From the Departments of Immunology and
§ Cancer Biology, Lerner Research Institute, The Cleveland
Clinic Foundation, Cleveland, Ohio 44195
Received for publication, January 17, 2003, and in revised form, February 21, 2003
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ABSTRACT |
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Recent studies show that a member of the
interleukin-1 (IL-1)/Toll receptor superfamily, Toll-like receptor 3 (TLR3), recognizes double-stranded RNA (dsRNA). Because of the
similarity in their cytoplasmic domains, IL-1/Toll receptors share
signaling components that associate with the IL-1 receptor, including
IL-1 receptor-associated kinase (IRAK), MyD88, and TRAF6. However, we
find that, in response to dsRNA, TLR3 can mediate the activation of
both NF The interleukin-1
(IL-1)1/Toll receptors play
essential roles in inflammation and innate immunity. The defining
feature of members of the superfamily is a TIR (Toll/IL-1 receptor)
domain on the cytoplasmic side of the receptors (1, 2). The first described members of this superfamily are in the IL-1R family. These
receptors contain three Ig domains in their extracellular regions
(3-5). The second group includes only one receptor so far, the single
Ig IL-1R-related molecule (SIGIRR), which has only a single
extracellular Ig domain (6). The last group in the superfamily is the
recently identified pattern recognition receptors, the Toll-like
receptors (TLRs), 10 members of which contain two major domains
characterized by extracellular leucine-rich repeats and an
intracellular Toll-like domain (2, 7-10). TLR4 has been genetically
identified as a signaling molecule essential for the responses to LPS,
a feature of Gram-negative bacteria (11). Mice with targeted disruption
of the TLR4 gene are LPS-unresponsive. Unlike TLR4, TLR2 responds to
mycobacteria, yeast, and Gram-positive bacteria (12-15). TLR9 has been
shown to recognize bacterial DNA (10), whereas TLR5 mediates the
induction of the immune response by bacterial flagellins (16). Recent
studies show that TLR3 recognizes dsRNA (17).
Much progress has been made in understanding the IL-1R-mediated
signaling. Upon IL-1 stimulation, the cytosolic proteins MyD88 (18-20)
and Tollip (21) are recruited to the receptor complex, which then
recruits serine-threonine kinases IRAK4 (IL-1 receptor-associated kinase 4)2 (22, 23) and IRAK
(24). Although IRAK is hyperphosphorylated, mediating the recruitment
of TRAF6 to the receptor complex (25), IRAK4 is probably the kinase
that functions upstream of and phosphorylates IRAK (22, 23). IRAK-TRAF6
then leaves the receptor complex to interact with TAK1, a member of the
MAP kinase kinase kinase family, and the two proteins that bind to it,
TAB1 and TAB2, on the membrane (25-27). TAK1 and TAB2 are
phosphorylated on the membrane followed by the formation and
translocation of TRAF6-TAK1-TAB1-TAB2 from the membrane to the cytosol
(25), where TAK1 is activated (25). Although genetic studies show that
IRAK is required for the IL-1-induced activation of TAK1 (28), in
vitro biochemical analyses reveal that TRAF6-mediated
ubiquitination may also play an important role in TAK1 activation (29).
The activation of TAK1 eventually leads to the activation of I Because the receptors in the IL-1/Toll receptor superfamily are similar
in their cytoplasmic domains, they share some signaling components that
function with the IL-1 receptor, including MyD88, IRAK4, IRAK, and
TRAF6, leading to the activation of NF TLR3-deficient mice show reduced responses to poly(dI·dC) and reduced
production of inflammatory cytokines, indicating that TLR3 mediates the
induction of immune response by dsRNA. Although TLR3 induces cytokine
production through a signaling pathway dependent on MyD88,
poly(dI·dC) can still induce the activation of NF Biological Reagents and Cell Culture--
Recombinant human
IL-1 Recombinant Plasmids and Stable
Transfection--
pE-selectin-luc, an NF Co-immunoprecipitation and Immunoblotting--
Cells, untreated
or treated with 100 units/ml IL-1, were lysed in a Triton-containing
lysis buffer (0.5% Triton X-100, 20 mM HEPES, pH 7.4, 150 mM NaCl, 12.5 mM In Vitro Phosphorylation Assay--
TAK1 and TAB2
immunoprecipitates were incubated with 1 µg of bacterially expressed
MKK6 in 20 µl of kinase buffer containing 10 mM HEPES, pH
7.4, 1 mM dithiothreitol, 5 mM
MgCl2, and 5 µCi of Subcellular Fractionation--
The method as described
previously (46) was used with minor modifications. Cells were lysed in
a hypotonic buffer (10 mM HEPES, pH 7.4, 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol)
and homogenized on ice with a Dounce homogenizer. Unlysed cells, cell
debris, and nuclei were removed by centrifugation at 1000 × g for 5 min. Soluble (supernatant, S-100) and particulate
(pellet, P-100) fractions were generated by centrifugation at
100,000 × g for 1 h at 4 °C. Particulate fractions were solubilized in the Triton-containing lysis buffer followed by centrifugation at 10,000 × g for 10 min.
