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B-Inducing Kinase Is a Common Mediator of IL-17-, TNF-
-, and IL-1ß-Induced Chemokine Promoter Activation in Intestinal Epithelial Cells1
Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B protein-DNA complexes consisting
of p65/p50 heterodimers in the rat intestinal epithelial cell line
IEC-6. The induction of NF-B correlated with the induction of CXC
and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a
synergistic fashion with IL-1ß to induce the NF-B site-dependent
CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells
was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent.
Furthermore, IL-17 induction of the CINC promoter could be inhibited by
kinase-negative mutants of NF-B-inducing kinase and IB
kinase-
. In addition to activation of the NF-B, IL-17 regulated
the activities of extracellular regulated kinase, c-Jun N-terminal
kinase, and p38 mitogen-activated protein kinases in IEC-6 cells.
Whereas the IL-17-mediated activation of extracellular regulated kinase
mitogen-activated protein kinases was mediated through
ras, c-Jun N-terminal kinase activation was dependent on
functional TRAF6. These data suggest that NF-B-inducing kinase
serves as the common mediator in the NF-B signaling cascades
triggered by IL-17, TNF-, and IL-1ß in intestinal epithelial
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-17 has been shown to induce NF-B consensus sequence binding in
mouse fibroblasts (5) and human macrophages (7). Subsequently, the
expression of several cytokines known to contain NF-B recognition
sites in their promoters was demonstrated to be regulated by IL-17.
These cytokines include IL-8, IL-6, and granulocyte CSF (3, 4, 5).
Furthermore, TNF- and IFN-
had an additive effect on the
stimulation of IL-6 expression by IL-17 (4), and the combination of
IL-17 and TNF- could stimulate the expression of
granulocyte-macrophage CSF by fibroblasts (4). However, the
IL-17-induced signal transduction events leading to the activation of
NF-B are not known. To understand the molecular basis of the
function of the IL-17R in intestinal epithelial cells we characterized
IL-17-induced signal transduction and transcriptional activation events
in the nontransformed rat intestinal epithelial cell line IEC-6.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant cytokines (IL-17, IL-1ß, and TNF-) were
obtained from R&D Systems (Minneapolis, MN). Polyclonal Abs against
extracellular regulated kinase-1
(ERK-1),4 ERK-2, c-Jun
N-terminal kinase-1 (JNK-1), p38, p50, and p65 were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Phosphotyrosine-specific p38 Ab
was obtained from New England Biolabs (Beverly, MA). Myelin basic
protein was purchased from Sigma (St. Louis, MO), and c-Jun was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Radiochemicals
([-32P]dCTP and [-32P]ATP) were
purchased from DuPont-New England Nuclear (Boston, MA).
Expression constructs
Dominant-negative expression constructs for TNF
receptor-associated factor-2 (TRAF287–501), TRAF6
(TRAF6289–522), NF-B-inducing kinase
(NIKKK429–430AA), and IB kinase- (IKK-) or
conserved helix loop helix ubiquitous kinase (CHUCKK44A)
were provided by David V. Goeddel. The TRAF287–501
construct lacks the N-terminus RING finger necessary for NF-B
activation (8). The TRAF6289–522 lacks the N-terminal
zinc-binding structure and has been shown to selectively inhibit
IL-1-activated, but not TNF--mediated, NF-B activation (9).
NIKKK429–430AA and CHUCKK44A are
kinase-negative mutants as previously described (10, 11). These
constructs were used in transfection assays at a concentration of 0.5
µg/ml. In experiments where these vectors were used, control cells
were transfected with an equal amount of empty pcDNA 3.1 vector
(Invitrogen, Carlsbad, CA). 5xGAL-Luc and GAL4-ELK-1 constructs have
been described previously (12) and were gifts from Anil K. Rustgi.
