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The Journal of Immunology, 1999, 162: 5337-5344.
Copyright © 1999 by The American Association of Immunologists

NF-{kappa}B-Inducing Kinase Is a Common Mediator of IL-17-, TNF-{alpha}-, and IL-1ß-Induced Chemokine Promoter Activation in Intestinal Epithelial Cells1

Masaaki Awane2, Pietro G. Andres2, Dan Jun Li and Hans-Christian Reinecker3

Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-{kappa}B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-{kappa}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-{kappa}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-{kappa}B-inducing kinase and I{kappa}B kinase-{alpha}. In addition to activation of the NF-{kappa}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-{kappa}B-inducing kinase serves as the common mediator in the NF-{kappa}B signaling cascades triggered by IL-17, TNF-{alpha}, and IL-1ß in intestinal epithelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IL-17 was originally identified as a rodent cDNA sequence termed CTLA-8 (cytotoxic T lymphocyte-associated Ag-8), from a T cell hybridoma that was derived from the fusion of a mouse cytotoxic T cell clone and a rat T cell lymphoma (1). The predicted amino acid sequence of CTLA-8 was 57% identical with that of herpesvirus saimiri gene 13 (2). Subsequently, a human IL-17 homologue was cloned (3). Expression of IL-17 was restricted to T lymphocytes activated with mAbs against CD3 and CD28 after addition of ionomycin (3) or PMA and ionomycin (4). Efforts to identify the receptor for CTLA-8 and HVS 13 resulted in the cloning of a unique single type I transmembrane receptor from a murine thymoma cell line with an open reading frame encoding 864 amino acids (5). The mouse and human IL-17R have no apparent homology with the catalytic domains of other growth factor receptors (5, 6). Northern blot analysis of mouse IL-17R mRNA expression demonstrated the expression of a single transcript of 3.7 kb in a wide variety of tissues and cell lines, including the nontransformed rat intestinal epithelial cell line IEC-6 (5).

IL-17 has been shown to induce NF-{kappa}B consensus sequence binding in mouse fibroblasts (5) and human macrophages (7). Subsequently, the expression of several cytokines known to contain NF-{kappa}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-{alpha} and IFN-{gamma} had an additive effect on the stimulation of IL-6 expression by IL-17 (4), and the combination of IL-17 and TNF-{alpha} 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-{kappa}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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and reagents

Human recombinant cytokines (IL-17, IL-1ß, and TNF-{alpha}) 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 ([{alpha}-32P]dCTP and [{gamma}-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-{kappa}B-inducing kinase (NIKKK429–430AA), and I{kappa}B kinase-{alpha} (IKK-{alpha}) 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-{kappa}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-{alpha}-mediated, NF-{kappa}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-{alpha} 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 [{alpha}-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-{alpha} (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 [{gamma}-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 [{gamma}-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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-17 activates the NF-{kappa}B subunits p65 and p50 in intestinal epithelial cells

IEC-6 cells were incubated with recombinant IL-17, IL-1ß, or TNF-{alpha} for 30 min, and EMSAs were performed to determine whether IL-17 activates NF-{kappa}B consensus sequence binding in intestinal epithelial cells. As demonstrated in Fig. 1Go, IL-17 up-regulated the binding of proteins to the NF-{kappa}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-{kappa}B oligonucleotides containing a wild-type, but not a mutant, NF–{kappa}B binding sequence (Fig. 1Go). However, the amount of NF-{kappa}B binding induced by IL-17 was lower than that induced by IL-1ß or TNF-{alpha} (Fig. 1Go).



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  		FIGURE 1. IL-17 induces the binding of p65/p50 heterodimers to the NF-{kappa}B consensus sequence in IEC-6 cells. EMSA using labeled double-stranded oligonucleotide encoding the NF-{kappa}B binding consensus sequence (wt*), cold competitor (wt), and mutant competitors (mut). Supershifts were conducted by a 30-min preincubation of nuclear extract at room temperature with polyclonal Abs recognizing p65 ({alpha}-p65) and p50 ({alpha}-p50).

