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IL-1 Induced Chemokine Production
Through the Association of Syk with TNF
Receptor-Associated Factor-6 in Nasal
Fibroblast Lines
Takechiyo Yamada, Shigeharu Fujieda, Shigeru Yanagi,
Hirohei Yamamura, Ryoko Inatome, Hideyuki Yamamoto,
Hideki Igawa and Hitoshi Saito
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 167:283-288; ;
doi: 10.4049/jimmunol.167.1.283
http://www.jimmunol.org/content/167/1/283
IL-1 Induced Chemokine Production Through the Association
of Syk with TNF Receptor-Associated Factor-6 in Nasal
Fibroblast Lines1
Takechiyo Yamada,* Shigeharu Fujieda,2* Shigeru Yanagi,† Hirohei Yamamura,†
Ryoko Inatome,† Hideyuki Yamamoto,* Hideki Igawa,* and Hitoshi Saito*
F
ibroblasts, a rich source of chemokines, interact with eosinophils and play a key role in the pathogenesis of airway
disease. The fibroblasts released the chemokine RANTES
or GM-CSF and recruited the infiltrating cells, including eosinophils (1). High amounts of RANTES were found in the nasal polyp
specimens (2). Cultured nasal fibroblasts expressed RANTES
mRNA and secreted RANTES protein (3). We established the human fibroblast lines showing high Syk expression and the lines
showing low Syk expression from pieces of nasal polyp (4), although Syk is widely expressed and plays an important role in
intracellular signal transduction in hemopoietic cells, B cells (5),
mast cells (6), platelets (7), eosinophils (8), and so on.
RANTES production requires the transcription factor NF-␬B
and the activation of mitogen-activated protein kinases (MAPKs;3
Refs. 9 –13). IL-1 induces the interaction of TNFR- associated
factor 6 (TRAF6) with IL-1R-associated kinase (IRAK), which is
recruited rapidly to the IL-1R after IL-1 induction, whereas
TRAF2 participates in TNF-␣ signaling (14, 15). Recently, Wong
et al. (16) demonstrated a signaling complex involving tyrosine
kinase c-Src and TRAF6. Additionally, Li et al. (17) revealed that
tyrosine kinase inhibitor substantially alleviated the cytokine gene
expression and decreased the level and activity of IRAK.
*Department of Otorhinolaryngology, Fukui Medical University, Fukui, Japan; and
†
Department of Biochemistry, Kobe University School of Medicine, Kobe, Japan.
Received for publication November 20, 2000. Accepted for publication April
23, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by grants-in-aid for Scientific Research from Ministry of
Education, Culture, Sports, Science and Technology, Japan, and by the Uehara
Foundation.
2
Address correspondence and reprint requests to Dr. Shigeharu Fujieda, Shimoaizuki,
Matsuoka, Fukui 910-1193, Japan. E-mail address: [email protected]
3
Abbreviations: MAPK, mitogen-activated protein kinase; TRAF, TNFR-associated
factor; IRAK, IL-1R-associated kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-related kinase; Akt, antiapoptotic serine/threonine kinase; SH, Src
homology.
Copyright © 2001 by The American Association of Immunologists
In the present study, we examined whether Syk is involved in
the RANTES production by IL-1 and TNF-␣ via TRAFs. We also
looked at the phosphorylation state of c-Jun N-terminal kinase
(JNK), extracellular signal-related kinase (ERK), and p38 MAPK
in response to IL-1 when Syk expression was artificially changed
by oligonucleotides and vectors. Further, IL-1-induced I␬B degradation was investigated as a reporter of NF-␬B-signaling, a transcription factor essential for RANTES expression (18, 19).
Materials and Methods
Reagents
Anti-Syk Ab, anti-c-Src Ab, anti-TRAF6 Ab, anti-TRAF 2 Ab, anti-JNK
Ab, anti-phosphorylated JNK Ab, anti-p38 Ab, anti-phosphorylated p38
Ab, anti-ERK Ab, and anti-phosphorylated ERK Ab were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant human IL-1␤
was obtained from Cistron Biotechnology (Pine Brook, NJ). Recombinant
human TNF-␣ and the ELISA kits for RANTES were purchased from
Biosource (Camarillo, CA). The adenovirus expression vector kit was purchased from Takara Biomedicals (Tokyo, Japan).