Transfection and Reporter Assays--
For stable transfections,
2 × 105 cells were seeded onto a 10-cm plate and
cotransfected the following day by the calcium phosphate method with 10 µg of each expression vector and 1 µg of pBabePuro vector. After
48 h, the cells were selected with 1 µg/ml puromycin until
clones appeared. For reporter assays, 2 × 105 cells
were transfected by the same procedure with 1 µg of pE-selectin-luc, 1 µg of pSV2- Gel Shift Assays--
An NF IRAK Is Not Involved in TLR3-mediated Signaling--
We have
previously taken a genetic approach to the study of
IL-1-dependent signaling pathways, through random
mutagenesis, generating IL-1-unresponsive cell lines lacking specific
components of the pathways. Mutant cell line I1A lacks both IRAK
protein and mRNA (43, 49). Neither NF TRAF6, TAK1, and TAB2 Are Recruited to TLR3 upon Poly(dI·dC)
Stimulation--
To investigate the molecular mechanism by which TLR3
mediates the activation of NF
To confirm the interactions between TRAF6, TAK1, and TAB2 and TLR3,
extracts from 293-TLR3 cells untreated or stimulated with poly(dI·dC)
were immunoprecipitated with anti-TRAF6 followed by Western analyses
with anti-Flag (M2), anti-TAK1, anti-TAB2, and anti-TRAF6. The results
show that TRAF6, TAK1, and TAB2 indeed form a complex at TLR3 upon
stimulation (Fig. 2D and Table I).
TLR3-mediated Signaling Leads to TAK1 Activation--
TAK1, a
member of the MAP kinase kinase kinase family, and two proteins that
bind to it, TAB1 and TAB2 (26, 27), have recently been shown to play a
critical role in the IL-1 pathway. TAK1 is activated upon IL-1
stimulation, which in turn leads to the activation of IKK complex and
MKK6 by an unknown mechanism, resulting in NF Poly(dI·dC) Induces the Translocation of TRAF6-TAK1-TAB2 from the
Membrane to the Cytosol, Interaction with PKR, and Activation of TAK1
in the Cytosol--
Previous studies showed that TAB2, associated with
TAK1-TAB1, is localized exclusively on the membrane before stimulation and translocates to the cytosol upon IL-1 stimulation (25, 27, 46). To
investigate the fate of TAB2 upon poly(dI·dC) stimulation, membrane
(P-100) and cytosolic (S-100) fractions from 293-TLR3 cells, untreated
or stimulated with poly(dI·dC) for different lengths of time, were
examined by the Western procedure with anti-TAB2. The data demonstrate
that TAB2 translocates from the membrane to the cytosol upon
poly(dI·dC) stimulation (Fig.
4A). Actin was used as a
cytosolic marker and a loading control.
We next examined where TAK1 is activated in response to poly(dI·dC)
stimulation. We examined whether TAK1 is activated upon its recruitment
to TLR3 upon poly(dI·dC) stimulation. Cell extracts from 293-TLR3
cells, stimulated with poly(dI·dC) or left untreated, were
immunoprecipitated with monoclonal anti-Flag (M2) antibody to pull down
the Flag-tagged TLR3 followed by an in vitro kinase assay
using MKK6 as a substrate. As shown in Fig. 2A, TAK1 is not
activated when it is bound to the receptor complex. We then examined
whether TAK1 is activated on the membrane or in the cytosol. Membrane
and cytosolic fractions prepared from 293-TLR3 cells were
immunoprecipitated with anti-TAK1 followed by an in vitro kinase assay using MKK6 as a substrate. As shown in Fig. 4B,
similar to how TAK1 is activated in IL-1-mediated signaling, the kinase activity of TAK1 is not activated on the membrane but only in the
cytosol upon poly(dI·dC) stimulation. In contrast, TAK1 and TAB2 are
also phosphorylated only in the cytosol upon poly(dI·dC) stimulation,
whereas they are phosphorylated on the membrane in response to IL-1
(25), suggesting that a different mechanism is likely to be involved in
activating TAK1 in response to poly(dI·dC) and to IL-1.
To identify the signaling components associated with TAK1, extracts
from 293-TLR3 cells treated with poly(dI·dC) were immunoprecipitated with anti-TAK1 followed by Western analyses with antibodies against candidate proteins, including TRAF6, TAB1, TAB2, IRAK4, and PKR. TRAF6
and PKR form a poly(dI·dC)-induced complex with TAK1 and TAB2,
suggesting the involvement of these components in TAK1 activation (Fig.
4C). We also found that a kinase-inactive PKR mutant
(DN-PKR) can efficiently inhibit TLR3-mediated NF On the basis of the results presented above, we have provided a
working model for TLR3-mediated signaling pathway (Fig.
5). Upon binding of dsRNA to TLR3, TRAF6,
TAK1, and TAB2 are recruited to the receptor to form a complex. The
TRAF6-TAK1-TAB2 complex then leaves the receptor and translocates to
the cytosol to interact with PKR, where TAK1 is phosphorylated and
activated. Activated TAK1 then leads to the phosphorylation and
activation of IKK and MKK6, resulting in the activation of NFB and mitogen-activated protein (MAP) kinases in
IL-1-unresponsive mutant cell lines, including IRAK-deficient I1A and
I3A cells, which are defective in a component that is downstream of
IL-1R but upstream of IRAK. These results clearly indicate that
TLR3 does not simply share the signaling components employed by the
IL-1 receptor. Through biochemical analyses we have identified an
IRAK-independent TLR3-mediated pathway. Upon binding of dsRNA to TLR3,
TRAF6, TAK1, and TAB2 are recruited to the receptor to form a complex,
which then translocates to the cytosol where TAK1 is phosphorylated and
activated. The dsRNA-dependent protein kinase (PKR) is also
detected in this signal-induced TAK1 complex. Kinase inactive
mutants of TAK1 (TAK1DN) and PKR (PKRDN) inhibit
poly(dI·dC)-induced TLR3-mediated NF
B activation,
suggesting that both of these kinases play important roles in this pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
kinase (IKK) by an unknown mechanism. Activated IKK phosphorylates
I
B proteins, which are degraded, releasing NF
B to activate
transcription in the nucleus (30-33). Activated TAK1 has also been
implicated in the IL-1-induced activation of MKK6 and JNK (26), leading
to the phosphorylation and activation of activating
transcription factor (ATF) and activating protein 1 (AP1), which
thereby also activates gene transcription.
