From the Department of Molecular Biology, Graduate
School of Science, Nagoya University, and CREST, Japan Science and
Technology Corporation, Chikusa-ku, Nagoya 464-8602, Japan,
¶ Chugai Pharmaceutical Co., Ltd., Fuji-Gotemba Research
Laboratories, 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513, Japan,
and the § Department of Environmental and Molecular
Toxicology, North Carolina State University,
Raleigh, North Carolina 27695-7633
Received for publication, July 24, 2002, and in revised form, February 24, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
TAK1, a member of the mitogen-activated kinase
kinase kinase (MAPKKK) family, participates in proinflammatory cellular
signaling pathways by activating JNK/p38 MAPKs and NF- TAK1 is a member of the mitogen-activated protein kinase
kinase kinase (MAPKKK)1
family that phosphorylates and activates MKK3, MKK4, MKK6, and MKK7
MAPKKs, which in turn activate the c-Jun N-terminal kinase (JNK) and
p38 MAPKs (1-3). We have recently demonstrated that TAK1 also
activates I Several lines of evidence suggest that TAK1 is a key molecule in
proinflammatory signaling pathways. Various proinflammatory cytokines and endotoxins activate the kinase activity of endogenous TAK1 (4, 9, 10). Overexpression of kinase-dead TAK1 inhibits IL-1- and TNF-induced activation of both JNK/p38 and NF- In this study, we screened for compounds that can inhibit TAK1 kinase
activity. This strategy resulted in the isolation of one natural
compound 5Z-7-oxozeaenol, a resorcylic lactone of fungal
origin. We found that 5Z-7-oxozeaenol inhibited the kinase activity of purified TAK1, whereas no significant inhibition of TAK1
activity was observed with structurally related compounds including
radicicol. 5Z-7-Oxozeaenol had no significant effect on the
kinase activities of other members of the MAPKKK family such as MEKK1
and ASK1. Exposure of cells to 5Z-7-oxozeaenol blocked IL-1-induced activation of TAK1, IKK, JNK, p38, and NF- Compounds, Reagents, and Cell Culture--
Zeaenol analog and
radicicol were prepared from the culture broth of fungal strain f6024
and f6065, respectively. Recombinant human IL-1 Immunoprecipitation and Immunoblotting--
Cells were washed
once with ice-cold phosphate-buffered saline and lysed in 0.3 ml of
0.5% Triton X-100 lysis buffer containing 20 mM HEPES (pH
7.4), 150 mM NaCl, 12.5 mM
Kinase Assay--
For screening TAK1 inhibitors, insect
expression vectors for TAK1 and TAB1 were co-infected into Sf9
cells. After 2 days of incubation, cell lysates were immunoprecipitated
with anti-TAK1 antibody (M-17). The immunoprecipitates were incubated
with various compounds and subsequently incubated with 2 µg of myelin
basic protein and 10 µCi of [ Reporter Gene Assay--
Assays for reporter gene activity were
performed as described (4). An Ig- Ear Swelling Assay--
Female BALB/c mice (6 weeks old) were
sensitized by applying 0.1 ml of picryl chloride (50 mg/ml) in an olive
oil/acetone solution (1:5, v/v) to the shaved abdomen of the mice at
day 0. Seven days later, 10 µl of picryl chloride solution (10 mg/ml) in olive oil was applied to each side of the right ear (PC challenge). At day 10, mice were resensitized with picryl chloride. At day 17, the
PC challenge was repeated (second PC challenge). Ten µl of 1 mg/ml
5Z-7-oxozeaenol or vehicle alone (ethanol) were painted on
each side of the right ear before and after the second PC challenge. The ear thickness was measured with calibrated digital thickness gauges
before and 24 h after the second PC challenge, and the difference
in thickness was calculated.
Screening for Inhibitors of TAK1 MAPKKK--
We have previously
shown that TAK1 has no kinase activity when expressed alone but is
activated when TAB1 is co-expressed (5, 7). To identify inhibitors of
TAK1, we developed an in vitro kinase assay system using
purified TAK1 and TAB1 proteins expressed in insect cells. We tested 90 compounds, including 59 compounds that have been reported to inhibit
protein kinases, 24 oxindole-related compounds, and 7 resorcylic acid
lactone-related compounds. Of these compounds, one resorcylic acid
lactone-related compound, 5Z-7-oxozeaenol, was found to be a
very potent inhibitor of TAK1, with an IC50 of 8 nM (Fig.
1A and Table
I). Other structurally related compounds
such as radicicol had little inhibitory activity, with an IC50 of >10
µM. The remaining 89 compounds did not exhibit any
effective inhibition of TAK1. These included oxindole protein-tyrosine kinase inhibitors (SU5402 and SU4984), staurosporine-related protein kinase C inhibitors, the platelet-derived growth factor receptor inhibitor AG1433, and the plant flavonoid apigenin. Two compounds, Ro092210 and L783277, with very similar structures to
5Z-7-oxozeaenol were previously demonstrated to inhibit MEK
kinase activity (17, 18). We examined the effect of
5Z-7-oxozeaenol on purified rat MEK1 kinase activity (Fig.
1B). 5Z-7-Oxozeaenol did inhibit MEK1 kinase
activity; however, the IC50 of 5Z-7-oxozeaenol required to
inhibit MEK1 is 411 nM, which is 50-fold higher than that
for TAK1.