Cell preparation
Nasal polyps were obtained during surgery from patients with chronic
paranasal rhinosinusitis. Chronic rhinosinusitis was strictly diagnosed via
endoscopic findings, paranasal sinus x-ray tomography, clinical history,
and symptoms by a specialist of otorhinolaryngology. Nasal polyps were
fluid-filled sacks formed from the upper part of the nasal cavity.
Human nasal fibroblast lines were grown from small pieces of nasal
polyp and regular passages (20). Fibroblasts were used at passage numbers
4 –10. No contamination of epithelial cells was confirmed by immunohistochemical examination with cytokeratin marker. There was wide variation
in the expression of Syk by nasal fibroblast lines as described previously
(4). Before starting the experiments, Western blot analysis was performed
to be determined Syk expression in fibroblast cell line. In this study, we
used the human fibroblast line with the highest Syk expression and the line
with the lowest Syk expression in our library. We could not find any difference in growth characteristics or morphology between fibroblast line
with the highest and that with the lowest Syk expression.
Cell stimulation and chemokine assay
The fibroblasts were stimulated by IL-1␤ and TNF-␣ in RPMI 1640 medium supplemented with 10% FCS and in humidified atmosphere of 10%
0022-1767/01/$02.00
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The fibroblasts stimulated by cytokines released the chemokine and recruited the infiltrating cells, including eosinophils, that play
a key role in the pathogenesis of airway disease. We established the human fibroblast lines showing high Syk expression and the
lines showing low Syk expression from pieces of nasal polyp. IL-1 induces the interaction of TNFR-associated factor (TRAF) 6 with
IL-1R-associated kinase, which is rapidly recruited to the IL-1R after IL-1 induction, whereas TRAF2 participates in TNF-␣signaling. In the present study, we found that Syk played a different role in IL-1- and TNF-␣-induced chemokine production
through a signaling complex involving Syk and TRAF6. Overexpression of wild-type Syk by gene transfer enhanced RANTES
production from nasal fibroblasts stimulated with IL-1. The decrease of Syk expression by the administration of Syk antisense
inhibited RANTES production in response to IL-1. However, the change of Syk expression did not affect RANTES production by
TNF-␣ stimulation. We concluded that Syk is required for the IL-1-induced chemokine production through the association with
TRAF-6 in fibroblasts of nasal polyps. The Journal of Immunology, 2001, 167: 283–288.
284
IL-1 CAUSED THE ASSOCIATION OF Syk WITH TRAF-6
CO2 in air at 37°C for 24 h. The supernatants were harvested and stored at
⫺80°C. Amounts of RANTES in the cell culture supernatant were measured with an ELISA kit. Measurements were performed according to the
manufacturers’ protocol.
Statview software (Abacus Concepts, Berkeley, CA) were used for all statistical analyses.
Coimmunoprecipitation
Results
Cells were collected by centrifugation and lysed in 1 ml of immunoprecipitation buffer (1% digitonin, 0.15 M NaCl, 5 mM ␩, 100 mM Na3VO4,
10 mg/ml leupeptin, 1 mM PMSF, and 50 mM Tris-HCl, pH 7.5). The
lysate was clarified and incubated with excess protein A-Sepharose 4B
(50% slurry). The cleared sample was immunoprecipitated with Abs and
protein A-Sepharose 4B at 4°C. The immune complexes were processed
for immunoblot as described above.
Gel electrophoresis and Western blots
The fibroblasts or immunoprecipitants were boiled with electrophoresis
sample buffer for 3 min and separated by SDS-PAGE. The separated proteins were transferred electrophoretically to polyvinylidene difluoride
membranes at the same protein concentration per lane. The membranes
were blocked at room temperature for 1 h in pH 7.4 PBS with 10% BSA
and were probed with Abs as described previously (4).