GAL4-ELK-1 contains the GAL4 DNA binding domain linked to the ELK-1
trans-activation domain. 5xGAL-Luc contains five consensus
GAL4 DNA binding sites (17-mer) subcloned into p20-Luc. HA-Ras-Asn-17
(RasN17) is a dominant-negative Ras construct that
preferentially bind GDP vs GTP and specifically interferes with the
function of endogenous Ras protein (13). The RasN17
construct was a gift from Timothy C. Wang. These constructs were used
in transfection assays at a concentration of 1 µg/ml.
CINC promoter luciferase reporter construct
Part (973 bp) of the 5'-flanking region of the rat CINC gene
(14) were amplified by PCR. The upper primer was flanked by a
BglII sequence: 5'-GGC AGA TCT AGA AGG CAT GGA AGT TCA ATA.
The lower primer was flanked by a HindIII sequence: 5'-GCG
CAA GCT TGC TCT GTT GGA GTG TGG. After the resulting PCR product was
digested with the restriction enzymes mentioned above, it was inserted
into pGL3 basic luciferase reporter vector (Promega, Madison, WI),
which had also been digested with BglII and
HindIII. To determine CINC promoter activation, IEC-6 cells
were transfected with 1 µg/ml CINC promoter construct and 24 h
later were stimulated with IL-17, IL-1ß, or TNF- for 12 h.
The cells were harvested using the lysis buffer provided in the
Luciferase Assay System Kit (Promega).
Cell culture and cell transfections
The nontransformed rat intestinal epithelial cell line IEC-6 was obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM (Cellgro, Mediatech, Herndon, VA) supplemented with 5% heat-inactivated FCS (Sigma), 100 mg/ml penicillin and streptomycin (Life Technologies, Gaithersburg, MD), and 0.1 U/ml insulin (Life Technologies). Cells were grown at 37°C in a 5% CO2 atmosphere within a humidified incubator. For transfections, IEC-6 cells were seeded onto six-well plates at a cell density of 5 x 105 cells/well on the day before transfection. All transfections were performed using 0.2 µg/ml of pSV-ß-galactosidase vector (Promega) as a control for transfection efficiency. The amount of transfected DNA was kept constant in all transfections. Transfections were performed over a 3-h period using a liposome-mediated transfection method (Lipofectamine-Plus, Life Technologies). Luciferase activity was measured by a luminometer (Analytical Luminescence Laboratory, San Diego, CA) and was expressed as relative light units normalized to ß-galactosidase activity in the same lysates measured with the luminescent ß-gal detection kit (Clontech, Palo Alto, CA). Statistical analysis was performed using Student’s t test.
Northern blotting
Total RNA was extracted from IEC-6 cells using Trizol reagent
(Life Technologies, Gaithersburg, MD). Poly(A)+ RNA was
isolated using the poly(A)+ tract system (Promega). Two
micrograms of poly(A)+ RNA samples were electrophoresed in
a 1% agarose formaldehyde gel and then transfered onto a nylon
membrane (Magna NT, Micron Separations, Westborough MA) by capillary
blotting. CINC and rat MCP-1 cDNAs were generated by RT-PCR from IEC-6
cells RNA (CINC upper primer, 5'-GGC AGA TCT GGA AGT TCC CGA GGT TCA
AA; lower primer, 5'-GGC GAA GCT TGG TGC TCT GTT GGA GTG TGG; rat MCP-1
upper primer, 5'-TCG GCT GGA GAA CTA CAA GAG; lower primer, 5'-AGG CAT
CAC ATT CCA AAT CA), subcloned into pCR 2.1 (Invitrogen), and
sequenced. cDNA probes were labeled with [-32P]dCTP by
a random hexamer priming method using the Rediprime Random Primer
Labeling Kit (Amersham Life Science, Arlington Heights, IL). Membranes
were hybridized in Quickhyb solution (Stratagene, La Jolla, CA) at
68°C for 1 h. The membranes were washed, and the blots were
analyzed by autoradiography.
EMSAs
IEC-6 cells grown on a 10-cm dish were starved overnight in
serum-free DMEM (Mediatech) and stimulated with IL-17 (100 ng/ml),
IL-1ß (10 ng/ml), or TNF- (100 ng/ml) for 30 min. Nuclear extracts
were prepared according to the protocol described by Schreiber et al.
