bp
of DNA nucleotides coding for the FLAG peptide (DYLDDDDL). The
Ad5I
BAA virus has been characterized and described previously
(20). Ad5LUC virus containing the luciferase gene and/or
Ad5GFP virus containing the green fluorescent protein were used as
control virus throughout the study.
IEC infection.
After cells were cultured to 80% confluence, they were infected with
the various adenoviral vectors in serum-free medium (Opti-MEM, GIBCO,
Grand Island, NY) for 16 h. Different multiplicities of infection
(0, 1:10, 1:50, and 1:100 IEC/virus particles) were tested to establish
experimental conditions for high expression of the transgene with no
cytotoxic effects. Cell viability estimated by trypan blue exclusion
was always >97%. The adenovirus was then washed off, fresh medium
containing serum was added, and cells were treated at the various time
points with human recombinant IL-1, TNF-
, Fas agonistic antibody,
or LT-
1
2.
RNA extraction and RT-PCR analysis.
RNA was isolated using TRIzol (GIBCO), reverse transcribed, and
amplified as described elsewhere (18). The PCR products (5 µl) were subjected to electrophoresis on 2% agarose gels containing Gel Star fluorescent dye (FMC, Philadelphia, PA). Fluorescent staining
was captured using an AlphaImager 2000 (AlphaInnotech, San Leandro,
CA). Negative controls included amplifications with no nucleic acid or
no reverse transcriptase. The IL-8 and -actin primers have been
previously described (18). The NIK primers used were
(5')5-CTGGCCTGTGTAGACAGCCAGA-3 (position 1091, NIK-A) and
(3')3-TAATCCACTGGCTTGAGTTTCTCA-5 (position 1381, NIK-B). The length of
the amplified product was 314 bp. To confirm the specificity and
identity of the amplified product, the DNA was sequenced at the
University of North Carolina, Chapel Hill, Automated Sequencing Facility on a model 377 DNA sequencer (Applied Biosystems Division, Perkin Elmer) using the ABI PRISM dye terminator cycle sequencing ready
reaction kit with AmpliTaq DNA polymerase FS (Applied Biosystems Division, Perkin Elmer).
Western blot analysis.
Uninfected or Ad5dnNIK- or Ad5dnIKK-infected cells were stimulated
with IL-1
or TNF-
(both at 5 ng/ml) for 0-60 min. The cells
were then lysed in 1× Laemmli buffer (18), and 20 µg of protein were subjected to electrophoresis on 10% SDS-polyacrylamide gels. Antiphosphoserine I
B
(New England Biolabs, Beverly, MA), anti-I
B
(Santa Cruz Biotechnology, Santa Cruz, CA), anti-HA (Boehringer Mannheim, Indianapolis, IN), anti-NIK (Santa Cruz Biotechnology), and anti-FLAG M2 (Eastman Kodak, New Haven, CT) antibodies were all used at 1:1,000 dilution. The specific
immunoreactive proteins were detected using the enhanced
chemiluminescence kit (ECL; Amersham), as described previously
(18).
Immunofluorescence analysis. HT-29 and HeLa cells were infected with Ad5dnNIK or left uninfected for 16 h. Cells were fixed with 100% ice-cold methanol, and dnNIK was detected using a mouse anti-HA antibody followed by incubation with an anti-mouse rhodamine-conjugated antibody, as described previously (20).
Nuclear extraction and electrophoretic mobility shift assay.
Uninfected or adenovirus-infected cells were stimulated with IL-1 or
TNF-
(both at 5 ng/ml) for 30 min or with Fas agonistic antibody for
6 h (100 ng/ml), and nuclear extracts were prepared as described
previously (18). Extracts (5 µg) were incubated with
radiolabeled double-stranded oligonucleotides specifying the consensus
sequence for class I major histocompatibility complex
B sites
(GGCTGGGGATTCCCCATCT). Protein-DNA complexes were then separated by
nondenaturating electrophoresis and visualized by autoradiography, as
described previously (18).
Cell death assay.
HT-29 cells (1 × 106) were grown in six-well plates
and at 80% of confluence were infected with Ad5IBAA or Ad5dnNIK for
16 h and then exposed to TNF-
(5 ng/ml) for 6 h. Cell
death was determined by counting the number of floating cells per well
with a hemocytometer, a technique commonly used to quantify cell death in HT-29 cells as well as other cell lines of epithelial origin (14, 17, 43). In addition, cell death was evaluated by DNA fragmentation analysis on agarose gels. After treatment, cells were
washed twice with PBS and then lysed in a hypotonic lysis buffer (10 mM
Tris, 1 mM EDTA, and 0.2% Triton X-100, pH 7.5). Proteinase K
digestion was performed for 16 h at 37°C. The DNA was then
extracted with phenol-chloroform-isoamyl alcohol (25:24:1), precipitated in ethanol, and resuspended in TE buffer (10 mM
Tris · HCl-1 mM EDTA). DNA was separated by electrophoresis on
a 1.5% agarose gel.