An adenovirus vector encoding Syk was constructed with the use of an
adenovirus expression kit (Takara). A 2.7-kb cDNA fragment containing
the entire coding sequence of Syk was ligated into the pAxCAwt cosmid
vector, which contains the modified chicken ␤-actin promoter with cytomegalovirus-IE enhancer (CAG promoter). Then, the recombinant adenovirus pAxCAwt-Syk was prepared by homologous recombination of the
expression cosmid cassette and parental viral genome and amplified to
achieve a stock with a titer of ⬃109 PFU/ml. A control recombinant adenovirus pAxCAwt-LacZ was used. Fibroblasts were equally infected with
10 multiplicity of infection of pAxCAwt-Syk or pAxCAwt-LacZ, respectively. Syk expression were detected from day 2 to day 8 after the infection.
Antisense oligodeoxynucleotides
Human nasal fibroblasts were cultured in RPMI 1640 in the presence
of phosphorothioate oligodeoxynucleotides (Nisshinbo Industries, Tokyo,
Japan) at 10 ␮M. The sequences used were as follows: antisense Syk,
CATGCTTCAGGGGCCGG; sense Syk, CCGGCCCTGAAGCATG (4, 7).
We obtained the human fibroblast line showing high Syk expression. Production of RANTES by the fibroblasts was measured by
ELISA. As shown in Fig. 1A, the levels of RANTES production by
the fibroblasts in the presence of IL-1␤ (1 ng/ml) or TNF-␣ (1
ng/ml) was significantly elevated compared with those in the absence of IL-1 or TNF-␣ ( p ⬍ 0.01).
To examine whether Syk plays a role in the RANTES production, we artificially regulated the Syk expression in the fibroblast
line with antisense oligodeoxynucleotides to Syk. As shown in Fig.
1B, human nasal fibroblasts exposed to 10 ␮M phosphorothionated
Syk antisense oligodeoxynucleotides for 6 h inhibited the expression of Syk protein, whereas the exposure of Syk sense oligodeoxynucleotides did not change Syk protein levels. We examined
c-Src expression in these fibroblasts with anti-c-Src Ab as a control. There were no differences in the levels of c-Src expression.
IL-1 failed to induce RANTES production from fibroblasts that
did not exhibit Syk protein (Fig. 1A). Sense oligodeoxynucleotides
did not alter IL-1-induced RANTES production. There was a significant difference in IL-1-induced RANTES production between
fibroblasts treated with Syk antisense oligodeoxynucleotides and
those with sense oligodeoxynucleotides ( p ⬍ 0.01).
In contrast, there was no difference in TNF-␣-induced RANTES
production between fibroblasts treated with Syk antisense oligodeoxynucleotides and those with sense oligodeoxynucleotides (Fig.
1A). Interestingly, the role of Syk for IL-1 signaling might be
different from that of TNF-␣ signaling in RANTES production.
Statistical analyses
IL-1-dependent association of Syk with TRAF6
Statistical analysis was performed using the Mann-Whitney U test and
Wilcoxon signed-ranks test to assess the differences in RANTES production levels. Macintosh computers (Apple Computer, Cupertino, CA) with
To investigate IL-1 or TNF-␣-induced signaling, we examined the
relationship between Syk and the TRAF family with digitonin as a
detergent in lysis buffer, which has been reported to maintain the
FIGURE 1. Syk antisense oligodeoxynucleotides attenuated IL-1-induced RANTES production from human nasal fibroblasts. A, Nasal fibroblasts with
high Syk expression (1 ⫻ 106/ml) were cultured in the presence of 10 ␮M Syk antisense, 10 ␮M Syk sense, or in the absence of oligodeoxynucleotide for
6 h and then stimulated with IL-1␤ (1 ng/ml) or TNF-␣ (1 ng/ml). Supernatants were harvested for analysis of RANTES secretion by ELISA 24 h after
the stimulation. The pretreatment of Syk antisense-inhibited RANTES production by fibroblasts stimulated with IL-1␤ (ⴱ, p ⬍ 0.01), but did not inhibit
those stimulated with TNF-␣. B, Nasal fibroblasts were transfected with Syk antisense, Syk sense, or no oligodeoxynucleotide. Harvested proteins (30 ␮g)
from cultured cells were reacted with anti-Syk Ab and anti-c-Src Ab and then processed for immunoblotting. The arrows indicate the positions of Syk and
c-Src.