To explore the mechanism for 5Z-7-oxozeaenol inhibition of
TAK1, we examined whether 5Z-7-oxozeaenol is competitive
with ATP. We incubated increasing concentrations of ATP and
5Z-7-oxozeaenol with purified TAK1 and subsequently assayed
kinase activity of TAK1. We found that the IC50 of
5Z-7-oxozeaenol required to inhibit TAK1 shifted to higher
values with increasing ATP concentrations (Fig. 1D),
suggesting that 5Z-7-oxozeaenol is a competitive inhibitor of ATP binding to TAK1. When 5Z-7-oxozeaenol was
preincubated with TAK1 for 30 min before the addition of ATP, the IC50
values of 5Z-7-oxozeaenol did not shift with increasing ATP
concentrations (Fig. 1D), suggesting that the binding of
5Z-7-oxozeaenol to TAK1 is either irreversible or very
slowly reversible. Thus, 5Z-7-oxozeaenol is likely to
irreversibly interact within the ATP binding site of TAK1, thereby
inhibiting the catalytic activity of TAK1.
5Z-7-Oxozeaenol Selectively Inhibits TAK1 Kinase
Activity--
To evaluate whether 5Z-7-oxozeaenol is a
specific inhibitor of TAK1 or if it more generally inhibits the MAPKKK
family, we tested the effect of 5Z-7-oxozeaenol on
bacterially expressed MEKK1 kinase activity in vitro (Fig.
1C). 5Z-7-Oxozeaenol had a weak effect on MEKK1
kinase activity. The IC50 of 5Z-7-oxozeaenol required to
inhibit MEKK1 was 268 nM. To further verify the effects of
5Z-7-oxozeaenol on MAPKKKs, we utilized ectopically
expressed MAPKKKs in 293 cells. TAK1 is known to be active when it is
co-expressed together with TAB1 (5, 7). When N-terminal truncated MEKK1 (MEKK1 5Z-7-Oxozeaenol Inhibits the IL-1 Signaling Pathway--
We have
previously demonstrated that TAK1 is involved in the IL-1 signaling
pathway (4). The observation that 5Z-7-oxozeaenol inhibits
TAK1 activity raised the possibility that this compound might be an
effective inhibitor of IL-1 signaling. Treatment of cells with IL-1
activates endogenous TAK1 activity and consequently stimulates the MAPK
cascade and IKK, leading to the activation of JNK/p38 MAPKs and
NF-
NF-
We next examined whether 5Z-7-oxozeaenol inhibits
IL-1-induced JNK/p38 activation. We pretreated 293-IL-1RI cells with
increasing amounts of 5Z-7-oxozeaenol and stimulated the
cells with IL-1 treatment. The activated JNK and p38 were detected with
anti-phospho-JNK and -p38 antibodies that specifically recognize the
dually phosphorylated activated forms of JNK1/JNK2 and p38,
respectively (Fig. 4A). IL-1-dependent JNK/p38 activation was abrogated with
treatment of 5Z-7-oxozeaenol in a dose-dependent
manner. The amount of 5Z-7-oxozeaenol required to inhibit
IL-1-induced JNK/p38 activation was in a similar range to that required
for NF-
To further examine the specificity of 5Z-7-oxozeaenol, we
tested the effect of 5Z-7-oxozeaenol on MAPK cascades
activated by several other stimuli. Hydrogen peroxide is a strong
stimulator of JNK/p38; however, it poorly activates TAK1 in 293-IL-1RI
cells,2 suggesting that TAK1
is not involved in this pathway. 293-IL-1RI cells were treated with
5Z-7-oxozeaenol for 30 min followed by hydrogen peroxide
simulation (Fig. 4B). No pronounced inhibition of either JNK
or p38 activation was observed in 5Z-7-oxozeaenol-treated cells. We also assayed UV- and EGF-induced ERK activation. UV and EGF
activate the MEK-ERK MAP kinase cascade, in which TAK1 does not
participate. 293-IL-1RI cells were pretreated with
5Z-7-oxozeaenol and stimulated with UV or EGF. The activated
ERK was detected with anti-phospho-ERK antibody that specifically
recognizes the dually phosphorylated activated forms of ERK1 and ERK2
(Fig. 4, C and D). 5Z-7-Oxozeaenol had
little effect on UV- or EGF-induced ERK activation even at a
concentration of 500 nM. These results suggest that
5Z-7-oxozeaenol selectively inhibits TAK1, thereby inhibiting IL-1-induced JNK/p38 activation in culture cells.
5Z-7-Oxozeaenol Inhibits IL-1-induced TAK1 Activation in Culture
Cells--
We then examined whether 5Z-7-oxozeaenol
inhibits kinase activity of endogenous TAK1 upon IL-1 stimulation. We
have previously observed that TAK1 is transiently activated around 2-5
min after IL-1 stimulation when 293-IL-1RI cells were treated with IL-1 (8). We treated 293-IL-1RI cells with various concentrations of
5Z-7-oxozeaenol prior to IL-1 stimulation. At 5 min after
IL-1 stimulation, cells were lysed, and endogenous TAK1 was
immunoprecipitated with anti-TAK1 antibody. The catalytic activity of
TAK1 was measured using MKK6 as a substrate (Fig.