(15). Consensus (5'-AGT TGA GGG GAC TTT CCC AGG) and mutant (5'-AGT TGA
GGC GAC TTT CCC AGG C) oligonucleotides were obtained from Santa Cruz
Biotechnology. Double-stranded oligonucleotides were end labeled with
[-32P]ATP using T4 polynucleotide kinase (New England
Biolabs). For competition and supershift assays, an 80-fold excess of
cold or mutant oligonucleotide or 2 µg of Ab was added to the
reaction, respectively. The reaction was conducted in a total volume of
20 µl, using 0.5 ng of labeled oligonucleotide, 10 µg of nuclear
protein extract, and 1 µg of poly(dI-dC) in 1x EMSA buffer (10 mM
Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 mM
EDTA, and 5% glycerol). The samples were loaded onto a 6%
nondenaturing polyacrylamide gel and run in 0.25x TBE buffer. The
resultant DNA-protein complexes were then detected by
autoradiography.
In vitro kinase assays and Western blot analysis
For in vitro kinase assays preconfluent intestinal epithelial
cells were starved for 12 h in serum-free medium to reduce
background activation. After stimulation with 100 ng/ml IL-17, cells
were lysed in lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 7.4),
300 mM NaCl, 2 mM EDTA, 2 mM EGTA, 10 mg/ml aprotinin, 200 mM PMSF, and
10 mg/ml leupeptin) containing phosphatase inhibitors (400 mM sodium
orthovanadate and 4 mM NaF). After 30 min on ice, cell lysates were
cleared by centrifugation at 12,000 x g for 20 min.
The protein concentration in each sample was quantified by the Bradford
method, and 200 µg of protein was used for immunoprecipitation. The
preclarified cell lysate in IP buffer were incubated with anti-MAP
kinase Abs and protein A-agarose (Oncogene, Cambridge, MA) overnight at
4°C. The immune complexes were washed three times with
immunoprecipitation buffer and twice with kinase reaction buffer (40 mM
HEPES (pH 7.0), 10 mM MgCl2, and 3 mM MnCl2).
In vitro kinase reactions were performed in the presence of 5 mCi of
[-32P]ATP and 2 µg of MAP kinase-specific substrate
(myelin basic protein for ERK1 and ERK2, c-Jun for JNK-1) at 30°C for
30 min. Reactions were terminated by boiling in Laemmli’s SDS sample
buffer. The samples were fractionated by SDS-PAGE and analyzed by
autoradiography.
For the detection of phosphorylated p38, IEC-6 cells were solubilized in lysis buffer, immunoprecipitation was conducted with anti-p38 Abs as described above, and immune complexes were electrophoresed through a 4–12% gradient SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). Detection of tyrosine-phosphorylated p38 with specific Abs (New England Biolabs) was conducted according to the manufacturer’s instructions. To confirm equal loading, the immunoblot was stripped with 62.5 mM Tris (pH 6.8)/2% SDS containing 10 mM 2-ME at 50°C for 1 h and reprobed with anti-p38 Abs.
Cell proliferation assay
IEC-6 cells were seeded into 96-well plates at a density of 5000 cells/well and cultured in DMEM with or without 5% FCS in the presence of various concentration of recombinant human IL-17 at 37°C for 48 h. Proliferation was assessed by the MTS (3-[4,5-dimethylthiazol-2-yl]-5[3-carboxymethoxyphenyl]-2[4-sulfophenyl]-2H-tetrazolium, inner salt) assay using the CellTiter 96 Aqueous kit (Promega) according to the manufacturer’s instructions. Each assay was performed in triplicate. Wells containing only medium without cells were subtracted as background from the raw absorbance values, and inhibition of proliferation was expressed as a percentage of IEC-6 cell proliferation without addition of IL-17.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B subunits p65 and p50 in intestinal
epithelial cells
IEC-6 cells were incubated with recombinant IL-17, IL-1ß, or
TNF- for 30 min, and EMSAs were performed to determine whether IL-17
activates NF-B consensus sequence binding in intestinal epithelial
cells. As demonstrated in Fig. 1
, IL-17
up-regulated the binding of proteins to the NF-B consensus sequence
in IEC-6 cells. The formation of the slowest migrating complex induced
by the addition of IL-17 was specifically inhibited by the addition of
an 80-fold excess of unlabeled NF-B oligonucleotides containing a
wild-type, but not a mutant, NF–B binding sequence (Fig. 1).