Luciferase assay.
HT-29 and HeLa cells were infected with a B-luciferase adenoviral
vector (Ad5
BLUC) alone or coinfected with Ad5dnNIK or control virus
(Ad5GFP) for 16 h. After infection, cells were washed in PBS, and
fresh medium containing serum was added before stimulation. Cells were
stimulated with TNF-
(0.05 ng/ml), IL-1
(0.5 ng/ml), and Fas
antibody (100 ng/ml). After stimulation with TNF-
and IL-1
for
8 h and with Fas antibody for 16 h, cells were harvested and
lysed as previously described (18).
B-dependent
luciferase activity was evaluated on a Monolight 2010 luminometer
(Analytical Luminescence Laboratory, San Diego, CA). Values are
expressed as means ± SE derived by triplicates of each condition tested.
Statistical analysis. Statistical significance was evaluated by the two-tailed Student's t-test for paired data. P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Selective role of NIK in Fas-, TNF--, and IL-1
-mediated
B-dependent transcriptional activity.
To determine whether NIK has a differential role in Fas, TNF-
and
IL-1
signaling to NF-
B in epithelial cells, we constructed adenoviral vectors encoding for a dnNIK (Ad5dnNIK). Adenoviral vectors
are not only suitable for gene therapy but are also a useful tool to
dissect endogenous signaling cascades in cells refractory to regular
lipid-based transfection (21). Immunofluorescence analysis
using an anti-HA antibody demonstrated that HA-tagged dnNIK was
expressed by >90% of Ad5dnNIK-infected HT-29 and HeLa cells (Fig.
1). We next investigated the role of NIK
in cytokine- and Fas death receptor-induced NF-
B activation in
the two different epithelial cells, HeLa and HT-29. Cells were infected
with an adenoviral vector encoding for the
B-luciferase gene
(Ad5
BLUC) alone or with Ad5dnNIK or the control virus and then were
stimulated with IL-1
, TNF-
, or Fas antibody. TNF-
, IL-1
,
and Fas antibody induced NF-
B-dependent transcriptional activity in
both cell lines, although Fas ligation induced lower activation than
cytokines (data not shown). To compare the ability of dnNIK to block
signal-induced NF-
B transcriptional activity, we established doses
of IL-1
, Fas antibody, and TNF-
that give similar levels of
NF-
B induction (data not shown). Figure
2 shows that IL-1- and TNF-induced
NF-
B transcriptional activity is significantly inhibited in
Ad5dnNIK-infected HeLa cells but not in Ad5dnNIK-infected HT-29 cells,
while Fas-induced NF-
B transcriptional activity is strongly blocked
in both cell lines.
|
|
Differential involvement of NIK in regulating phosphorylation of
IB
induced by cytokines and Fas death receptor in HT-29 and HeLa
cells.
NIK has been positioned as a proximal kinase involved in IKK
activation; therefore, a dominant-negative molecule should inhibit cytokine-induced I
B
phosphorylation. To test this hypothesis, HeLa and HT-29 cells were infected with Ad5dnNIK and stimulated with
TNF-
, IL-1
, or Fas antibody for various times, then
phosphorylation at serine-32 of I
B
was analyzed by Western
blotting. Because phosphorylated I
B
is an unstable intermediate
because of its rapid proteasome-mediated degradation, cells were
preincubated with the proteasome inhibitor MG-132 to allow accumulation
of phosphorylated I
B
. As shown in Fig.
3, TNF-
, IL-1
, and Fas antibody
induced I
B
phosphorylation in both HeLa and HT-29 cells (compare
lanes 2-4 with lane 1), although the
kinetics of phosphorylation were slower (>30 min) in Fas- than in
cytokine-stimulated cells (<10 min). In accordance with data presented
in Fig. 2, Ad5dnNIK strongly reduced IL-1
- and TNF-
-induced
I
B
phosphorylation in HeLa cells, but only marginally in HT-29
cells (Fig. 3), whereas Fas-induced phosphorylation of I
B
was
blocked in both cell lines. Overall, this suggests that NIK has a
critical role in regulating phosphorylation of I
B
in Fas
signaling to NF-
B in both epithelial cell lines.
|
Differential ability of NIK in regulating cytokine- and Fas-induced
NF-B DNA binding activity and gene expression in HT-29 and HeLa
cells.