5A, upper
panel). Treatment of the cells with
5Z-7-oxozeaenol inhibited kinase activity of endogenous TAK1. The IC50 of 5Z-7-oxozeaenol to inhibit endogenous TAK1
was 65 nM (Fig. 5A), which is correlated
with the IC50 to inhibit NF-
We next tested whether the 5Z-oxozeaenol-mediated inhibition
of TAK1 in culture cells is reversible or irreversible. We treated 293-IL-1RI cells with 100 nM 5Z-7-oxozeaenol for
30 min and then incubated for an additional 30 min without
5Z-7-oxozeaenol. The cells were subsequently stimulated with
IL-1, and the catalytic activity of endogenous TAK1 was measured (Fig.
5B). 5Z-7-Oxozeaenol significantly inhibited TAK1
kinase activity even after 5Z-7-oxozeaenol was removed from
the culture medium. These results suggest that 5Z-7-oxozeaenol irreversibly binds to and inhibits TAK1 in
293-IL-1RI cells, consistent with the result showing that
5Z-7-oxozeaenol irreversibly inhibits ATP binding to TAK1
in vitro (Fig. 1D). Thus, it is likely that
5Z-7-oxozeaenol, when added into the culture medium,
inhibits TAK1 activity by irreversibly inhibiting the binding of ATP to TAK1.
We also investigated the time course of activation of TAK1, IKK, JNK,
and p38 upon IL-1 stimulation. In this assay, 293-IL-1RI cells were
treated with 500 nM 5Z-7-oxozeaenol for 30 min
to completely inhibit kinase activity of TAK1 and then stimulated with
IL-1 (Fig. 6). Cells were harvested at 3 and 12 min post-IL-1 stimulation, and the lysates were
immunoprecipitated with anti-TAK1 followed by in vitro
kinase assay (Fig. 6A). 5Z-7-Oxozeaenol
treatment abolished IL-1-induced activation of TAK1. We have previously shown that autophosphorylation of TAK1 upon IL-1 stimulation is essential for its activation (7). TAK1 autophosphorylation can be
detected on SDS-PAGE as slowly migrating TAK1 bands (Fig. 6A, lower panel). We observed that
5Z-7-oxozeaenol inhibited IL-1-induced autophosphorylation
of TAK1. We have also previously demonstrated that endogenous TAK1
constitutively interacts with TAB1 (7). The amount of coprecipitated
TAB1 in TAK1 immunoprecipitates was not changed with treatment of
5Z-7-oxozeaenol (Fig. 6A, lower panel), suggesting that 5Z-7-oxozeaenol did not
interfere with interaction of TAK1 with TAB1. IKK activity was measured
using GST-I Effects of 5Z-7-Oxozeaenol on Inflammation--
IL-1 is a
proinflammatory cytokine that induces the expression of many genes
that up-regulate inflammation (23). One such gene product is
cyclooxgenase 2 (COX-2), which catalyzes the production of
prostaglandin (24). We tested the effect of 5Z-7-oxozeaenol on IL-1-induced COX-2 production (Fig.
7A). The level of COX-2 proteins was increased after IL-1 treatment, whereas no increase was
detected when cells were pretreated with 5Z-7-oxozeaenol, even in the presence of IL-1. Thus, 5Z-7-oxozeaenol inhibits
production of inflammation mediators.
We next examined whether 5Z-7-oxozeaenol could suppress
inflammation in vivo. For this experiment, we used
PC-induced ear swelling as a model for COX-2-mediated inflammation. The
ear swelling system has been widely used as a model for allergic
cutaneous diseases and inflammatory skin disorders (25, 26). Indeed, it
has been shown that inhibitors of COX-2 block PC-induced ear swelling
(27). Furthermore, reduction of IL-1 production have also been shown to
block ear swelling induced by PC (28), suggesting that IL-1 signaling
is involved in this disorder. When 5Z-7-oxozeaenol was
administrated to the PC-challenged ear, ear swelling was reduced by up
to 50% of that of the control ear treated with vehicle (Fig. 7B). Thus, 5Z-7-oxozeaenol is able to prevent
inflammation, probably through inhibiting TAK1 activity.
Our screening for a TAK1 kinase inhibitor identified a natural
compound, 5Z-7-oxozeaenol, a fungal resorcylic acid lactone that has been previously reported to inhibit endotoxin-induced production of TNF (29). 5Z-7-Oxozeaenol is also able to
inhibit anisomycin-induced JNK/p38 activation (30). However, the
mechanisms underlying these inhibitory effects had been unclear. Here
we demonstrate that 5Z-7-oxozeaenol specifically inhibits
the catalytic activity of TAK1. Since TAK1 is activated upon treatment
with various endotoxins and stresses, it is likely that
5Z-7-oxozeaenol might inhibit TAK1 activity activated by
endotoxin and anisomycin, thereby reducing TNF production and JNK/p38 activation.