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Preparation of recombinant adenoviruses
Syk antisense oligodeoxynucleotides attenuated IL-1-induced
RANTES production from human nasal fibroblasts
The Journal of Immunology
285
The effect of reduced Syk expression on the phosphorylation of
JNK, p38, and ERK
Because IL-1 was reported to cause the activation of JNK (14, 15),
we investigated IL-1-induced phosphorylation of JNK, ERK, and
p38 MAPK. To analyze whether Syk plays some roles in regulating the phosphorylation of these molecules in response to IL-1, the
fibroblasts were exposed to Syk sense or Syk antisense oligodeoxynucleotides, and then treated with IL-1 for 10 min. As shown
in Fig. 3, cell lysates were subjected to immunoblotting with anti-
FIGURE 2. IL-1-dependent association of Syk with TRAF6. A, Fibroblasts with high Syk expression were cultured without (lanes 1 and 3), or
with 1 ng/ml IL-1␤ (lane 2) and TNF-␣ (lane 4) for 10 min. The cells were
collected and lysed in 1 ml of immunoprecipitation buffer. The sample was
immunoprecipitated with anti-TRAF6 Ab (lanes 1 and 2) and anti-TRAF2
Ab (lanes 3 and 4). The sample then was blotted with anti-Syk and anti-Src
Ab. The same samples were blotted with anti-TRAF6 Ab (lanes 1 and 2)
and anti-TRAF2 Ab (lanes 3 and 4). B, After the treatment as shown in A,
the same amount of lysate (30 ␮g) before immunoprecipitation was applied
to each lane and blotted with anti-Syk Ab, anti-c-Src Ab, anti-TRAF6 Ab,
and anti-TRAF2 Ab.
FIGURE 3. The effect of reduced Syk expression on the phosphorylation of JNK, p38, and ERK. Nasal fibroblasts with high Syk expression
were transfected with 10 ␮M Syk sense (lanes 1 and 2) or antisense (lanes
3 and 4) for 6 h, and then stimulated without IL-1␤ (lanes 1 and 3) or with
1 ng/ml IL-1␤ (lanes 2 and 4) for 10 min. The same amount of lysate (30
␮g) was applied to each lane and blotted with anti-phosphorylated JNK Ab
(top) and anti-JNK1 Ab (bottom) as shown in Fig. 3A, with antiphosphorylated p38 Ab (top) and with anti-p38 Ab (bottom) in Fig. 3B,
with anti-phosphorylated ERK Ab (top) and with anti-ERK Ab (bottom) in
Fig. 3C, and with anti-Syk Ab in Fig. 3D. The antisense treatment decreased IL-1-induced JNK and p38 phosphorylation in nasal fibroblasts.
phosphorylated JNK Ab, anti-phosphorylated p38 Ab, and antiphosphorylated ERK Ab.
JNK was phosphorylated and activated after treatment with IL-1
for 10 min because the anti-phosphorylated JNK Ab reacted with
active JNK1 phosphorylated on threonine 183 and tyrosine 185
(Fig. 3A, top; Ref. 4). JNK1 phosphorylation was almost maximal
within 10 min and remained elevated throughout the 30-min incubation of IL-1 (data not shown). Phosphorylation of p38 also
occurred by treatment with IL-1, with the anti-phosphorylated p38
Ab reacting with active p38 phosphorylated on tyrosine-182 (Fig.
3B, top).
The treatment with Syk sense oligodeoxynucleotides had no effect on the JNK and p38 phosphorylation induced by IL-1. However, IL-1 failed to induce JNK phosphorylation of the fibroblasts
treated with Syk antisense oligodeoxynucleotides (Fig. 3A). Syk
antisense oligodeoxynucleotides attenuated p38 phosphorylation
induced by IL-1 (Fig. 3B) The semiquantitative densitometric
analysis in p38 phosphorylation showed that the value in the Syk
antisense-treated fibroblast was half of that in the Syk sensetreated fibroblast (0.38 vs 0.77).