We next used HT-29 and HeLa cells to further characterize the role of
NIK in cytokine- and Fas-induced NF-
B-dependent gene expression and
DNA binding activity. As shown in Fig.
4A, all three stimuli,
TNF-
, IL-1
, and Fas antibody, induced NF-
B DNA binding
activity, although Fas ligation induced only a modest increase. In
agreement with Fig. 2A, Ad5dnNIK blocked Fas
antibody-induced NF-
B DNA binding activity but only weakly blocked
IL-1
- or TNF-
-induced NF-
B DNA binding activity in HT-29 cells
(Fig. 4A). In contrast, TNF-
-induced NF-
B DNA binding
activity was strongly inhibited in Ad5dnNIK-infected HeLa cells (Fig.
4B). As in HT-29 cells, Fas-induced NF-
B DNA binding
activity was also blocked in Ad5dnNIK-infected HeLa cells (Fig.
4B). To rule out the possibility that dnNIK failed to
overcome the strong activation of NF-
B by cytokines in HT-29 cells
but was efficient in blocking the weaker Fas signal, we next performed
a TNF dose response in HT-29 cells. As shown in Fig. 4C,
Ad5dnNIK failed to prevent NF-
B DNA binding activity even at low TNF
concentrations, suggesting that the lack of inhibition is not related
to the extent of NF-
B activation. This is in agreement with a recent
report showing that TNF-
- and IL-1
-induced NF-
B DNA binding
activity is NIK independent in mouse embryonic fibroblasts (MEF) isolated from NIK
/
mice (61).
Western blot analysis revealed equal expression of dnNIK in infected
cells (Fig. 4C, bottom). LT-
1
2
has been shown to induce NF-
B activation in HT-29 cells
(30). Figure 4D shows that Fas-induced NF-
B
DNA binding activity is inhibited by Ad5dnNIK, whereas the LT
R
signal is minimally affected. Interestingly LT
R-induced NF-
B DNA
binding activity is not blocked in cells isolated from
NIK
/
mice, further confirming the validity of our in
vitro approach through delivery of a dominant-negative molecule.
|
|
|
Increased apoptosis in Ad5IBAA- but not
Ad5dnNIK-infected HT-29 cells.
Blockade of NF-
B has been shown to sensitize cells to
TNF-
-induced apoptosis (58). Therefore, we
compared the effect of Ad5I
BAA delivering a superrepressor of
NF-
B (20) and Ad5dnNIK on TNF-
-stimulated HT-29
cells. Interestingly, cell detachment increased in Ad5I
BAA-infected
TNF-
-stimulated HT-29 cells as early as 6 h but not in
Ad5dnNIK-infected cells, as seen by morphological analysis (Fig.
7A) and by counting floating
cells (Fig. 7B), a widely used method for evaluation of cell
death in cells of epithelial origin (14, 17, 43).
Moreover, DNA laddering was increased in Ad5I
BAA-infected, but not
in Ad5dnNIK-infected, HT-29 cells stimulated with TNF-
for 6 h.
This may suggest that the protective role of NF-
B is still present
in conditions where NIK function is impaired.
|
IKK is critical for Fas- and cytokine-induced NF-
B activation
and IL-8 gene expression in HT-29 and HeLa cells.
We next investigated whether in both cell lines Fas and cytokine
signaling to NF-
B proceed through the IKK complex. We first compared
the level of I
B
phosphorylation in cells infected with an
adenoviral vector delivering a kinase-deficient mutant of IKK
(Ad5dnIKK
). TNF-
, IL-1
, and Fas antibody induced I
B
phosphorylation in HeLa cells (Fig.
8A) and HT-29 cells (Fig.
8B), in accordance with data presented in Fig. 3.
Interestingly, as opposed to Ad5dnNIK, a blockade at the level of
IKK
by Ad5dnIKK
strongly reduced I
B
phosphorylation in both
cell lines and in all the conditions tested. Moreover, Ad5dnIKK
strongly inhibited Fas- and IL-1
-induced NF-
B DNA binding
activity in HT-29 (Fig. 9) and HeLa (data
not shown) cells. In accordance with blockade of NF-
B activation, cytokine- and Fas antibody-induced IL-8 mRNA accumulation was blocked
in Ad5dnIKK
-infected HT-29 cells (Fig.