B (22, 34-36). Although
studies with MyD88-null mice have demonstrated that MyD88 functions as
a general adaptor for the IL-1/Toll receptor superfamily (13, 20, 37),
these studies also revealed a MyD88-independent pathway that leads to
NF
B and JNK activation in TLR4-dependent signaling.
MyD88 adaptor-like protein (Mal/TIRAP), which was implicated in the
activation of the MyD88-independent signaling pathway (38, 39), has now
clearly been shown to function for the MyD88-dependent
pathway via TLR2 and TLR4 (40, 41). PKR-deficient mice exhibit reduced
responses to different TLR ligands and reduced production of
proinflammatory cytokines in response to LPS, suggesting that PKR is an
intermediary in TLR signaling (42). These studies indicate that the
IL-1/Toll receptors probably mediate much more complex signaling
pathways than simply sharing the same signaling components of the IL-1 pathway.
B and MAP kinases
in MyD88-deficient macrophages, suggesting that both
MyD88-dependent and -independent pathways may be involved in TLR3-mediated signaling. However, the detailed molecular mechanism by which TLR3 transduces the signal is unclear. In this study, we took
both biochemical and genetic approaches to the study of TLR3-mediated
signaling. We report here that TLR3-mediated activation of NF
B and
MAP kinases is through an IRAK-independent pathway employing the
signaling components TLR3-TRAF6-TAK1-TAB2-PKR.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
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DISCUSSION
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was provided by the National Cancer Institute. Poly(dI·dC)
was purchased from Amersham Biosciences. Anti-IRAK4 polyclonal
antibody was kindly provided by Dr. Holger Wesche (Tularik, South San
Francisco, CA). Anti-MyD88 polyclonal antibody was from Stressgen
(Victoria, Canada). Anti-TRAF6, anti-IRAK, anti-PKR, and anti-actin
polyclonal antibodies were from Santa Cruz (Santa Cruz Biotechnology).
Rabbit anti-TAK1, anti-TAB1, and anti-TAB2 polyclonal antibodies were
kindly provided by Dr. Kunihiro Matsumoto (26, 27). Antibodies against
phospho-JNK, phospho-p42/44, and phospho-p38 were from Cell Signaling
Technology (Beverly, MA). Anti-Flag (M2) was from Sigma. Anti-HA was
from Upstate (Charlottesville, VA). 293-TK/Zeo cells, I1A and
I3A (43), were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, penicillin G (100 µg/ml),
and streptomycin (100 µg/ml).
B-dependent
E-selectin-luciferase reporter plasmid, was described by Schindler and
Baichwal (44). Dominant negative TAK1 (TAK1DN-K66W) was a kind gift
from Dr. Kunihiro Matsumoto (Nagoya University, Japan). PKRDN
(K296R) was previously described (45). For stable
transfections, 2 × 105 cells were seeded onto a 10-cm
plate and cotransfected the following day by the calcium phosphate
method with 10 µg of each expression vector and 1 µg of pBabePuro.
After 48 h, the cells were selected with 1 µg/ml puromycin until
clones appeared.
-glycerophosphate, 1.5 mM MgCl2, 10 mM NaF, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mM EGTA, 20 µM aprotinin, 1 mM
phenylmethylsulfonyl fluoride). Cell extracts were incubated with 1 µg of antibody or preimmune serum (negative control) for 2 h
followed by a 2-h incubation with 20 µl of protein A-Sepharose beads
(pre-washed and resuspended in phosphate-buffered saline at a 1:1
ratio). After incubation the beads were washed four times with lysis
buffer, separated by SDS-PAGE, transferred to Immobilon-P membranes
(Millipore), and analyzed by immunoblotting.
32P-labeled ATP (3000 Ci/mmol) at 25 °C for 2 min. Samples were resolved by SDS-PAGE,
transferred to Immobilon-P membranes, and visualized by
autoradiography. The membranes were also analyzed by immunoblotting.
-gal, and 100 ng of each expression
construct. After 48 h, the cells were split onto two 35-mm plates
and the next day were stimulated with poly(dI·dC) for 4 h before
harvest. Luciferase and
-galactosidase activities were determined by
using the luciferase assay system and chemiluminescent reagents from
Promega (Madison, WI).
B binding site
(5'-GAGCAGAGGGAAATTCCGTAACTT-3') from the IP-10 gene was used as a
probe (47). Complementary oligonucleotides, end-labeled with
polynucleotide kinase (Roche Applied Science) and
32P-labeled ATP, were annealed by slow cooling.
Approximately 20,000 cpm of probe were used per assay (48). Whole cell
extracts were used for the assay. The binding reaction was carried out
at room temperature for 20 min in a total volume of 20 µl containing
20 mM HEPES buffer, pH 7.0, 10 mM KCl, 0.1%
Nonidet P-40, 0.5 mM dithiothreitol, 0.25 mM
phenylmethylsulfonyl fluoride, and 10% glycerol.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B nor JNK is activated in
IL-1-treated I1A cells, but these responses are restored in I1A-IRAK
cells, indicating that IRAK is required for both (49). Mutant I3A, also
deficient in both IL-1-induced NF
B and JNK activation, is defective
in a component upstream of IRAK but downstream of the IL-1 receptor
(43, 49). I1A and I3A mutant cells were used to examine whether these
upstream signaling components of the IL-1 pathway are also required for
TLR3-mediated signaling. Transfection of TLR3 into the parental
293-TK/Zeo (293-TLR3) cells rendered them sensitive to
poly(dI·dC)-induced NF
B activation (49). Interestingly,
poly(dI·dC) also induced NF
B activation in I1A and I3A mutant
cells transfected with TLR3 (I1A-TLR3 and I3A-TLR3), indicating that
IRAK and the component deficient in I3A cells are not required for
TLR3-mediated NF
B activation (Fig.