However, the amount of NF-B binding induced by IL-17 was lower than
that induced by IL-1ß or TNF- (Fig. 1).
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B consensus sequence binding proteins are
activated by IL-17 in intestinal epithelial cells, we performed
supershift assays with specific Abs recognizing the p65 and p50 NF-B
subunits. The NF-B binding complexes initiated by the stimulation of
IL-17 in intestinal epithelial cells were completely supershifted with
Abs recognizing either p65 (Fig. 1
, lane 5) or p50 (Fig. 1, lane 6). In contrast, NF-B binding complexes formed in
response to IL-1ß (Fig. 1, lane 9) and TNF- (Fig. 1, lane 12) were not completely shifted by anti-p50 Abs,
suggesting the involvement of additional NF-B subunits. These
findings demonstrate that the NF-B binding complexes observed after
IL-17 stimulation in IEC-6 cells are composed of p65/p50
heterodimers. IL-17 induces CXC and CC chemokine mRNA expression in intestinal epithelial cells
The activation of NF-B leads to a coordinated increase in the
expression of many genes whose products mediate and regulate
inflammatory and immune responses. NF-B has been shown to be
involved in the induction of chemokine expression (16). Furthermore,
IL-17 has been shown to induce IL-8 expression in fibroblasts (4). We
therefore determined whether the observed induction of NF-B results
in activation of chemokine gene transcription in IEC-6 cells by IL-17.
Northern blot analysis demonstrated that IL-17 was able to rapidly
up-regulate the mRNA expression of transcripts encoding the rat CXC
chemokine CINC, a homologue of the human GRO- (17) as well as
transcripts encoding for the rat CC chemokine MCP-1 (18) in a dose- and
time-dependent (Fig. 2B)
fashion (Fig. 2, A and B). CINC and rat MCP-1
mRNA expression was differently regulated by IL-17. IL-17 stimulation
induced a strong CINC mRNA expression within 30 min (Fig. 2A), while induction of MCP-1 mRNA by IL-17 was observed
1 h after stimulation (Fig. 2B).
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To determine whether IL-17 is able to augment IL-1-mediated
induction of chemokine expression, we transfected IEC-6 cells with a
CINC promoter luciferase reporter construct, stimulated 24 h later
with IL-17 or IL-1ß alone or in combination and assayed for
luciferase activity after an additional 24 h. As demonstrated in
Fig. 3, 100 ng/ml IL-17 was able to
induce a 6-fold increase in CINC promoter activity, and 10 ng/ml
IL-1ß was able to stimulate a 12-fold increase. However, much lower
concentrations of IL-17 were sufficient to stimulate up-regulation of
CINC promoter activity when IEC-6 cells were costimulated with
suboptimal concentrations of IL-1ß. In the presence of 0.1 ng/ml of
IL-1ß, even 1 ng/ml of IL-17 was enough to induce a 5-fold increase
in CINC promoter activity (Fig. 3). The induction of CINC promoter
activity by IL-17 in IEC-6 cells was less than that observed with
stimulation by either IL-1ß or TNF-.