10A). In addition, TNF-
-
and Fas antibody-induced IL-8 mRNA accumulation was also blocked in
Ad5dnIKK
-infected HeLa cells (Fig. 10B). Although IL-1
induced
B-dependent transcriptional activity (Fig. 2B),
it failed to induce significant IL-8 mRNA expression (unpublished
observation). Together, these data demonstrate that NIK is an essential
component of Fas signaling to NF-
B but is dispensable for IL-1
-
and TNF-
-induced NF-
B activation. On the other hand, cytokine-
and Fas-initiated signals converge on the IKK complex and utilize the
IKK
catalytic subunit to induce NF-
B activity.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Numerous unrelated signals such as IL-1, TNF,
lipopolysaccharide, and the viral protein Tax induce NF-
B activity
through IKK kinase (63). An important question is whether
these various inducers utilize diverse or common transmitting pathways
to relay the signal to IKK. NIK has been proposed as an upstream kinase activated by diverse stimuli and inducing the IKK complex, and, as
such, it may represent a potent therapeutic target for disorders involving NF-
B dysregulation.
Our data indicate that NIK is obligatory for NF-B activation
induced by Fas ligation in HeLa and HT-29 cells, since a
dominant-negative molecule blocks I
B
phosphorylation, NF-
B DNA
binding activity,
B-dependent gene expression, and IL-8 mRNA
accumulation. Recently, it has been reported that LT-
, but not TNF,
utilizes NIK to activate the NF-
B pathway (34). Our
data show that TNF-, IL-
-, and LT-
-induced NF-
B DNA binding
activity is not blocked in Ad5dnNIK-infected HT-29 cells. This suggests
that the role of NIK in the NF-
B pathway is restricted to the Fas
signaling cascade, at least in HT-29 and HeLa cells. During the
preparation/submission of our manuscript, a study has demonstrated that
IL-1
-, TNF-, and LT-
-induced NF-
B DNA binding activity
proceeds through a NIK-independent mechanism in MEF or B cells isolated
from NIK-deficient mice (61). Therefore, our findings that
cytokine-induced NF-
B activation is NIK independent in HT-29 cells
are in line with this new study. In addition, we demonstrated for the
first time that Fas signaling to the NF-
B pathway is NIK dependent.
Although Fas ligation has been shown to induce B-dependent
transcription and IL-8 gene expression (3, 8, 11, 15, 16, 32, 45,
50), the signaling cascade responsible for NF-
B activity has
not been established yet. Our study shows that the Fas signaling
cascade to NF-
B activation is blocked by Ad5dnNIK and Ad5dnIKK
.
From these data, an emerging pathway for Fas signaling to NF-
B
includes the obligatory role of NIK and IKK
. It is still unclear how
Fas signals to NIK and IKK proteins. The Fas cytoplasmic tail is able
to recruit Fas-associated death domain and the receptor-interacting protein (RIP) (57). Recently, it has been shown that TNF
receptor 1 utilizes RIP to mediate IKK activation (12).
Therefore, similar to TNF type1 receptor, Fas may utilize RIP to
activate IKK. Although it remains to be demonstrated whether RIP is
necessary for Fas-mediated NF-
B activation, a potential pathway
could involve RIP, NIK, and IKK
. It remains to be seen whether cells
isolated from NIK-deficient mice display an impaired Fas signaling to
NF-
B.
The role of Fas in intestinal homeostasis is not well understood. However, this pathway may participate in mucosal injury by reducing intestinal barrier function through induction of cell apoptosis and/or by impairing epithelial barrier function (1). Therefore, our finding that NIK is selectively involved in Fas signal transduction in IEC provides an interesting checkpoint to modulate this pathway.
The failure of Ad5dnNIK to completely block IL-1- and
TNF-
-induced NF-
B activation in HT-29 cells strongly suggests the existence of an alternate route of activation. Mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) kinase-1 (MEKK1) has been postulated to be a strong inducer of IKK
kinase activity (26, 27, 38, 40). Recently, it was reported that cells isolated from MEKK1
/
mice display a functional
NF-
B activation after IL-1
and TNF-
stimulation
(62). In addition, significant IKK and NF-
B activity was detected in cells where both MEKK1 and NIK signals were blocked (49). This suggests the existence of a signaling pathway
to NF-
B utilized by IL-1
and TNF-
that is independent of NIK
and MEKK1. Potential candidate kinases include p38 kinase,
phosphoinositide 3-kinase, protein kinase B (Akt), MEKK2, MEKK3, and
the atypical protein kinase C, which are all capable of NF-
B
activation (7, 25, 44, 47, 52, 55, 65). Interestingly,
phosphoinositide 3-kinase inhibition by pharmacological blockade does
not prevent TNF-
-induced NF-
B activation (59). From
our data, we suggest that different stimuli target various intermediate
kinases to activate NF-
B and that NIK is part of a multiple network
of kinases but is not a critical component of IKK activation. It is
still unclear why NIK is obligatory for Fas signaling to NF-
B but
dispensable for TNF-
or IL-1
. The differences may relate to the
pleiotrophic nature of IL-1 and TNF signaling, which involve a
multitude of kinases as opposed to the more linear Fas signaling.