TAK1 is a multifunctional protein kinase involved not only in the IL-1
signaling pathway but also in the transforming growth factor-B. To identify
drugs that prevent inflammation, we screened inhibitors of TAK1
catalytic activity. We identified a natural resorcylic lactone of
fungal origin, 5Z-7-oxozeaenol, as a highly potent
inhibitor of TAK1. This compound did not effectively inhibit the
catalytic activities of the MEKK1 or ASK1 MAPKKKs, suggesting that
5Z-7-oxozeaenol is a selective inhibitor of TAK1. In cell
culture, 5Z-7-oxozeaenol blocked interleukin-1-induced
activation of TAK1, JNK/p38 MAPK, I
B kinases, and NF-
B, resulting
in inhibition of cyclooxgenase-2 production. Furthermore, in
vivo 5Z-7-oxozeaenol was able to inhibit picryl
chloride-induced ear swelling. Thus, 5Z-7-oxozeaenol blocks proinflammatory signaling by selectively inhibiting TAK1 MAPKKK.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B kinases (IKKs), ultimately leading to activation of the
transcription factor NF-
B (4). TAK1 participates in proinflammatory
cellular signaling pathways such as the interleukin-1 (IL-1) pathway by
activating both JNK/p38 MAPKs and IKKs. Exposure of cells to IL-1
induces the interaction between endogenous TAK1 and TRAF6
(tumor necrosis factor (TNF)
receptor-associated factor 6), a molecule essential for IL-1 activation of both
JNK/p38 and NF-
B. This interaction in turn leads to TAK1 activation.
We have previously identified two TAK1-binding proteins, TAB1 and TAB2 (5, 6). When ectopically co-expressed, TAB1 augments the kinase
activity of TAK1, indicating that TAB1 functions as an activator of
TAK1 (5, 7). TAB2 functions as an adaptor linking TAK1 to TRAF6 by
directly binding to both, thereby mediating TAK1 activation in the IL-1
signaling pathway (6, 8).
B (4, 10).
The Drosophila homolog of TAK1 was recently identified as an
essential molecule for host defense signaling in Drosophila (11). Furthermore, the TAK1 gene-silencing study using the small interfering RNA method defined that TAK1 is essential for both IL-1- and TNF-induced NF-
B activation in mammalian cells (12). Therefore, it can be expected that inhibition of TAK1 activity may be
effective in preventing inflammation and tissue destruction promoted by
proinflammatory cytokines.
B.
Furthermore, 5Z-7-oxozeaenol inhibited IL-1-induced
production of cyclooxygenase-2 and relieved ear swelling induced by
picryl chloride. These results suggest that 5Z-7-oxozeaenol
blocks proinflammatory signaling by selectively inhibiting TAK1
MAPKKK.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(Roche Applied
Science), recombinant human TNF
(Roche Applied Science), and
epidermal growth factor (EGF) (BD Biosciences) were used. The following
antibodies were used: anti-TAK1 polyclonal antibody M-17 (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), anti-FLAG monoclonal antibody M2
(Sigma), anti-phosphoextracellular signal-regulated kinase (ERK)
(Thr-202/Tyr-204) polyclonal antibody (Cell Signaling), anti-ERK
polyclonal antibody (Cell Signaling), anti-phospho-JNK
(Thr-183/Tyr-185) monoclonal antibody (Cell Signaling), anti-JNK
polyclonal antibody FL (Santa Cruz Biotechnology), anti-phospho-p38 (Thr-180/Tyr-182) polyclonal antibody (Cell Signaling), anti-p38 polyclonal antibody (Cell Signaling), anti-IKK
polyclonal antibody H-744 (Santa Cruz Biotechnology), and anti-cyclooxygenase-2 polyclonal antibody M-19 (Santa Cruz Biotechnology). The rabbit anti-TAK1 and
anti-TAB1 polyclonal antibodies (4) were also used to immunoprecipitate and/or detect endogenous TAK1 and TAB1 in 293-IL-1RI cells (13). Expression vectors for FLAG-TAK1, FLAG-MEKK1
N, FLAG-ASK1,
NF-
B-interacting kinase, and FLAG-IKK
were described
previously (4, 14-16). Purified MEKK1 and MEK1 were purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY). 293-IL-1RI cells and
mouse embryonic fibroblast cells were maintained in high glucose
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, penicillin G (100 units/ml) and streptomycin (100 µg/ml). For
the transfection studies, cells (1 × 106) were plated
in 10-cm dishes, transfected with a total of 10 µg of DNA containing
various expression vectors by the calcium phosphate precipitate method,
and incubated for 24-36 h before stimulation.
-glycerophosphate, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 2 mM
dithiothreitol, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, and 20 µM aprotinin.
Cellular debris was removed by centrifugation at 10,000 × g for 5 min. Proteins from cell lysates were
immunoprecipitated with 1 µg of various antibodies and 20 µl of
protein G-Sepharose (Amersham Biosciences). The immune complexes were
washed three times with wash buffer containing 20 mM HEPES
(pH 7.4), 500 mM NaCl, and 10 mM
MgCl2, and once with rinse buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, and 10 mM MgCl2 and suspended in 30 µl of rinse
buffer. For immunoblotting, the immunoprecipitates or whole cell
lysates were resolved on SDS-PAGE and transferred to Hybond-P membranes
(Amersham Biosciences). The membranes were immunoblotted with various
antibodies, and the bound antibodies were visualized with horseradish
peroxidase-conjugated antibodies against rabbit or mouse IgG using the
ECL Western blotting system (Amersham Biosciences).