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To determine whether the synergistic regulation of CINC promoter
activity by IL-17 is dependent on the presence of functional IL-1ß
and/or TNF- signal transduction pathways in IEC-6 cells, these
pathways were selectively blocked with dominant-negative
TRAF6289–522 or dominant-negative
TRAF287–501. As demonstrated in Fig. 4 the expression of dominant-negative
TRAF6289–522 significantly reduced the induction of IL-17
and IL-1ß, but not TNF--induced CINC promoter activity, in IEC-6
cells. By contrast, the expression of dominant-negative
TRAF287–501 did not alter IL-17- or
IL-1ß-dependent CINC promoter activation, but reduced CINC promoter
activation induced by TNF- (Fig. 4), demonstrating that the
dominant-negative effect of the TRAF6 mutant was specific. Transfection
of IEC-6 cells with TRAF6289–522 or
TRAF287–501 dominant-negative expression constructs did
not significantly alter the baseline activity of the reporter construct
(Fig. 4).
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B-inducing kinase NIK and the IB kinase IKK-
IL-1 as well as TNF- signaling pathways converge at NIK and use
IKK-, originally designated CHUK, as the common activator of NF-B
(9, 10). To assess whether IL-17-induced chemokine promoter activation
in intestinal epithelial cells requires functional NIK and IKK-, we
transfected IEC-6 cells with kinase negative mutants of NIK
(NIKKK429–430AA) and IKK- (CHUCKK44A). As
demonstrated in Fig. 5,
NIKKK429–430AA significantly decreased the CINC promoter
activity induced by IL-17, TNF-, and IL-1ß in IEC-6 cells.
Furthermore, activation of the CINC promoter in IEC-6 cells was
dependent on IKK-, as expression of the catalytic inactive
CHUCKK44A mutant significantly diminished the CINC promoter
activity in response to IL-17, IL-1ß, and TNF- (Fig. 5). Thus,
IL-17-mediated signal transduction shares the utilization of MAP3K NIK
and the IB kinase IKK- with IL-1ß and TNF- in activation
of the CINC promoter in IEC-6 cells.
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B subunit p65 is NIK
dependent
As demonstrated in Fig. 6, the
inhibition of IL-17-mediated CINC promoter activation by the expression
of kinase-negative NIKKK429–430AA mutant correlates
with the inhibition of NF-B activation in IEC-6 cells. IEC-6 cells
were transfected with empty mock control vector or dominant-negative
NIKKK429–430AA, and expression of the NF-B subunit p65
was determined in nuclear extracts without or after stimulation for 30
min with 100 ng/ml TNF-, 10 ng/ml IL-1ß, or 100 ng/ml IL-17 by
Western blot analysis (Fig. 6A). Densitometric
quantification revealed that the expression of the kinase-negative NIK
mutant reduced the nuclear translocation of p65 protein induced by
TNF-, IL-1ß, and IL-17 by 39.4, 38.4, and 45.4%, respectively
(Fig. 6B). These experiments suggest NIK as a common
mediator in IL-17 as well as IL-1ß and TNF- signal transduction
pathways leading to NF-B activation in IEC-6 cells.
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Signal transduction events induced by TNF- and IL-1ß
include the activation of MAP-kinases (11, 19, 20, 21). We therefore
assessed the ability of IL-17 to activate ERK-1, ERK-2, JNK-1, and p38
in IEC-6 cells. The activation of ERK and JNK MAP-kinases was
determined by in vitro immune complex kinase assays. IL-17 stimulation
resulted in a biphasic regulation of ERK-1 activity in IEC-6 cells
(Fig. 7A). ERK-1 activity was
down-regulated within 5 min followed by an increase in ERK-1 activity,
which peaked 15 min after stimulation with 100 ng/ml IL-17 (Fig. 7A). The same pattern of initial down-regulation and
subsequent up-regulation of kinase activity was observed for ERK-2
activity by IL-17 in IEC-6 cells (Fig. 7B). IL-17 activated
the JNK-mediated phosphorylation of c-Jun in intestinal epithelial
cells (Fig. 7C). In contrast to the activation of ERK MAP
kinase activation, IL-17 induced a gradual increase in JNK-1 activation
that peaked at 15 min (Fig. 7C). Immunoprecipitation of p38
followed by Western blot analysis with a specific Ab that recognizes
activated p38 phosphorylated at Tyr182 was used to
determine whether IL-17 and IL-1ß are able to activate p38 MAP kinase
in IEC-6 cells. As demonstrated in Fig. 8, IL-17 and IL-1ß were also able to
induce the phosphorylation of p38 in IEC-6 cells. The phosphorylation
of p38 was transiently up-regulated after 15 min of stimulation with
100 ng/ml of IL-17 (Fig. 8).