1A). Although IRAK is
necessary for IL-1-induced JNK, MAP kinases p42/44, and p38 activation
(49), poly(dI·dC) can efficiently induce the activation of these
kinases in I1A-TLR3 (Fig. 1, B and C), showing
that IRAK is not required for TLR3-mediated JNK, MAP kinases, p42/44,
and p38 activation. Moreover, poly(dI·dC) induced IL-8 gene
expression in both 293-TLR3 and I1A-TLR3 cells, indicating that
TLR3-mediated gene expression is also IRAK-independent (Fig. 1D). Although the above described results clearly show that
IRAK is not necessary for TLR3-mediated signaling, it is important to
examine whether IRAK is at all involved in the pathway. Whereas IRAK is
hyperphosphorylated and ubiquitinated upon IL-1 stimulation (49), IRAK
is not modified upon poly(dI·dC) stimulation, indicating that IRAK
does not participate in the TLR3-mediated dsRNA signaling pathway (Fig.
1E).
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Fig. 1.
IRAK is not involved in TLR3-mediated
signaling. A, NF B gel shift assay. Parental
293-TK/Zeo (293) and IL-1-unresponsive mutants (I1A and I3A)
untransfected or transfected with Flag-TLR3 (293-TLR3, I1A-TLR3, and
I3A-TLR3), untreated (
) or stimulated with either IL-1 (100 units/ml)
for the indicated times or poly(dI·dC) (0.1 mg/ml) for 2 h, and
harvested for an NF
B gel shift assay. B, activation of
MAP kinases. Extracts of 293-TLR3 and I1A-TLR3 cells, untreated or
stimulated with poly(dI·dC) for the indicated times, were analyzed by
Western procedure with antibodies against phospho-JNK,
phospho-p42/44, and phospho-p38. C, poly(dI·dC)-induced
JNK activation is TLR3-dependent. I1A and I1A-TLR3 cells
were untreated or stimulated with poly(dI·dC) (0.1 mg/ml) for the
indicated times followed by Western analyses with antibodies against
phospho-JNK. D, TLR3-mediated IL-8 gene expression. RNA
samples prepared from 293-TLR3 and I1A-TLR3, untreated or stimulated
with poly(dI·dC) for the indicated times, were subjected to Northern
analysis and probed with IL-8 cDNA. E, IRAK
modification. 293-TLR3 cells were untreated or stimulated with either
IL-1 (100 units/ml) or poly(dI·dC) (0.1 mg/ml) for the indicated
times followed by Western analyses with anti-IRAK and
anti-phospho-I
B.
B and MAP kinases, we first identified the signaling components that are recruited to TLR3 upon poly(dI·dC) stimulation. Cell extracts from 293-TLR3 cells, stimulated with poly(dI·dC) or left untreated, were immunoprecipitated with
monoclonal anti-Flag (M2) antibody to pull down the Flag-tagged TLR3,
followed by Western analyses with antibodies against signaling
components that are implicated in pathways mediated by IL-1/Toll
receptors, including MyD88, Mal/TIRAP, IRAK4, IRAK, TRAF6, TAK1, and
TAB2. Among these components, TRAF6, TAK1, and TAB2 form a complex with TLR3 upon poly(dI·dC) stimulation, indicating that TLR3 recruits these proteins to mediate signaling (Fig.
2A and Table I). Although MyD88, IRAK4, and IRAK are recruited to
IL-1R (Fig. 2B), these IL-1R-proximal components are not recruited to TLR3, suggesting that
they are probably not utilized by TLR3 to mediate signaling. Because
Mal/TIRAP has been shown to play a role in the TLR2- and TLR4-mediated
pathway, we also examined whether this adapter molecule is recruited to
TLR3 upon poly(dI·dC) stimulation. However, we fail to detect any
interaction between TLR3 and Mal/TIRAP before or after poly(dI·dC)
stimulation (Fig. 2C and Table I). Taken together, the
results show that TLR3 recruits TRAF6, TAK1, and TAB2 upon
poly(dI·dC) stimulation but not MyD88, MAL/TIRAP, IRAK4, or IRAK,
forming a unique receptor complex that differs from the other members
of the IL-1/Toll receptor superfamily. This conclusion is consistent
with the above described genetic experiments in IL-1-unresponsive
mutants, in which IL-1R-proximal signaling components including IRAK
are shown to be dispensable for TLR3-mediated signaling.
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Fig. 2.
Poly(dI·dC)-induced recruitment of TRAF6,
TAK1, and TAB2 to TLR3. A, poly(dI·dC)-induced TLR3
immune complex. Extracts of 293-TLR3 (Flag-tagged), untreated or
stimulated with poly(dI·dC) (0.1 mg/ml) for the indicated times, were
immunoprecipitated with anti-Flag followed by Western analyses with
antibodies against the Flag tag (TLR3), MyD88, IRAK4, IRAK, TRAF6,
TAK1, and TAB2. The immune complex immunoprecipitated with anti-Flag
(Flag-TLR3) was also followed by in vitro kinase assay
with His-MKK6 as a substrate. IP,
immunoprecipitation. B, IL-1-induced IL-1R
immune complex. Extracts of 293-TLR3, untreated or stimulated
with IL-1 (100 units/ml) for the indicated times, were
immunoprecipitated with anti-IL-1R followed by Western analyses
with antibodies against IL-1R, MyD88, IRAK4, IRAK, and TRAF6.