In contrast, ERK was constantly phosphorylated on tyrosine 204
before the stimulation in the fibroblast lines and was not changed
by stimulation of IL-1 (Fig. 3C). Syk antisense oligodeoxynucleotides did not affect the phosphorylation of ERK in the fibroblasts.
Wild-type Syk vector transfection magnified IL-1-induced
RANTES production by nasal fibroblasts
We also established the human fibroblast line showing low Syk
expression and transfected expression vector from wild-type Syk
into the fibroblasts. As shown in Fig. 4A, there was no difference
in the spontaneous RANTES production in the absence of IL-1 or
TNF-␣ among the fibroblasts transfected with the different vectors.
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associations after cell lysis (21). It has been determined that endogenous Syk, c-Src, and TRAF6 form a signaling complex in
nasal fibroblasts after simulation of IL-1.
As shown in Fig. 2A, immunoblot analysis revealed the existence of Syk in the immune complex precipitated by anti-TRAF6
Ab. IL-1 stimulation substantially increased the association of
TRAF6 with Syk in nasal fibroblasts. Also, we found the existence
of c-Src in the immune complex precipitated by anti-TRAF6 Ab
after the stimulation of IL-1. In contrast, TNF-␣ stimulation
caused no association of TRAF2 with Syk or with c-Src (Fig. 2A).
When samples were immunoprecipitated with TRAF6 or TRAF2
and blotted with corresponding TRAF6 or TRAF2, respectively,
there were no differences in the levels of TRAF6 and TRAF2 immunoprecipitates before and after stimulation. Also, we found no
association of TRAF6 with Syk in the fibroblasts after TNF-␣stimulation (data not shown).
Fig. 2B proved the existence of endogenous Syk, c-Src, TRAF6,
and TRAF2 in the fibroblasts. No differences were found in the
expression levels of these molecules with the samples before
immunoprecipitation.
286
IL-1 CAUSED THE ASSOCIATION OF Syk WITH TRAF-6
Although TNF-␣ caused the high levels of RANTES production
from the fibroblasts showing low Syk expression, IL-1 induced
small amounts of RANTES production from the same fibroblasts.
However, the fibroblasts transfected with the wild-type Syk vector
produced significantly higher levels of RANTES after stimulation
with IL-1 than those transfected with the control vector did ( p ⬍
0.01). Wild-type Syk vector transfection did not affect the TNF␣-induced RANTES production by nasal fibroblasts. No difference
was found in the morphology and cell growth among four fibroblasts even after the transfection of virus vector (data not shown).
The transfection of wild-type Syk vector enhanced the expression of Syk on day 2, whereas the control vector did not do so (Fig.
4B). We examined c-Src expression with anti-c-Src Ab in these
fibroblasts transfected with vectors. There were no differences in
the levels of c-Src expression, as shown in Fig. 4B.
The effect of increased Syk expression on the phosphorylation of
JNK, p38, and ERK
After the fibroblasts showing low Syk expression were transfected
with wild-type Syk vector or control vector, we investigated IL1-induced phosphorylation of JNK, p38 MAPK, and ERK. The
fibroblasts were transfected with wild-type Syk vector or control
vector and then treated with IL-1 for 10 min. The cell lysates were
subjected to immunoblotting with anti-phosphorylated JNK Ab,
anti-phosphorylated p38 Ab, and anti-phosphorylated ERK Ab
(Fig. 5).
The phosphorylation of JNK after IL-1 stimulation occurred
more in the fibroblasts transfected with wild-type Syk vector than
in the fibroblasts transfected with control vector (Fig. 5A, top). The
transfection with wild-type Syk vector also amplified the p38phosphorylation of the fibroblasts after stimulation with IL-1 for
10 min from 0.32 to 0.72 by semiquantitative densitometric analysis (Fig. 5B, top). However, ERK was phosphorylated without the
stimulation in the fibroblasts transfected with wild-type Syk vector
or control vector (Fig. 5C, top). Wild-type Syk vector did not affect
ERK phosphorylation after IL-1 stimulation.