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Since IL-17 induced a biphasic regulation of ERK-1 and ERK-2
activity, we determined whether the IL-17-dependent regulation of ERK
MAP kinases results in the downstream activation of ELK-1. We used a
fusion containing the DNA binding domain of the yeast transcription
factor GAL4 linked to the carboxyl-terminal transcriptional activation
domain of ELK-1. A reporter gene containing five GAL4 binding sites
upstream of a minimal promoter linked to luciferase was used to assess
activation of the GAL4 fusion protein. As demonstrated in Fig. 9, IL-17 and IL-1ß stimulated ELK-1
trans-activation in IEC-6 cells. IL-17- as well as
IL-1ß-induced activation of ELK-1 could be inhibited by
cotransfection of dominant-negative RasN17 (Fig. 9).
This inhibition was specific, as the dominant negative
RasN17 did not alter the baseline activity of the reporter
construct in IEC-6 cells (Fig. 9). These results are consistent with an
IL-17-dependent ERK MAP kinase activation in IEC-6 cells.
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B signal
transduction pathway
TNF- activation of the JNK/stress-activated protein kinase
(SAPK) pathway bifurcates from the NF-B pathway at the level of
TRAF2 (10). We therefore determined whether the activation of JNK-1 by
IL-17 in IEC-6 cells is linked to the TRAF-NIK signal transduction
pathway. As demonstrated in Fig. 10,
A and B, expression of dominant
TRAF6289–522 (Fig. 10, lanes 6–10) inhibited
the IL-17-mediated activation of JNK-1 after 15 min (Fig. 10, lane 9) by 52.6% compared with that of mock-transfected
IEC-6 cells (Fig. 10, lane 19). In contrast, the expression
of either TRAF287–501 (Fig. 10, lanes 1–5) or
kinase-deficient NIK (NIKKK429–430AA; Fig. 10, lanes 11–15) did not alter the IL-17-induced activation of
JNK1 in IEC-6 cells. These experiments demonstrate that JNK-1
activation by IL-17 may require the presence of functional TRAF6 but is
independent of NIK.
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The activation of MAP kinases has been linked to the
regulation of proliferation in different cell populations (21). We
therefore determined whether human IL-17 is able to regulate the
proliferation of the nontransformed rat intestinal epithelial cell line
IEC-6 (Fig. 11). As shown in Fig. 11, the proliferation of IEC-6 cells was significantly decreased up to 47%
by human IL-17 in a dose-dependent manner in the absence of serum.
IL-17 did not significantly alter the proliferation rate of IEC-6 cells
when cultured continuously in the presence of 5% FCS (Fig. 9).
Therefore, growth factors contained in the serum may be able to
compensate for the antiproliferative effect of IL-17 in synchronized
IEC-6 cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and subsequent chemokine
expression in intestinal epithelial cell line IEC-6. NF-B is a
ubiquitously expressed transcription factor with particular importance
in immune and inflammatory responses (22). The DNA-binding NF-B
family members share a Rel homology domain that is responsible for DNA
binding, nuclear localization, and protein dimerization. DNA-binding
members of NF-B/Rel include p65 (RelA), p50 (NF-B1), c-Rel, p52
(NF-B2), and RelB (I-Rel). These different forms of NF-B probably
activate different sets of target genes as demonstrated by the
different phenotypes of knockout mice for the different NF-B
subunits (23). In unstimulated cells, NF-B is bound in the cytoplasm
to the family of IB proteins, which prevents NF-B from entering
the nuclei. Upon stimulation, IB proteins are phosphorylated by
IB kinases and rapidly degraded (24, 25), followed by the
translocation of NF-B into the nucleus. Although it had been
demonstrated that IL-17 induced NF-B consensus sequence binding
complexes in fibroblasts, the induced NF-B proteins had not been
identified (5). IL-17 induced NF-B heterodimers were supershifted by
Abs directed against p65 or p50, whereas the NF-B consensus binding
activity induced by IL-1ß and TNF- in IEC-6 cells may encompass
additional NF-B subunits.