C, poly(dI·dC)-induced TLR3 immune complex in 293-TLR3
cells transfected with HA-tagged MAL/TIRAP. Extracts of 293-TLR3
(Flag-tagged) transfected with HA-tagged Mal/TIRAP, untreated or
stimulated with poly(dI·dC) (0.1 mg/ml) for the indicated
times, were immunoprecipitated with anti-Flag followed by Western
analyses with antibodies against the Flag tag (TLR3), TAK1, and HA tag
(HA-Mal). D, poly(dI·dC)-induced TRAF6 immune complex.
Extracts of 293-TLR3 (Flag-tagged), untreated or stimulated with
poly(dI·dC) (0.1 mg/ml) for the indicated times, were
immunoprecipitated with anti-TRAF6, followed by Western analyses with
antibodies against TRAF6, the Flag-tag (TLR3), TAK1, and TAB2.
WCE, whole cell extracts.
Interactions between different signal proteins in responding to
poly(dI·dC) treatment
B and MAP kinase
activation (29). The fact that TAK1 is recruited to TLR3 upon
poly(dI·dC) stimulation suggests that this kinase may play an
important role in TLR3-mediated pathway. To investigate the function of
TAK1 in poly(dI·dC)-induced TLR3-mediated NF
B activation, we
examined TAK1 kinase activity upon poly(dI·dC) stimulation. Whole
cell extracts from 293-TLR3 cells untreated or stimulated with
poly(dI·dC) were immunoprecipitated with anti-TAK1 antibody followed
by an in vitro kinase assay using bacterially expressed MKK6
as a substrate. TAK1 is highly activated in both wild type (293-TLR3)
and IRAK-deficient (I1A-TLR3) cells upon poly(dI·dC) treatment (Fig.
3A), indicating that TAK1 is
likely to be involved in this IRAK-independent TLR3-mediated signaling. In support of this conclusion, we found that a kinase-inactive TAK1
mutant (DN-TAK1) can effectively inhibit TLR3-mediated NF
B activation, suggesting that TAK1 is probably involved in this pathway
(Fig. 3B). As expected, DN-IRAK failed to inhibit
poly(dI·dC)-induced NF
B activation, serving as a negative control
for DN-TAK1.
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Fig. 3.
Activation of TAK1. A, TAK1
kinase assay. Extracts of 293-TLR3 cells (293) and I1A-TLR3
(I1A) cells, untreated or stimulated with poly(dI·dC) (0.1 mg/ml) or IL-1 (100 units/ml) for the indicated times, were
immunoprecipitated with anti-TAK1 followed by in vitro
kinase assay using His-MKK6 as a substrate and Western analysis with
anti-TAK1. B, TAK1 dominant negative mutant. Increasing
amounts of DN-TAK1 and DN-IRAK (500 ng, 1 and 3 µg) were
co-transfected with E-selectin luciferase reporter construct (1 µg)
into 293-TLR3 cells. The transfected cells were either untreated or
stimulated with poly(dI·dC) for 4 h followed by a luciferase
reporter assay. Vector DNA (3 µg) and E-selectin luciferase reporter
construct (1 µg) were transfected into 293-TLR3 cells as a control.
Data are presented as the -fold induction of luciferase activity in the
poly(dI·dC)-treated cells. Shown are the averages and standard
deviation from three independent experiments.
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Fig. 4.
TAK1 is activated in the cytosol.
A, poly(dI·dC)-induced translocation of TAB2. 293-TLR3
cells were untreated or stimulated with poly(dI·dC) (0.1 mg/ml) for
the indicated times and fractionated into membrane (P-100)
and cytosolic (S-100) fractions followed by Western analyses
with anti-TAB2 and anti-actin. Whole cell extracts (WCE)
from 293-TLR3 cells, untreated or stimulated with poly(dI·dC), were
also examined by Western immunoblotting with anti-TAB2. B,
TAK1 activation. 293-TLR3 cells were untreated or stimulated with
poly(dI·dC) (0.1 mg/ml) for the indicated times and fractionated into
membrane and cytosolic fractions followed by immunoprecipitation with
anti-TAK1. The immunoprecipitates were subjected to in vitro
kinase assay using His-MKK6 as a substrate and Western analyses with
anti-TAB2 and anti-TAK1. C, TAK1 complex. Extracts of
293-TLR3 untreated or stimulated with poly(dI·dC) (0.1 mg/ml) for the
indicated times were immunoprecipitated with anti-TAK1 followed by
Western analyses with antibodies against TAK1, TAB2, TRAF6, and PKR. WCE, whole cell extracts. IP,
immunoprecipitation. D, PRK dominant negative mutant.
Increasing amounts of DN-PKR (500 ng, 1 µg, and 3 µg) were
co-transfected with E-selectin luciferase reporter construct (1 µg)
into 293-TLR3 cells. The transfected cells were either untreated or
stimulated with poly(dI·dC) for 4 h followed by a luciferase
reporter assay. Vector DNA (3 µg) and E-selectin luciferase reporter
construct (1 µg) were transfected into 293-TLR3 cells as a control.
Data are presented as the -fold induction of luciferase activity in the
poly(dI·dC)-treated cells. Shown are the averages from three
independent experiments.
B activation,
suggesting that PRK is probably involved in this pathway (Fig.