-32P]ATP (3,000 Ci/mmol) in 10 µl of the kinase buffer containing 10 mM
HEPES (pH 7.4), 1 mM dithiothreitol, 5 mM
MgCl2 at 30 °C for 5 min. Samples were separated by 10%
SDS-PAGE, and 32P incorporated into myelin basic protein
was quantified with a bioimage analyzer (FUJIX BAS2000). The catalytic
activity of MEK1 was determined by activation of ERK2 (Upstate
Biotechnology) to phosphorylate myelin basic protein according to the
manufacturer's procedure. The catalytic activity of MEKK1 was measured
with 2 µg of myelin basic protein as a substrate in the kinase
buffer. For subsequent kinase assays, immunoprecipitates were incubated with 5 µCi of [
-32P]ATP (3,000 Ci/mmol) and 1 µg
of bacterially expressed MKK6 or GST-I
B
-(1-72) in 10 µl
of the kinase buffer at 25 °C for 2 min. Samples were separated by
10% SDS-PAGE and visualized by autoradiography.
-luciferase reporter was used to
measure NF-
B-dependent transcription. A plasmid
containing the
-galactosidase gene under the control of the
-actin promoter (pAct-
-galactosidase) was used for normalizing
transfection efficiency.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 1.
Inhibition of TAK1 activity by
5Z-7-oxozeaenol. A, TAK1 and TAB1 proteins
purified from insect cells were used for in vitro kinase
assays in the presence of 5Z-7-oxozeaenol or radicicol. The
percentage inhibition of kinase activity is shown. B,
purified rat MEK1 was used for in vitro kinase assays in the
presence of 5Z-7-oxozeaenol. The percentage inhibition of
kinase activity is shown. C, bacterially expressed MEKK1 was
used for in vitro kinase assays in the presence of
5Z-7-oxozeaenol. The percentage inhibition of kinase
activity is shown. D, TAK1 and TAB1 proteins purified from
insect cells were incubated with various concentrations of ATP together
with 5Z-7-oxozeaenol or incubated with
5Z-7-oxozeaenol for 30 min before the addition of ATP
(preincubation). IC50 values shown are means from three independent
experiments (n = 3). ND, not done.
Inhibitory activity of compounds on
TAK1
N) or the full-length ASK1 is overexpressed in 293 cells, they are catalytically active (19, 20). FLAG-tagged TAK1
together with TAB1, FLAG-MEKK1
N, or FLAG-ASK1 was expressed in 293 cells, and each kinase was immunoprecipitated with anti-FLAG antibody. We measured their abilities both to autophosphorylate themselves and to
phosphorylate MAPKK MKK6 (Fig. 2). In
this assay, 5Z-7-oxozeaenol inhibited autophosphorylation of
TAK1 and TAK1 activity to phosphorylate MKK6 at concentrations of
30-300 nM. By contrast, no inhibitory effect of
5Z-7-oxozeaenol was observed on MEKK1 or ASK1. The kinase activity of another MAPKKK, MEKK4, was also not inhibited by
5Z-7-oxozeaenol at concentrations as high as 500 nM (data not shown). Thus, 5Z-7-oxozeaenol is
potent and selective inhibitor of TAK1.
View larger version (38K):
[in a new window]
Fig. 2.
Effect of 5Z-7-oxozeaenol on
MAPKKKs. 293 cells were transfected with expression vectors for
FLAG-TAK1, TAB1, FLAG-MEKK1 N, FLAG-ASK1, and FLAG-IKK
as
indicated. Cell extracts were immunoprecipitated with anti-FLAG
antibody. The immunoprecipitates were treated with various
concentrations of 5Z-7-oxozeaenol and subjected to kinase
reactions with bacterially expressed MKK6. The autophosphorylation of
TAK1, MEKK1
N, and ASK1 and phosphorylation of MKK6 are shown as
indicated. The kinase activity of IKK
was assayed using bacterial
expressed GST-I
B as a substrate.
B, respectively. To verify if 5Z-7-oxozeaenol can
inhibit IL-1 signaling, we test for the effect of
5Z-7-oxozeaenol on NF-
B-dependent
transcriptional activation induced by IL-1 (Fig.
3A). We found that treatment
of cells with 5Z-7-oxozeaenol effectively inhibited
IL-1-induced activation of NF-
B.
View larger version (20K):
[in a new window]
Fig. 3.
Effect of 5Z-7-oxozeaenol on
NF- B activation. A, 293 cells were transfected with an
Ig-
-luciferase reporter and pAct-
-galactosidase, together with
empty vector or expression vectors encoding TAK1, TAB1, Tax, or
NF-
B-interacting kinase (NIK) as indicated. The indicated
amounts of 5Z-7-oxozeaenol or the same volumes of
Me2SO were added to culture medium upon transfection and at
12 and 24 h after transfection. Cells transfected with empty
vector were treated with IL-1 (5 ng/ml) or TNF (10 ng/ml). Following a
36-h incubation, luciferase activity was determined and normalized to
the levels of
-galactosidase activity. The percentage stimulation of
5Z-7-oxozeaenol-treated samples relative to the untreated
controls is shown. Stimulation relative to control transfection without
IL-1 treatment was as follows: IL-1, 79-fold; TNF, 241-fold; TAK1 plus
TAB1, 160-fold; Tax, 51-fold; NF-
B-interacting kinase
(NIK), 133-fold. B, 293 cells were transfected
with an Ig-
-luciferase reporter and pAct-
-galactosidase, together
with expression vectors encoding TAK1 and TAB1. Luciferase activity was
determined and normalized to the levels of
-galactosidase activity.