However, regardless of the intermediate kinases used by IL-1
and
TNF-
, a blockade imposed by Ad5dnIKK
totally prevented NF-
B
signaling and IL-8 gene expression. Thus, although the signal from the
cell surface receptor may lead to redundant and alternate routes, these different pathways converge on IKK
to activate NF-
B. This
indicates that IKK
, but not NIK, represents a potential target for
therapeutic intervention targeting the NF-
B pathway in many cell
types, including IEC.
In summary, we have demonstrated that NIK is an essential signaling
molecule for Fas-mediated NF-B activity. However, the role of NIK in
IL-1
- or TNF-
-induced NF-
B activity appears to be signal and
cell type specific. These findings may help design new therapeutic
targets for inflammatory disorders.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institutes of Health Grant ROI-DK-47700 to C. Jobin.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: C. Jobin, Div. of Digestive Diseases and Nutrition, CB# 7038, Glaxo Bldg., University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7038 (E-mail: Job{at}med.unc.edu).
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.
First published February 27, 2002;10.1152/ajpcell.00166.2001
Received 2 April 2001; accepted in final form 21 February 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abreu, MT,
Palladino AA,
Arnold ET,
Kwon RS,
and
McRoberts JA.
Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells.
Gastroenterology
119:
1524-1536,
2000[ISI][Medline].
2.
Abreu-Martin, MT,
Palladino AA,
Faris M,
Carramanzana NM,
Nel AE,
and
Targan SR.
Fas activates the JNK pathway in human colonic epithelial cells: lack of a direct role in apoptosis.
Am J Physiol Gastrointest Liver Physiol
276:
G599-G605,
1999
3.
Abreu-Martin, MT,
Vidrich A,
Lynch DH,
and
Targan SR.
Divergent induction of apoptosis and IL-8 secretion in HT-29 cells in response to TNF- and ligation of Fas antigen.
J Immunol
155:
4147-4154,
1995[Abstract].
4.
Awane, M,
Andres PG,
Li DJ,
and
Reinecker HC.
NF-B-inducing kinase is a common mediator of IL-17-, TNF-
-, and IL-1
-induced chemokine promoter activation in intestinal epithelial cells.
J Immunol
162:
5337-5344,
1999
5.
Baker, SJ,
and
Reddy EP.
Modulation of life and death by the TNF receptor superfamily.
Oncogene
17:
3261-3270,
1998[ISI][Medline].
6.
Barnes, PJ,
and
Karin M.
Nuclear factor-B, a pivotal transcription factor in chronic inflammatory diseases.
N Engl J Med
336:
1066-1071,
1997
7.
Berghe, W,
Plaisance S,
De Brosscher K,
Schmitz ML,
Fiers W,
and
Haegeman G.
p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-B p65 transactivation mediated by tumor necrosis factor.
J Biol Chem
273:
3285-3290,
1998
8.
Cheema, ZF,
Wade SB,
Sata M,
Walsh K,
Sohrabja F,
and
Miranda RC.
Fas/Apo (apoptosis)-1 and associated proteins in the differentiating cerebral cortex: induction of caspase-dependent cell death and activation of NF-B.
J Neurosci
19:
1754-1770,
1999
9.
Chen, F,
Castranova V,
Shi X,
and
Demers LM.
New insights into the role of nuclear factor-B, a ubiquitous transcription factor in the initiation of diseases.
Clin Chem
45:
7-17,
1999
10.
Cohen, L,
Henzel WJ,
and
Baeuerle PA.
IKAP is a scaffold protein of the IB kinase complex.
Nature
395:
292-296,
1998[ISI][Medline].
11.
Depraetere, V,
and
Golstein P.
Fas and other cell death signaling pathways.
Semin Immunol
9:
93-107,
1997[Medline].
12.
Devin, A,
Cook A,
Lin Y,
Rodriguez Y,
Kelliher M,
and
Liu ZG.
The distinct roles of TRAF2 and RIP in IKK activation by TNF-R1: TRAF-2 recruits IKK to TNF-R1 while RIP mediates IKK activation.
Immunity
12:
419-429,
2000[ISI][Medline].
13.
Garceau, N,
Kosaka Y,
Masters S,
Hambor J,
Shinkura R,
Honjo T,
and
Noelle RJ.
Lineage-restricted function of nuclear factor-B-inducing kinase (NIK) in transducing signals via CD40.
J Exp Med
191:
381-386,
2000
14.
Giardina, C,
Boulares H,
and
Inan MS.
NSAID and butyrate sensitize a human colorectal cancer cell line to TNF- and Fas ligation: the role of reactive oxygen species.