4D).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and
MAP kinase.
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Fig. 5.
Model for TLR3-mediated signaling.
See "Discussion."
Although both IL-1R and TLR3 activate TAK1, the upstream signaling
events that lead to their activation are quite different (Fig. 5). In
this study, we have clearly shown that IRAK is not required for
TLR3-mediated signaling. Instead, TLR3 recruits TRAF6-TAK1-TAB2 to form
a receptor complex. One important question is how TRAF6-TAK1-TAB2 are
recruited to TLR3 upon poly(dI·dC) stimulation. TRAF6 is often recruited to receptors through TRAF binding sites that reside on the
receptors (50). However, such TRAF binding sites are absent in the
cytoplasmic domain of TLR3. Therefore, an adapter molecule(s) is likely
to be required to mediate the interaction of TRAF6-TAK1-TAB2 with the
TIR domain of TLR3. Although MyD88 and Mal/TIRAP are the two logical
candidates, several lines of evidence suggest that they are probably
not involved in TLR3-mediated signaling. First, poly(dI·dC) can still
induce activation of NFB and MAP kinases in MyD88-deficient
macrophages (17). Second, we failed to detect any interaction of MyD88
and Mal/TIRAP with TLR3 upon poly(dI·dC) stimulation. Third, although
MyD88 is the adapter that recruits IRAK in the signaling
pathways mediated by other IL-1/Toll receptors, both MyD88 and
Mal/TIRAP fail to activate NF
B in IRAK-null cells2 (49),
suggesting that both MyD88 and Mal/TIRAP mediate downstream signaling
through IRAK. Importantly, our results clearly show that IRAK is not
involved in TLR3-mediated signaling, implying that MyD88 and
Mal/ TIRAP may also not be required for this pathway. Finally,
although TLR3 fails to induce the production of some cytokines in
MyD88-deficient macrophages (17), it could very well be because of the
involvement of a poly(dI·dC)-induced secondary pathway. Therefore,
novel adapter molecules are probably required for TLR3 to recruit
TRAF6-TAK1-TAB2. Recently, Yamamoto et al. (51) and Oshiumi
et al. (52) reported a novel TIR domain-containing molecule,
named TRIF (or TIACM-1). The dominant negative mutant TRIF inhibited
TLR3-dependent activation gene expression, and TRIF
associates with TLR3, suggesting that TRIF is a likely candidate adapter to recruit TRAF6-TAK1-TAB2 to TLR3.
As mentioned above, we have proved definitively that IRAK is not at all
involved in TLR3-mediated signaling. Now the question is whether any of
the other IRAK family members are involved in this pathway. There are
three more IRAK-like molecules, including IRAK2, IRAKM, and IRAK4. The
expression of IRAK2 and IRAKM is extremely low or non-existent
in 293-TLR3 cells where the TLR3-mediated signaling is intact, strongly
suggesting that these two IRAKs are not required for the pathway. IRAK4
has been shown to be the kinase that phosphorylates IRAK upon IL-1
treatment, whereas IRAK is not phosphorylated upon poly(dI·dC)
stimulation. Furthermore, IRAK4 fails to activate NFB in IRAK-null
cells, confirming that IRAK4 functions through IRAK2 (23).
The fact that IRAK does not participate in TLR3-mediated signaling
suggests that IRAK4 is probably not involved in this pathway either.
Moreover, we could not detect any interaction of IRAK4
with TLR3 or with other signaling components upon
poly(dI·dC) stimulation. Taken together, these results suggest that
IRAK4 is probably not involved directly in TLR3-mediated signaling. Although it was shown that poly(dI·dC)-mediated cytokine production is reduced in IRAK4-deficient mouse embryonic fibroblasts (22), TLR3-mediated early signaling events have not been reported in IRAK4-deficient macrophages and mouse embryonic fibroblasts. The observed defect could be because of the effect of a
poly(dI·dC)-induced secondary pathway.
PKR has been implicated in different stress-induced signaling pathways
including dsRNA signaling to NFB activation (45, 53), although the
precise function of PKR in these signaling pathways remains
controversial (53, 54). PKR has also been shown to interact with
Mal/TIRAP in TLR4-mediated signaling, although it is not required for
LPS-induced interferon-
production (55). However, we did not detect
any interaction of PKR with TLR3 (Table I). Instead, we detected
signal-dependent interaction of PKR with the TAK1 complex
(TRAF6-TAK1-TAB2), providing evidence for the involvement of PKR in
TLR3-mediated signaling. It is important to note that TAK1 is both
phosphorylated and activated in response to poly(dI·dC) stimulation.
Therefore, it is possible that PKR may contribute to the activation
process of TAK1. Alternatively, PKR might function downstream of TAK1.
The precise function of PKR in TLR3-mediated signaling will likely
require the identification of all components of this pathway(s).
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ACKNOWLEDGEMENTS |
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We thank Dr. Jun Ninomiya-Tsuji and Dr. Kunihiro Matsumoto for dominant negative mutant TAK1 and antibodies against TAK1 and TAB2. We thank Dr. Holger Wesche for providing us with the anti-IRAK4 antibody.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 600020 (to X. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Immunology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-8706; Fax: 216-444-9329; E-mail: Lix@ccf.org.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M300562200
2 Z. Jiang and X. Li, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: IL-1, interleukin-1; IL-1R, IL-1 receptor; TIR, Toll/IL-1 receptor; TLR, Toll-like receptor; LPS, lipopolysaccharide; dsRNA, double-stranded RNA; IRAK, IL-1 receptor-associated kinase; TRAF6, tumor necrosis factor receptor-associated factor 6; MAP, mitogen-activated protein; MKK, MAP kinase kinase; JNK, c-Jun NH2-terminal kinase; PKR, dsRNA-dependent protein kinase; HA, hemagglutinin.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bowie, A., and O'Neill, L. A. (2000) J. Leukocyte Biol. 67, 508-514[Abstract] |
2. |
Rock, F. L.,
Hardiman, G.,
Timans, J. C.,
Kastelein, R. A.,
and Bazan, J. F.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
588-593 |
3. |
Mitcham, J. L.,
Parnet, P.,
Bonnert, T. P.,
Garka, K. E.,
Gerhart, M. J.,
Slack, J. L.,
Gayle, M. A.,
Dower, S. K.,
and Sims, J. E.