FIGURE 5. The effect of increased Syk expression on the phosphorylation of JNK, p38, and ERK. Nasal fibroblasts with low Syk expression
were transfected with control vector (lanes 1 and 2) or wild-type Syk
vector (lanes 3 and 4) for 48 h and then stimulated without IL-1␤ (lanes 1
and 3) or with 1 ng/ml IL-1␤ (lanes 2 and 4) for 10 min. The same amount
of lysate (30 ␮g) was applied to each lane and blotted with antiphosphorylated JNK Ab (top) and with anti-JNK1 Ab (bottom) as shown in
A, with anti-phosphorylated p38 Ab (top) and with anti-p38 Ab (bottom) in
B, with anti-phosphorylated ERK Ab (top) and with anti-ERK Ab (bottom)
in C, and with anti-Syk Ab in D. Stimulation of IL-1 induced JNK phosphorylation and enhanced p38 phosphorylation in fibroblast transfected
with wild-type Syk vector.
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FIGURE 4. Wild-type Syk vector transfection magnified IL-1-induced RANTES production by nasal fibroblasts. A, After the transfection with wild-type
Syk vector or control vector or no vector into the fibroblasts with low Syk expression for 48 h, the cells were incubated in the presence or absence of IL-1␤
(1 ng/ml) or TNF-␣ (1 ng/ml), and supernatants were harvested for analysis of RANTES secretion by ELISA. IL-1-induced RANTES levels from fibroblasts
transfected with wild-type Syk vector were significantly higher than those from fibroblasts transfected with control vector (ⴱ, p ⬍ 0.01), whereas there was
no difference between TNF-␣-induced RANTES levels from fibroblasts transfected with wild-type Syk vector and those from fibroblasts transfected with
control vector. B, Nasal fibroblasts with low Syk expression are used in this experiment. The prepared proteins (30 ␮g) were processed and reacted with
anti-Syk Ab or anti-c-Src Ab. Anti-Syk immunoblotting showed the increased Syk expression in the fibroblasts transfected with wild-type Syk vector but
did not in those transfected with control vector.
The Journal of Immunology
FIGURE 6. The effect of increased Syk expression on the degradation
of I␬B. The fibroblasts with low Syk expression were transfected with
control vector (lanes 1 and 2), or wild-type Syk vector (lanes 3 and 4) for
48 h, and then stimulated without IL-1␤ (lanes 1 and 3), or with 1 ng/ml
IL-1␤ (lanes 2 and 4) for 20 min. The same amount of lysate (30 ␮g) was
applied to each lane and blotted with I␬B␣ as shown in A, and with antiSyk Ab in B. The degradation of I␬B␣ after IL-1 stimulation was shown in
the fibroblast transfected with wild-type Syk vector.
Because IL-1 was reported to cause the activation of NF-␬B (17),
we investigated the effects of Syk expression on the degradation of
I␬B-␣, which regulates NF-␬B (Fig. 6). The fibroblasts showing
low Syk expression were transfected with wild-type Syk vector or
control vector and then stimulated with IL-1. IL-1 induced the
degradation of I␬B-␣ in the fibroblasts transfected with wild-type
Syk vector, although it did not induce any degradation I␬B-␣ in
those transfected with control vector, suggesting that IL-1 induced
NF-␬B activation in the fibroblast showing high Syk expression.
Discussion
Although Syk expression was usually found in hemopoietic cells,
mouse embryonic fibroblasts and rat hepatocytes recently were
reported to express Syk (22, 23). We found that some human fibroblast lines highly expressed Syk protein (4). In this study, IL-1
failed to induce RANTES production by fibroblasts that did not
exhibit Syk, and TNF-␣ induced RANTES production in an independent manner on the Syk-expression level. We also demonstrated that IL-1-signaling was associated with Syk and TRAF6,
although TNF-␣ caused no association of TRAF2 of Syk. The
artificial change of Syk expression affected JNK-phosphorylation
and p38-phosphorylation by IL-1. The signal pathway of Syk -JNK
or Syk-p38 connects with NF-␬B activation in RANTES gene
expression.