Biochim Biophys Acta
1448:
425-438,
1999[ISI][Medline].
15.
Hagimoto, N,
Kuwano K,
Kawasaki M,
Yoshimi M,
Kaneko Y,
Kunitake R,
Maeyama T,
Tanaka T,
and
Hara N.
Induction of interleukin-8 secretion and apoptosis in bronchiolar epithelial cells by fas ligation.
Am J Respir Cell Mol Biol
21:
436-445,
1999
16.
Hardy, S,
Kitamura M,
Harris-Stansil T,
Dal Y,
and
Phipps L.
Construction of adenovirus vectors through Cre-lox recombination.
J Virol
71:
1842-1849,
1997[Abstract].
17.
Heerdt, BG,
Houston MA,
and
Augenlicht LH.
Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines.
Cancer Res
54:
3288-3293,
1994[Abstract].
18.
Jobin, C,
Haskill S,
Mayer L,
Panja A,
and
Sartor RB.
Evidence for an altered regulation of IB
degradation in human colonic epithelial cells.
J Immunol
158:
226-234,
1997[Abstract].
19.
Jobin, C,
Holt L,
Bradham CA,
Streetz K,
Brenner DA,
and
Sartor RB.
TRAF-2 is involved in both IL-1- and TNF-
-signaling cascade leading to NF-
B activation and IL-8 expression in human intestinal epithelial cells.
J Immunol
162:
4447-4454,
1999
20.
Jobin, C,
Panja A,
Hellerbrand C,
Iimuro Y,
Didonato J,
Brenner DA,
and
Sartor RB.
Inhibition of proinflammatory molecule production by adenovirus-mediated expression of an NF-B super-repressor in human intestinal epithelial cells.
J Immunol
160:
410-418,
1998
21.
Jobin, C,
and
Sartor RB.
The IB/NF-
B system: a key determinant of mucosal inflammation and protection.
Am J Physiol Cell Physiol
278:
C451-C462,
2000
22.
Jobin, C,
and
Sartor RB.
NF-B signaling proteins as therapeutic targets for inflammatory bowel diseases.
Inflamm Bowel Dis
6:
206-213,
2000[ISI][Medline].
23.
Karin, M.
The beginning of the end: IB kinase (IKK) and NF-
B activation.
J Biol Chem
274:
27399-27342,
1999
24.
Karin, M,
and
Delhase M.
JNK or IKK, AP-1 or NF-B, which are the targets for MEK kinase 1 action?
Proc Natl Acad Sci USA
95:
9067-9069,
1998
25.
Lallena, MJ,
Diaz-Meco MT,
Bren G,
Paya CV,
and
Moscat J.
Activation of IB kinase
by protein kinase C isoforms.
Mol Cell Biol
19:
2180-2188,
1999
26.
Lee, FS,
Hagler J,
Chen ZJ,
and
Maniatis T.
Activation of the IB
kinase complex by MEKK1, a kinase of the JNK pathway.
Cell
88:
213-222,
1997[ISI][Medline].
27.
Lee, FS,
Peters RT,
Dang LC,
and
Maniatis T.
MEKK1 activates both IB kinase
and I
B kinase
.
Proc Natl Acad Sci USA
95:
9319-9324,
1998
28.
Lin, X,
Mu Y,
Cunningham ET,
Marcu KB,
Geleziunas R,
and
Greene WC.
Molecular determinants of NF-B-inducing kinase action.
Mol Cell Biol
18:
5899-5907,
1998
29.
Ling, L,
Cao Z,
and
Goeddel DV.
NF-B-inducing kinase activates IKK-
by phosphorylation of Ser-176.
Proc Natl Acad Sci USA
95:
3792-3797,
1998
30.
Mackay, F,
Majeau GR,
Hochman PS,
and
Browning JL.
Lymphotoxin- receptor triggering induces activation of the nuclear factor
B transcription factor in some cell types.
J Biol Chem
271:
24934-24938,
1996
31.
Makris, C,
Godfrey VL,
Krahn-Senftleben G,
Takahashi T,
Roberts JL,
Schwarz T,
Feng L,
Johnson RS,
and
Karin M.
Female mice heterozygous for IKK/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti.
Mol Cell
5:
969-979,
2000[ISI][Medline].
32.
Malinin, NL,
Boldin MP,
Kovalenko AV,
and
Wallach D.
MAP3K-related kinase involved in NF-B induction by TNF, CD95 and IL-1.
Nature
385:
540-544,
1997[ISI][Medline].
33.
Matsumoto, M,
Iwamasa K,
Rennert PD,
Yamada T,
Suzuki R,
Matsushima A,
Okabe M,
Fujita S,
and
Yokoyama M.