(1996)
J. Biol. Chem.
271,
5777-5783 |
4. |
Parnet, P.,
Garka, K. E.,
Bonnert, T. P.,
Dower, S. K.,
and Sims, J. E.
(1996)
J. Biol. Chem.
271,
3967-3970 |
5. | Lovenberg, T. W., Crowe, P. D., Liu, C., Chalmers, D. T., Liu, X. J., Liaw, C., Clevenger, W., Oltersdorf, T., De Souza, E. B., and Maki, R. A. (1996) J. Neuroimmunol. 70, 113-122[CrossRef][Medline] [Order article via Infotrieve] |
6. | Thomassen, E., Renshaw, B. R., and Sims, J. E. (1999) Cytokine 11, 389-399[CrossRef][Medline] [Order article via Infotrieve] |
7. | Medzhitov, R., Preston-Hurlburt, P., and Janeway, C. A., Jr. (1997) Nature 388, 394-397[CrossRef][Medline] [Order article via Infotrieve] |
8. | Takeuchi, O., Kawai, T., Sanjo, H., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Takeda, K., and Akira, S. (1999) Gene 231, 59-65[CrossRef][Medline] [Order article via Infotrieve] |
9. | Chuang, T. H., and Ulevitch, R. J. (2000) Eur. Cytokine Netw. 11, 372-378[Medline] [Order article via Infotrieve] |
10. | Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. (2000) Nature 408, 740-745[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Poltorak, A.,
He, X.,
Smirnova, I.,
Liu, M. Y.,
Huffel, C. V.,
Du, X.,
Birdwell, D.,
Alejos, E.,
Silva, M.,
Galanos, C.,
Freudenberg, M.,
Ricciardi-Castagnoli, P.,
Layton, B.,
and Beutler, B.
(1998)
Science
282,
2085-2088 |
12. | Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K., and Akira, S. (1999) Immunity. 11, 443-451[Medline] [Order article via Infotrieve] |
13. |
Takeuchi, O.,
Kaufmann, A.,
Grote, K.,
Kawai, T.,
Hoshino, K.,
Morr, M.,
Muhlradt, P. F.,
and Akira, S.
(2000)
J. Immunol.
164,
554-557 |
14. | Underhill, D. M., Ozinsky, A., Hajjar, A. M., Stevens, A., Wilson, C. B., Bassetti, M., and Aderem, A. (1999) Nature 401, 811-815[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Underhill, D. M.,
Ozinsky, A.,
Smith, K. D.,
and Aderem, A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14459-14463 |
16. | Hayashi, F., Smith, K. D., Ozinsky, A., Hawn, T. R., Yi, E. C., Goodlett, D. R., Eng, J. K., Akira, S., Underhill, D. M., and Aderem, A. (2001) Nature 410, 1099-1103[CrossRef][Medline] [Order article via Infotrieve] |
17. | Alexopoulou, L., Holt, A. C., Medzhitov, R., and Flavell, R. A. (2001) Nature 413, 732-738[CrossRef][Medline] [Order article via Infotrieve] |
18. | Lord, K. A., Hoffman-Liebermann, B., and Liebermann, D. A. (1990) Oncogene 5, 1095-1097[Medline] [Order article via Infotrieve] |
19. | Wesche, H., Henzel, W. J., Shillinglaw, W., Li, S., and Cao, Z. (1997) Immunity 7, 837-847[Medline] [Order article via Infotrieve] |
20. | Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K., and Akira, S. (1998) Immunity 9, 143-150[Medline] [Order article via Infotrieve] |
21. | Burns, K., Clatworthy, J., Martin, L., Martinon, F., Plumpton, C., Maschera, B., Lewis, A., Ray, K., Tschopp, J., and Volpe, F. (2000) Nat. Cell Biol. 2, 346-351[CrossRef][Medline] [Order article via Infotrieve] |
22. | Suzuki, N., Suzuki, S., Duncan, G. S., Millar, D. G., Wada, T., Mirtsos, C., Takada, H., Wakeham, A., Itie, A., Li, S., Penninger, J. M., Wesche, H., Ohashi, P. S., Mak, T. W., and Yeh, W. C. (2002) Nature 416, 750-756[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Li, S.,
Strelow, A.,
Fontana, E. J.,
and Wesche, H.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
5567-5572 |
24. | Cao, Z., Xiong, J., Takeuchi, M., Kurama, T., and Goeddel, D. V. (1996) Nature 383, 443-446[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Jiang, Z.,
Ninomiya-Tsuji, J.,
Qian, Y.,
Matsumoto, K.,
and Li, X.
(2002)
Mol. Cell. Biol.