The percentage stimulation of 5Z-7-oxozeaenol-treated
samples relative to the untreated controls is shown. The IC50 value of
5Z-7-oxozeaenol to inhibit NF-
B activation by
overexpression of TAK1 and TAB1 is shown.
B is activated through several pathways including human T-cell
leukemia virus Tax protein and TNF pathways. TAK1 is implicated in
TNF-induced NF-
B activation (10, 12), whereas MEKK1 is involved in
Tax-induced NF-
B activation (21). NF-
B can also be activated in
the absence of extracellular signals by overexpression of TAK1 and TAB1
together or by NF-
B-interacting kinase alone (4, 22). To examine
whether the effect of 5Z-7-oxozeaenol is specific to NF-
B
activation mediated by TAK1, cells were treated with TNF or transfected
TAK1, TAB1, Tax, or NF-
B-interacting kinase expression vectors. We
found that 5Z-7-oxozeaenol treatment effectively inhibited
activation of NF-
B induced by TNF and overexpression of TAK1 and
TAB1, whereas it had marginal inhibitory effect on activation of
NF-
B induced by overexpression of Tax or NF-
B-interacting kinase
(Fig. 3A). These results suggest that
5Z-7-oxozeaenol specifically inhibits NF-
B activation by
blocking TAK1 activity. The IC50 value of 5Z-7-oxozeaenol
required to inhibit NF-
B activation by overexpression of TAK1 and
TAB1 was 83 nM (Fig. 3B). This IC50 is 10-fold
higher than that required to inhibit purified TAK1 in vitro
(Fig. 1A).
B inhibition.
View larger version (45K):
[in a new window]
Fig. 4.
Effect of 5Z-7-oxozeaenol on
activation of MAPKs. A, 293-IL-1RI cells were pretreated
with increasing concentrations of 5Z-7-oxozeaenol for 30 min
and stimulated with IL-1 (5 ng/ml) for 10 min. Cell lysates were
immunoblotted (IB) with anti-phospho-JNK, anti-JNK,
anti-phospho-p38, and anti-p38. B, 293-IL-1RI cells were
pretreated with increasing concentrations of 5Z-7-oxozeaenol
for 30 min and stimulated with hydrogen peroxide
(H2O2) (500 µM) for 30 min. Cell
lysates were immunoblotted with anti-phospho-JNK, anti-JNK,
anti-phospho-p38, and anti-p38. C, 293-IL-1RI cells were
pretreated with increasing concentrations of 5Z-7-oxozeaenol
for 30 min, irradiated with UV-B (20 J/m2), and
subsequently incubated for 30 min. Cell lysates were immunoblotted with
anti-phospho-ERK and anti-ERK. D, 293-IL-1RI cells were
pretreated with increasing concentrations of
5Z-7-oxozeaenol for 30 min and stimulated with EGF (50 ng/ml) for 10 min. Cell lysates were immunoblotted with
anti-phospho-ERK and anti-ERK.
B and JNK/p38 activation (Fig. 3
and 4).
View larger version (35K):
[in a new window]
Fig. 5.
5Z-7-Oxozeaenol inhibits kinase
activity of endogenous TAK1. A, 293-IL-1RI cells were
pretreated with various concentrations of 5Z-7-oxozeaenol
for 30 min and then stimulated with IL-1 (5 ng/ml) for 5 min. Cell
lysates were immunoprecipitated with anti-TAK1. The immunoprecipitates
were subjected to kinase assay with MKK6 as a substrate
(upper panel). The amounts of immunoprecipitated
TAK1 are shown in the lower panel. B,
293-IL-1RI cells were pretreated with 100 nM
5Z-7-oxozeaenol (Z) or the same volume of
Me2SO (D) for 30 min (0-30) followed
by additional preincubation with 100 nM
5Z-7-oxozeaenol (Z) or the same volume of Me2SO
(D) for 30 min (30-60). The cells were
subsequently treated with 5 ng/ml IL-1 for 5 min. Cell lysates were
immunoprecipitated with anti-TAK1. The immunoprecipitates were
subjected to a kinase assay with MKK6 (upper
panel). The amounts of immunoprecipitated TAK1 are shown
in the lower panel. IB,
immunoblot.
B as a substrate (Fig. 6B).
5Z-7-Oxozeaenol treatment inhibited 70-80% of the kinase
activity of the IL-1-induced IKK activity. Since
5Z-7-oxozeaenol had no inhibitory effect on kinase activity of IKK itself (Fig. 2), 5Z-7-oxozeaenol presumably inhibits
IL-1-induced activation of IKK by inhibiting TAK1 activity. We also
observed that 5Z-7-oxozeaenol abolished IL-1-induced
activation of JNK and p38 (Fig. 6, C and D).
Taken together, our results indicate that 5Z-7-oxozeaenol
inhibits the IL-1 signaling pathways that normally lead to activation
of both NF-
B and JNK/p38 by inhibiting TAK1.
View larger version (46K):
[in a new window]
Fig. 6.
Effect of 5Z-7-oxozeaenol on
IL-1-induced activation of endogenous TAK1, IKK, JNK, and p38.