IL-17 stimulated the expression of the rat CXC chemokine CINC and the
rat CC chemokine MCP-1 in IEC-6 cells. CINC may be a functional
homologue of IL-8 in the rat system, in which IL-8 has not been
identified (26, 27, 28). The sequence of CINC is more closely related to
human gro gene (melanoma growth-stimulating activity)
products than to human IL-8. The gro gene-derived chemokines
GRO-, GRO-ß, and GRO- all have chemotactic activities for human
neutrophils (29). Rat MCP-1 is the functional homologue of human MCP-1
(18). IL-17 induced the expression of CINC as well as MCP-1 mRNA
expression in IEC-6 cells.
CINC mRNA expression was rapidly up-regulated within 30 min by IL-17, whereas MCP-1 mRNA expression followed 1 h later. A similar early induction of IL-8 and a delayed up-regulation of MCP-1 mRNA have been observed in THP-1 cells after stimulation with LPS or TPA (30).
Intestinal epithelial cells have been shown to express the chemokines
IL-8, MCP-1, and epithelial cell-derived neutrophil activator-78 in
inflammatory bowel disease (31, 32, 33, 34). In addition, intestinal epithelial
cells express elevated chemokine mRNA levels during the development of
colitis in IL-2-deficient mice (35). Furthermore, a number of
tumor-derived human intestinal epithelial cell lines respond to
bacterial stimuli or immune regulatory cytokines with NF-B
activation and subsequent chemokine expression (36, 37, 38, 39).
NF-B-regulated gene transcription by IL-17 has been demonstrated in
conjunction with TNF- and IFN- (4). We therefore determined
whether IL-17 is able to synergize with IL-1ß in the induction of
chemokine promoter activation in IEC-6 cells. Regulation of the CINC
promoter has been shown to be NF-B dependent (40). IL-17 activated
the CINC promoter, confirming that the up-regulation of CINC mRNA
expression by IL-17 in IEC-6 cells is due to the induction of gene
transcription. IL-17-induced CINC promoter activity did not reach the
levels seen after stimulation with IL-1ß in IEC-6 cells. However, low
concentrations of IL-17 were able to induce CINC promoter activity in
IEC-6 cells when costimulated with a concentration of IL-1ß that by
itself did not induce promoter activity. These data suggest that IL-17
might be able to augment IL-1ß-induced cellular responses in
intestinal epithelial cells.
Subsequently, we determined whether the costimulatory effect of IL-17
and IL-1ß was due to activation of different or a shared pathway.
IL-1ß and TNF- are among the best characterized inducers of
NF-B activation (41, 42). Although IL-1ß and TNF- initiate
signaling cascades leading to NF-B activation via distinct families
of cell surface receptors (43), both pathways use members of the TRAF
family of adapter proteins as signal transducers (8, 9, 44).
TNF--induced NF-B activation requires TRAF2 (8). In contrast,
TRAF6 participates in NF-B activation by IL-1ß (9). Our data
demonstrate that IL-17-mediated CINC promoter activation is TRAF6
dependent but not TRAF2 dependent. Inhibition of the IL-17-induced CINC
promoter activation by dominant-negative NIK correlated with the
reduced nuclear localization of the NF-B subunit p65. Although
dominant-negative TRAF6 did not completely abrogate IL-17-induced CINC
promoter activity and nuclear translocation of p65, these data suggest
that the IL-17R uses signaling mediators involved in the IL-1R
signaling cascade. Furthermore, IL-17-mediated activation of the CINC
promoter was dependent on NIK and IKK- events downstream of TRAF6.