22,
7158-7167 |
26. | Ninomiya-Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J., Cao, Z., and Matsumoto, K. (1999) Nature 398, 252-256[CrossRef][Medline] [Order article via Infotrieve] |
27. | Takaesu, G., Kishida, S., Hiyama, A., Yamaguchi, K., Shibuya, H., Irie, K., Ninomiya-Tsuji, J., and Matsumoto, K. (2000) Mol. Cell 5, 649-658[Medline] [Order article via Infotrieve] |
28. |
Takaesu, G.,
Ninomiya-Tsuji, J.,
Kishida, S.,
Li, X.,
Stark, G. R.,
and Matsumoto, K.
(2001)
Mol. Cell. Biol.
21,
2475-2484 |
29. | Wang, C., Deng, L., Hong, M., Akkaraju, G. R., Inoue, J., and Chen, Z. J. (2001) Nature 412, 346-351[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Mercurio, F.,
Zhu, H.,
Murray, B. W.,
Shevchenko, A.,
Bennett, B. L.,
Li, J.,
Young, D. B.,
Barbosa, M.,
Mann, M.,
Manning, A.,
and Rao, A.
(1997)
Science
278,
860-866 |
31. | Regnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. (1997) Cell 90, 373-383[Medline] [Order article via Infotrieve] |
32. |
Woronicz, J. D.,
Gao, X.,
Cao, Z.,
Rothe, M.,
and Goeddel, D. V.
(1997)
Science
278,
866-869 |
33. | Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252[Medline] [Order article via Infotrieve] |
34. | Akira, S., Takeda, K., and Kaisho, T. (2001) Nat. Immunol. 2, 675-680[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Jacinto, R.,
Hartung, T.,
McCall, C.,
and Li, L.
(2002)
J. Immunol.
168,
6136-6141 |
36. |
Hacker, H.,
Vabulas, R. M.,
Takeuchi, O.,
Hoshino, K.,
Akira, S.,
and Wagner, H.
(2000)
J. Exp. Med.
192,
595-600 |
37. | Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999) Immunity 11, 115-122[Medline] [Order article via Infotrieve] |
38. | Fitzgerald, K. A., Palsson-McDermott, E. M., Bowie, A. G., Jefferies, C. A., Mansell, A. S., Brady, G., Brint, E., Dunne, A., Gray, P., Harte, M. T., McMurray, D., Smith, D. E., Sims, J. E., Bird, T. A., and O'Neill, L. A. (2001) Nature 413, 78-83[CrossRef][Medline] [Order article via Infotrieve] |
39. | Horng, T., Barton, G. M., and Medzhitov, R. (2001) Nat. Immunol. 2, 835-841[CrossRef][Medline] [Order article via Infotrieve] |
40. | Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuchi, O., Kobayashi, M., Fujita, T., Takeda, K., and Akira, S. (2002) Nature 420, 324-329[CrossRef][Medline] [Order article via Infotrieve] |
41. | Horng, T., Barton, G. M., Flavell, R. A., and Medzhitov, R. (2002) Nature 420, 329-333[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Goh, K. C.,
deVeer, M. J.,
and Williams, B. R.
(2000)
EMBO J.
19,
4292-4297 |
43. |
Li, X.,
Commane, M.,
Jiang, Z.,
and Stark, G. R.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
4461-4465 |
44. | Schindler, U., and Baichwal, V. R. (1994) Mol. Cell. Biol. 14, 5820-5831[Abstract] |
45. | Kumar, A., Haque, J., Lacoste, J., Hiscott, J., and Williams, B. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6288-6292[Abstract] |
46. |
Qian, Y.,
Commane, M.,
Ninomiya-Tsuji, J.,
Matsumoto, K.,
and Li, X.
(2001)
J. Biol. Chem.
276,
41661-41667 |
47. | Majumder, S., Zhou, L. Z., Chaturvedi, P., Babcock, G., Aras, S., and Ransohoff, R. M. (1998) J. Neurosci. Res. 54, 169-180[CrossRef][Medline] [Order article via Infotrieve] |
48. | Kessler, D. S., Veals, S. A., Fu, X. Y., and Levy, D. E. (1990) Genes Dev. 4, 1753-1765[Abstract] |
49. |
Li, X.,
Commane, M.,
Burns, C.,
Vithalani, K.,
Cao, Z.,
and Stark, G. R.
(1999)
Mol. Cell. Biol.
19,
4643-4652 |
50. | Ye, H., Arron, J. R., Lamothe, B., Cirilli, M., Kobayashi, T., Shevde, N. K., Segal, D., Dzivenu, O. K., Vologodskaia, M., Yim, M., Du, K., Singh, S., Pike, J. W., Darnay, B. G., Choi, Y., and Wu, H. (2002) Nature 418, 443-447[CrossRef][Medline] [Order article via Infotrieve] |
51. |
Yamamoto, M.,
Sato, S.,
Mori, K.,
Hoshino, K.,
Takeuchi, O.,
Takeda, K.,
and Akira, S.
(2002)
J. Immunol.
169,
6668-6672 |
52. | Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T., and Seya, T. (2003) Nat. Immunol. 4, 161-167[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Zamanian-Daryoush, M.,
Mogensen, T. H.,
DiDonato, J. A.,
and Williams, B. R.
(2000)
Mol. Cell. Biol.
20,
1278-1290 |
54. |
Iordanov, M. S.,
Wong, J.,
Bell, J. C.,
and Magun, B. E.
(2001)
Mol. Cell. Biol.
21,
61-72 |
55. | Toshchakov, V., Jones, B. W., Perera, P. Y., Thomas, K., Cody, M. J., Zhang, S., Williams, B. R., Major, J., Hamilton, T. A., Fenton, M. J., and Vogel, S. N. (2002) Nat. Immunol. 3, 392-398[CrossRef][Medline] [Order article via Infotrieve] |