293-IL-1RI cells were pretreated with 500 nM
5Z-7-oxozeaenol for 30 min and then stimulated with IL-1 (5 ng/ml) for the indicated times. A, cell lysates were
immunoprecipitated with anti-TAK1. The immunoprecipitates were
subjected to kinase assay with MKK6 as a substrate (upper
panel). The immunoprecipitated TAK1 and coprecipitated TAB1
were immunoblotted (IB) with anti-TAK1 and anti-TAB1,
respectively, in the lower panel. B,
cell lysates were immunoprecipitated with anti-IKK , and the in
vitro kinase assay was performed with GST-I
B (upper
panel). The amounts of immunoprecipitated IKK
were
detected by immunoblotting with anti-IKK
(lower
panel). C, whole cell lysates were immunoblotted
with anti-phospho-JNK and anti-JNK. D, whole cell lysates
were immunoblotted with anti-phospho-p38 and anti-p38.
View larger version (26K):
[in a new window]
Fig. 7.
Effects of 5Z-7-oxozeaenol on
inflammation. A, effect of 5Z-7-oxozeaenol on
IL-1-induced COX-2 production. Mouse embryonic fibroblast cells were
pretreated with 5Z-7-oxozeaenol (500 nM) for 30 min and stimulated with IL-1 (5 ng/ml). After a 17-h incubation, whole
cell lysates were immunoblotted (IB) with anti-COX-2
(upper panel) and anti-TAK1 (lower
panel). B, effect of 5Z-7-oxozeaenol
on picryl chloride-induced ear swelling. Mice were repeatedly
sensitized by picryl chloride and subsequently challenged with picryl
chloride (PC challenge) on the ear twice. Ten µl of
5Z-7-oxozeaenol (1 mg/ml) or vehicle alone (ethanol) were
painted on each ear 2 h before and 4 h after the second PC
challenge (total of twice) (upper panel) or every
hour three times before and 4 h after the second PC challenge
(total of four times) (bottom panel). The ear
thickness was measured 24 h after the second PC challenge. Results
shown are means ± S.D. from five mice.
family signaling pathway (3, 31). Furthermore, we have recently found
that TAK1 is involved in a MAP kinase-like pathway that negatively
regulates the Wnt signaling pathway (32-34). Since
5Z-7-oxozeaenol is a highly potent and selective inhibitor of TAK1, this compound will be a useful tool for studies on these signal transduction pathways. Furthermore, in this study, we show that
when applied topically, 5Z-7-oxozeaenol significantly
reduces the level of PC-induced ear swelling. These results suggest
that 5Z-7-oxozeaenol might be a useful therapeutic agent for
allergic cutaneous disorders such as allergic contact dermatitis and
atopic dermatitis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank H. Ichijo, S. Ohno, H. Saito, and E. Nishida for materials and M. Lamphier for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from Advanced Research on Cancer from the Ministry of Education, Culture, and Science of Japan; the Asahi Glass Foundation; Daiko Foundation; the Uehara Foundation; and the Yamanouchi Foundation for Research on Metabolic Disorders (to K. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-52-789-3000; Fax: 81-52-789-2589 or 81-52-789-3001; E-mail:
g44177a@nucc.cc.nagoya-u.ac.jp.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M207453200
2 J. Ninomiya-Tsuji, unpublished result.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MAPKKK, MAPKK
kinase;
MAPKK, MAPK kinase;
MAPK, mitogen-activated protein kinase;
IL-1, interleukin-1;
TNF, tumor necrosis factor;
JNK, c-Jun N-terminal
kinase;
IKK, IB kinase;
EGF, epidermal growth factor;
ERK, extracellular signal-regulated kinase;
PC, picryl chloride;
COX-2, cyclooxgenase 2;
GST, glutathione S-transferase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Moriguchi, T.,
Kuroyanagi, N.,
Yamaguchi, K.,
Gotoh, Y.,
Irie, K.,
Kano, T.,
Shirakabe, K.,
Muro, Y.,
Shibuya, H.,
Matsumoto, K.,
Nishida, E.,
and Hagiwara, M.
(1996)
J. Biol. Chem.
271,
13675-13679 |
2. |
Shirakabe, K.,
Yamaguchi, K.,
Shibuya, H.,
Irie, K.,
Matsuda, S.,
Moriguchi, T.,
Gotoh, Y.,
Matsumoto, K.,
and Nishida, E.
(1997)
J. Biol. Chem.
272,
8141-8144 |
3. | Yamaguchi, K., Shirakabe, K., Shibuya, H., Irie, K., Oishi, I., Ueno, N., Taniguchi, T., Nishida, E., and Matsumoto, K. (1995) Science 270, 2008-2011[Abstract] |
4. | 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] |
5. | Shibuya, H., Yamaguchi, K., Shirakabe, K., Tonegawa, A., Gotoh, Y., Ueno, N., Irie, K., Nishida, E., and Matsumoto, K. (1996) Science 272, 1179-1182[Abstract] |
6. | 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] |
7. |
Kishimoto, K.,
Matsumoto, K.,
and Ninomiya-Tsuji, J.
(2000)
J. Biol. Chem.
275,
7359-7364 |
8. |
Takaesu, G.,
Ninomiya-Tsuji, J.,
Kishida, S.,
Li, X.,
Stark, G. R.,
and Matsumoto, K.
(2001)
Mol. Cell. Biol.
21,
2475-2484 |
9. | Irie, T., Muta, T., and Takeshige, K. (2000) FEBS Lett. 467, 160-164[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Sakurai, H.,
Miyoshi, H.,
Toriumi, W.,
and Sugita, T.