In IEC-6 cells, dominant-negative mutants of NIK and IKK- both
inhibited CINC promoter activation by IL-17 as well as the activation
induced by IL-1ß and TNF-. Downstream of TRAF family members,
IL-1ß and TNF- NF-B activating signaling pathways have been
shown to converge at the MAP3 kinase-related serine threonine kinase
NIK (10, 42, 44). NIK was identified as a TRAF2-interacting protein
(45), but was subsequently shown to associate with additional members
of the TRAF family, including TRAF6 (10). NIK itself does not appear to
be an IB-kinase (11), but recently IKK- has been identified as
IB kinase that associates directly with and is activated by NIK
(11, 46). Our data suggest that IL-17-mediated induction of chemokine
transcription also uses NIK as a signal transducer in the activation of
NF-B through IKK-.
IL-17-induced signal transduction in IEC-6 cells resulted in the
regulation of three different families of MAP kinases. Most recently it
has become clear that the regulation of MAP kinases may be linked to
the NF-B activation pathways. Transient expression of TRAF6 in 293
cells has been shown to stimulate ERK MAP-kinase (29). Furthermore,
TRAF proteins have been demonstrated to be at the bifurcation of JNK
and NF-B activation by TNF- (10). In addition, p38 MAP kinase can
be activated by IL-1 (20). Recently it was demonstrated that the
activation of the IL-6 promoter NF-B site by TNF- may depend on
coactivation of ERK and p38 MAP kinases (47). Therefore, activation of
ERK, JNK/SAPK, and p38 MAP kinases by IL-17 in IEC-6 cells may follow
shared signaling events linked to the NF-B activation pathway.
In our studies IL-17 stimulation resulted in initial down-regulation of
ERK-MAP kinase activity followed by strong up-regulation, suggesting
overlapping of two different activation pathways. However, IL-17 is
able to induce ELK-1 activation. Therefore, the up-regulation of ERK
activity by IL-17 cells may be a functional important signal for
transcriptional activation. Furthermore, the IL-17-induced ELK-1
activation was Ras dependent, indicating the induction of a specific
signal transduction pathway. By contrast, the IL-17-mediated induction
of the JNK/SAPK pathway in IEC-6 cells depends on the presence of
functional TRAF6 and is therefore linked to the NF-B-activating
signal transduction. Since c-Jun forms the AP-1 transcription factor as
a homodimer or heterodimer with c-Fos, the activation of c-Jun by IL-17
may contribute to the activation of chemokine promoters (48).
IL-17 inhibited the proliferation of synchronized IEC-6 cells. However, the presence of serum could compensate for this inhibitory effect. Mouse IL-17 has been shown to stimulate the proliferation of mouse T cells in the presence of costimuli (3), but no proliferative response was observed after stimulation with human IL-17 in human T or B cells (4). Therefore, IL-17 may be able to regulate cell proliferation in a cell type-specific manner.
The ability of IL-17 to induce NF-B activity with subsequent
up-regulation of chemokine promoter activity and mRNA expression in
intestinal epithelial cells and the ability of IL-17 to act in
synergistic fashion with IL-1ß suggest that IL-17 may have a
proinflammatory role in intestinal immune responses. The signal
transduction pathways of IL-17 and IL-1 converge in the activation of
TRAF6 and use NIK and IKK- in the activation of the
NF-B-dependent CINC promoter. Our data support the concept that NIK
is a central upstream mediator of NF-B activation by extracellular
signals.
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Footnotes |
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2 M.A. and P.G.A. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Hans-Christian Reinecker, Gastrointestinal Unit, Jackson Building R711, Massachusetts General Hospital, 32 Fruit St., Boston, MA. E-mail address:
4 Abbreviations used in this paper: ERK, extracellular regulated kinase; JNK, c-Jun NH2-terminal kinase; TRAF, TNF receptor-associated factor; NIK, NF-B-inducing kinase; IKK, IB kinase; SAPK, stress-activated protein kinase; CHUCK, conserved helix loop helix ubiquitous kinase; MCP-1, monocyte chemoattractant protein-1.
Received for publication August 24, 1998. Accepted for publication February 8, 1999.
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