Involvement of distinct cellular compartments in the abnormal lymphoid organogenesis in lymphotoxin--deficient mice and alymphoplasia (aly) mice defined by the chimeric analysis.
J Immunol
163:
1584-1591,
1999
34.
Matsushima, A,
Kaisho T,
Rennert PD,
Nakano H,
Kurosawa K,
Uchida D,
Takeda K,
Akira S,
and
Matsumoto M.
Essential role of nuclear factor (NF)-B-inducing kinase and inhibitor of
B (I
B) kinase
in NF-
B activation through lymphotoxin
receptor, but not through tumor necrosis factor receptor I.
J Exp Med
193:
631-636,
2001
35.
Mercurio, F,
Zhu H,
Murray BW,
Shevchenko A,
Bennett BL,
Li JW,
Young DB,
Barbosa M,
Mann M,
Manning A,
and
Rao A.
IKK-1 and IKK-2: cytokine-activated IB kinases essential for NF-
B activation.
Science
278:
860-866,
1997
36.
Miagkov, AV,
Kovalenko DV,
Brown CE,
Didsbury JR,
Cogswell JP,
Stimpson SA,
Baldwin AS,
and
Makarov SS.
NF-B activation provides the potential link between inflammation and hyperplasia in the arthritic joint.
Proc Natl Acad Sci USA
95:
13859-13864,
1998
37.
Miyawaki, S,
Nakamura Y,
Suzuka H,
Koba M,
Yasumizu R,
Ikehara S,
and
Shibata Y.
A new mutation, aly, that induces a generalized lack of lymph nodes accompanied by immunodeficiency in mice.
Eur J Immunol
24:
429-434,
1994[ISI][Medline].
38.
Nakano, H,
Shindo M,
Sakon S,
Nishinaka S,
Mihara M,
Yagita H,
and
Okumura K.
Differential regulation of IB kinase
and
by two upstream kinases, NF-
B-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1.
Proc Natl Acad Sci USA
95:
3537-3542,
1998
39.
Natoli, G,
Costanzo A,
Moretti F,
Fulco M,
Balsano C,
and
Levrero M.
Tumor necrosis factor (TNF) receptor 1 signaling downstream of TNF receptor-associated factor 2.
J Biol Chem
272:
26079-26082,
1997
40.
Nemoto, S,
DiDonato JA,
and
Lin A.
Coordinate regulation of IB kinases by mitogen-activated protein kinase kinase kinase 1 and NF-
B-inducing kinase.
Mol Cell Biol
18:
7336-7343,
1998
41.
Ninomiya-Tsuji, J,
Kishimoto K,
Hiyama A,
Inoue JI,
Cao Z,
and
Matsumoto K.
The kinase TAK1 can activate the NIK-IB as well as the MAP kinase cascade in the IL-1 signaling.
Nature
398:
252-256,
1999[ISI][Medline].
42.
O'Neill, LAJ,
and
Greene C.
Signal transduction pathways activated by IL-1 receptor family: ancient machinery in mammals, insects, and plants.
J Leukoc Biol
63:
650-657,
1998[Abstract].
43.
Ossina, NK,
Cannas A,
Powers VC,
Fitzpatrick PA,
Knight JD,
Gilbert JR,
Shekhtman EM,
Tomei LD,
Umansky SR,
and
Kiefer MC.
Interferon- modulates a p53-independent apoptotic pathway and apoptosis-related gene expression.
J Biol Chem
272:
16351-16357,
1997
44.
Ozes, ON,
Mayo LD,
Gustin JA,
Pfeffer SR,
Pfeffer LM,
and
Donner DB.
NF-B activation by tumour necrosis factor requires the Akt serine-threonine kinase.
Nature
401:
82-85,
1999[ISI][Medline].
45.
Ponton, A,
Clement MV,
and
Stamenkovic I.
The CD95 (APO/Fas) receptor activates NF-B independently of its cytotoxic function.
J Biol Chem
271:
8991-8995,
1996
46.
Regnier, CH,
Song HY,
Gao X,
Goeddel DV,
Cao Z,
and
Rothe M.
Identification and characterization of an IB kinase.
Cell
90:
373-383,
1997[ISI][Medline].
47.
Romashkova, JA,
and
Makarov SS.
NF-B is a target of AKT in anti-apoptotic PDGF signalling.
Nature
401:
86-90,
1999[ISI][Medline].
48.
Rothwarf, DM,
Zandi E,
Natoli G,
and
Karin M.
IKK-g is an essential regulatory subunit of the I
B kinase complex.
Nature
395:
297-300,
1998[ISI][Medline].
49.