(1999)
J. Biol. Chem.
274,
10641-10648 |
11. |
Vidal, S.,
Khush, R. S.,
Leulier, F.,
Tzou, P.,
Nakamura, M.,
and Lemaitre, B.
(2001)
Genes Dev.
15,
1900-1912 |
12. | Takaesu, G., Surabhi, R. M., Park, K. J., Ninomiya-Tsuji, J., Matsumoto, K., and Gaynor, R. B. (2003) J. Mol. Biol. 326, 105-115[CrossRef][Medline] [Order article via Infotrieve] |
13. | Cao, Z., Henzel, W. J., and Gao, X. (1996) Science 271, 1128-1131[Abstract] |
14. | Hirai, S., Izawa, M., Osada, S., Spyrou, G., and Ohno, S. (1996) Oncogene 12, 641-650[Medline] [Order article via Infotrieve] |
15. |
Mochida, Y.,
Takeda, K.,
Saitoh, M.,
Nishitoh, H.,
Amagasa, T.,
Ninomiya-Tsuji, J.,
Matsumoto, K.,
and Ichijo, H.
(2000)
J. Biol. Chem.
275,
32747-32752 |
16. |
Woronicz, J. D.,
Gao, X.,
Cao, Z.,
Rothe, M.,
and Goeddel, D. V.
(1997)
Science
278,
866-869 |
17. | Williams, D. H., Wilkinson, S. E., Purton, T., Lamont, A., Flotow, H., and Murray, E. J. (1998) Biochemistry 37, 9579-9585[CrossRef][Medline] [Order article via Infotrieve] |
18. | Zhao, A., Lee, S. H., Mojena, M., Jenkins, R. G., Patrick, D. R., Huber, H. E., Goetz, M. A., Hensens, O. D., Zink, D. L., Vilella, D., Dombrowski, A. W., Lingham, R. B., and Huang, L. (1999) J. Antibiot. 52, 1086-1094[Medline] [Order article via Infotrieve] |
19. |
Ichijo, H.,
Nishida, E.,
Irie, K.,
ten Dijke, P.,
Saitoh, M.,
Moriguchi, T.,
Takagi, M.,
Matsumoto, K.,
Miyazono, K.,
and Gotoh, Y.
(1997)
Science
275,
90-94 |
20. | Yan, M., Dai, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and Templeton, D. J. (1994) Nature 372, 798-800[Medline] [Order article via Infotrieve] |
21. | Yin, M. J., Christerson, L. B., Yamamoto, Y., Kwak, Y. T., Xu, S., Mercurio, F., Barbosa, M., Cobb, M. H., and Gaynor, R. B. (1998) Cell 93, 875-884[Medline] [Order article via Infotrieve] |
22. | Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature 385, 540-544[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Dinarello, C. A.
(1996)
Blood
87,
2095-2147 |
24. | Smith, W. L., DeWitt, D. L., and Garavito, R. M. (2000) Annu. Rev. Biochem. 69, 145-182[CrossRef][Medline] [Order article via Infotrieve] |
25. | Krueger, G. G., and Stingl, G. (1989) J. Invest. Dermatol. 92, 32-51 |
26. | Weston, W. L. (1976) Ann. Allergy 37, 346-352[Medline] [Order article via Infotrieve] |
27. | Lavaud, P., Rodrigue, F., Carre, C., Touvay, C., Mencia-Huerta, J. M., and Braquet, P. (1991) J. Invest. Dermatol. 97, 101-105[Abstract] |
28. | Goto, Y., Inoue, Y., Tsuchiya, M., Isobe, M., Ueno, T., Uchi, H., Furue, M., and Hayashi, H. (2000) Int. Arch. Allergy Immunol. 123, 341-348[CrossRef][Medline] [Order article via Infotrieve] |
29. | Rawlins, P., Mander, T., Sadeghi, R., Hill, S., Gammon, G., Foxwell, B., Wrigley, S., and Moore, M. (1999) Int. J. Immunopharmacol. 21, 799-814[CrossRef][Medline] [Order article via Infotrieve] |
30. | Takehana, K., Sato, S., Kobayasi, T., and Maeda, T. (1999) Biochem. Biophys. Res. Commun. 257, 19-23[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Shibuya, H.,
Iwata, H.,
Masuyama, N.,
Gotoh, Y.,
Yamaguchi, K.,
Irie, K.,
Matsumoto, K.,
Nishida, E.,
and Ueno, N.
(1998)
EMBO J.
17,
1019-1028 |
32. | Ishitani, T., Ninomiya-Tsuji, J., Nagai, S., Nishita, M., Meneghini, M., Barker, N., Waterman, M., Bowerman, B., Clevers, H., Shibuya, H., and Matsumoto, K. (1999) Nature 399, 798-802[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Ishitani, T.,
Ninomiya-Tsuji, J.,
and Matsumoto, K.
(2003)
Mol. Cell. Biol.
23,
1379-1389 |
34. |
Ishitani, T.,
Kishida, S.,
Hyodo-Miura, J.,
Ueno, N.,
Yasuda, J.,
Waterman, M.,
Shibuya, H.,
Moon, R. T.,
Ninomiya-Tsuji, J.,
and Matsumoto, K.
(2003)
Mol. Cell. Biol.
23,
131-139 |