The IL-1 and TNF-␣ signals are transduced through TRAF6 and
TRAF2, respectively (15). IL-1 induced the interaction of TRAF6
with IRAK, although IRAK did not associate with TRAF2 after the
stimulation of IL-1 (14). The engagement of cytokine receptors
with TRAF6 activates the antiapoptotic serine/threonine kinase
(Akt/protein kinase B) through a signaling complex involving Src
family kinases and TRAF6 (16). Tyrosine kinases could regulate
the cytokine gene expression and IRAK activity (17). We revealed
that IL-1 induced chemokine production from the nasal fibroblasts
through a signaling complex involving Syk and TRAF6. IL-1 also
induced the interaction of c-Src with TRAF6. Src homology (SH)
3 domains contain proline-rich motifs containing PXXP consensus
sequence (24), and TRAF6 contains a SH3-interacting sequence
(16). Therefore, Src family kinases have SH3 domains with the
ability to interact with TRAF6. In human platelets, Syk associated
transiently with c-Src, and Src family kinases participated in the
signaling (25). In our system, Syk could take an important role
with Src family kinases and TRAF6 in IL-1 signaling. However,
we cannot exclude the possible interaction with unknown adaptor
proteins to mediate this signaling.
IL-1 stimulated transcription factors activating protein-1 and
NF-␬B through activation of JNK and the I␬B kinase (15). In
response to IL-1, MAPK was activated in a concentration-dependent manner, and its activation was attenuated by tyrosine kinase
inhibitor (13). In this report, we demonstrated that IL-1 induced
the phosphorylation of JNK mainly and p38 partially in nasal fibroblast lines. RANTES mRNA expressions were induced through
the activation of MAPKs (11–13) or through JNK and NF-␬B
kinase cascades in macrophages (26). The Syk-generated signal
cooperates to enhance Rac-induced JNK activation in T lymphocytes (27). MAPKs activation was compromised in the macrophages of Syk⫺/⫺ mice after Fc-␥ receptor stimulation (28). The
cytokine production was produced by an early tyrosine phosphorylation of Syk in murine resident peritoneal macrophages (29).
Here, we found that Syk expression mainly affected JNK activation in response to IL-1 in nasal fibroblast lines. Transcriptional
activation of the human RANTES promoter was dependent on specific activating protein-1, which was regulated by JNK (26). Syk
has two SH2, instead of SH3, and the C-terminal SH2 domain of
Syk was required for induction of JNK-activation (30).
Regulation of RANTES chemokine gene expression requires
NF-␬B through I␬B (9). Recently, Das (31) reported that JNK
mediated NF-␬B activation after degradation of I␬B. We demonstrated that Syk was related to JNK phosphorylation and degradation of I␬B. Phosphoinositide 3-kinase and Akt were involved in
NF-␬B signaling (32). TRAF6 induced Akt activation through Src
family kinases (16). Syk also is required for the activation of the
phosphoinositide 3-kinase and Akt in B cells (33).
Toll-like receptor has been proven to mediate LPS-induced cellular signaling (34). TRAF6 mediated both IL-1- and LPS-induced
signaling. However, TRAF2 did not mediate in the IL-1- and LPSsignaling while it was involved in TNF-signaling (35). We also found
that pretreatment of antisense oligodeoxynucleotides to Syk inhibited
RANTES production by fibroblasts stimulated with LPS (4).
The pathogenetic findings of nasal polyps shows an increase in
infiltrating cells including eosinophils (36). Eosinophils and fibroblasts strongly interacted with each other in the pathogenesis of
airway diseases, because fibroblasts are a rich source of chemokines, cytokines, and inflammatory mediators. Recently, Stenton et
al. (37) reported that Syk antisense oligodeoxynucleotides delivered by aerosol to lungs in vivo depressed Syk expression and
pulmonary inflammation. In conclusion, Syk proteins are endogenously expressed in human nasal fibroblasts. Syk antisense oligodeoxynucleotides depressed Syk expression and chemokine production in nasal fibroblasts that originated from nasal polyps. The
regulation of Syk expression may prove useful as one of the strategies for the treatment of airway diseases such as asthma or nasal
polyps.
Acknowledgments
We thank I. Funatsu for excellent technical assistance.
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