Russo, MP,
Bradham CA,
Bennett BL,
Manning AM,
Brenner DA,
and
Jobin C.
IB kinase
(IKK
), but not NF-
B-inducing kinase (NIK) or IKK
, represents a therapeutic target for modulation of Fas, IL-1
and TNF
-mediated NF-
B activation in intestinal epithelial cells (Abstract).
Gastroenterology
118:
A587,
2000.
50.
Russo, MP,
Mehta NP,
Keku TO,
Sartor RB,
and
Jobin C.
Increased susceptibility to Fas-mediated apoptosis in differentiated HT-29 cells independent of its effects on NF-B activation and IL-8 secretion (Abstract).
Gastroenterology
118:
A820,
2000.
51.
Sakurai, H,
Miyoshi H,
Toriumi W,
and
Sugita T.
Functional interactions of transforming growth factor -activated kinase 1 with I
B kinases to stimulate NF-
B activation.
J Biol Chem
274:
10641-10648,
1999
52.
Sanz, L,
Sanchez P,
Lallena MJ,
Diaz-Meco MT,
and
Moscat J.
The interaction of p62 with RIP links the atypical PKCs to NF-B activation.
EMBO J
18:
3044-3053,
1999
53.
Schmidt-Supprian, M,
Bloch W,
Courtois G,
Addicks K,
Israel A,
Rajewsky K,
and
Pasparakis M.
NEMO/IKK-deficient mice model incontinentia pigmenti.
Mol Cell
5:
981-992,
2000[ISI][Medline].
54.
Shinkura, R,
Kitada K,
Matsuda F,
Tashiro K,
Ikuta K,
Suzuki M,
Kogishi K,
Serikawa T,
and
Honjo T.
Alymphoplasia is caused by a point mutation in the mouse gene encoding NF-B-inducing kinase.
Nat Genet
22:
74-77,
1999[ISI][Medline].
55.
Sizemore, N,
Leung S,
and
Stark GR.
Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-B p65/RelA subunit.
Mol Cell Biol
19:
4798-4805,
1999
56.
Tak, PP,
and
Firestein GS.
NF-B: a key role in inflammatory diseases.
J Clin Invest
107:
7-11,
2001
57.
Wallach, D,
Varfolomeev EE,
Malinin NL,
Goltsev YV,
Kovalenko AV,
and
Boldin MP.
Tumor necrosis factor receptor and Fas signaling mechanisms.
Annu Rev Immunol
17:
331-367,
1999[ISI][Medline].
58.
Wang, CY,
Mayo MW,
and
Baldwin AS, Jr.
TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-B.
Science
274:
784-787,
1996
59.
Weaver, SA,
Russo MP,
Wright KL,
Kolios G,
Jobin C,
Robertson DA,
and
Ward SG.
Regulatory role of phosphatidylinositol 3-kinase on TNF--induced cyclooxygenase 2 expression in colonic epithelial cells.
Gastroenterology
120:
1117-1127,
2001[ISI][Medline].
60.
Woronicz, JD,
Gao X,
Cao Z,
Rothe M,
and
Goeddel DV.
IB kinase-
: NF-
B activation and complex formation with I
B kinase-
and NIK.
Science
278:
866-869,
1997
61.
Yin, L,
Wu L,
Wesche H,
Arthur CD,
White JM,
Goeddel DV,
and
Schreiber RD.
Defective lymphotoxin- receptor-induced NF-
B transcriptional activity in NIK-deficient mice.
Science
291:
2162-2165,
2001
62.
Yujiri, T,
Ware M,
Widmann C,
Oyer R,
Russel D,
Chan E,
Zaitsu Y,
Clarke P,
Tyler K,
Oka Y,
Fanger GR,
Henson P,
and
Johnson GL.
MEK kinase 1 gene disruption alters cell migration and c-Jun NH2-terminal kinase regulation but does not cause a measurable defect in NF-B activation.
Proc Natl Acad Sci USA
97:
7272-7277,
2000
63.
Zandi, E,
and
Karin M.
Bridging the gap: composition, regulation, and physiological function of the IB kinase complex.
Mol Cell Biol
19:
4547-4551,
1999
64.
Zandi, E,
Rothwarf DM,
Belhase M,
Hayakama M,
and
Karin M.
The IB kinase complex (IKK) contains two kinase subunits, IKK
and IKK
, necessary for I
B phosphorylation and NF-
B activation.
Cell
91:
243-252,
1997[ISI][Medline].
65.
Zhao, Q,
and
Lee FS.
Mitogen-activated protein kinase/ERK kinase kinase 2 and 3 activate nuclear factor-B through I
B kinase-
and I
B kinase-
.
J Biol Chem
274:
8355-8358,
1999