 
To determine which NF-{kappa}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-{kappa}B subunits. The NF-{kappa}B binding complexes initiated by the stimulation of IL-17 in intestinal epithelial cells were completely supershifted with Abs recognizing either p65 (Fig. 1Go, lane 5) or p50 (Fig. 1Go, lane 6). In contrast, NF-{kappa}B binding complexes formed in response to IL-1ß (Fig. 1Go, lane 9) and TNF-{alpha} (Fig. 1Go, lane 12) were not completely shifted by anti-p50 Abs, suggesting the involvement of additional NF-{kappa}B subunits. These findings demonstrate that the NF-{kappa}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-{kappa}B leads to a coordinated increase in the expression of many genes whose products mediate and regulate inflammatory and immune responses. NF-{kappa}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-{kappa}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-{alpha} (17) as well as transcripts encoding for the rat CC chemokine MCP-1 (18) in a dose- and time-dependent (Fig. 2GoB) fashion (Fig. 2Go, 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. 2GoA), while induction of MCP-1 mRNA by IL-17 was observed 1 h after stimulation (Fig. 2GoB).



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  		FIGURE 2. IL-17 stimulates CXC and CC chemokine mRNA expression in IEC-6 cells. Northern blot analysis of CINC mRNA expression in mRNA from IEC-6 cells (2 µg of poly(A)+ RNA/lane) demonstrates a concentration-dependent (A) and time-dependent (B) regulation of CINC and MCP-1 mRNA expression by IL-17. IEC-6 cells were stimulated with the indicated amount of IL-17 (A) or with 100 ng/ml of IL-17 for the indicated time periods (B).

 
IL-17 and IL-1ß synergistically induce CINC promoter activity in IEC-6 cells

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. 3Go, 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. 3Go). 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-{alpha}.



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  		FIGURE 3. IL-17 synergizes with IL-1ß in the activation of the CINC promoter. IEC-6 cells were transiently transfected with a wild-type CINC promoter luciferase reporter construct together with a ß-galactosidase reporter to access transfection efficiency and 24 h later were stimulated for 24 h with IL-17 and IL-1ß, individually or in combination, at the indicated concentrations. Similar results were obtained in three independent experiments (*, p < 0.001).

 
CINC promoter activation by IL-17 in IEC-6 cells is TRAF6 dependent

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-{alpha} 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. 4Go the expression of dominant-negative TRAF6289–522 significantly reduced the induction of IL-17 and IL-1ß, but not TNF-{alpha}-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-{alpha} (Fig. 4Go), 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. 4Go).



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  		FIGURE 4. Selective inhibition of IL-17-induced CINC promoter activity by TRAF6289–522 in IEC-6 cells. IEC-6 cells were transiently transfected with a control vector, dominant-negative TRAF287–501, or dominant-negative TRAF6289–522 together with the CINC promoter luciferase reporter construct and the ß-galactosidase reporter. After 24 h transfected IEC-6 cells were stimulated for 24 h with 100 ng/ml TNF-{alpha}, 10 ng/ml IL-1ß, or 100 ng/ml IL-17, and luciferase activity was determined and normalized for transfection efficiency. One representative experiment of three conducted is shown (¶, p < 0.01; #, p < 0.001; *, p < 0.005).

 
IL-17-induced CINC promoter activation is dependent on the NF-{kappa}B-inducing kinase NIK and the I{kappa}B{alpha} kinase IKK-{alpha}

IL-1 as well as TNF-{alpha} signaling pathways converge at NIK and use IKK-{alpha}, originally designated CHUK, as the common activator of NF-{kappa}B (9, 10). To assess whether IL-17-induced chemokine promoter activation in intestinal epithelial cells requires functional NIK and IKK-{alpha}, we transfected IEC-6 cells with kinase negative mutants of NIK (NIKKK429–430AA) and IKK-{alpha} (CHUCKK44A). As demonstrated in Fig. 5Go, NIKKK429–430AA significantly decreased the CINC promoter activity induced by IL-17, TNF-{alpha}, and IL-1ß in IEC-6 cells. Furthermore, activation of the CINC promoter in IEC-6 cells was dependent on IKK-{alpha}, as expression of the catalytic inactive CHUCKK44A mutant significantly diminished the CINC promoter activity in response to IL-17, IL-1ß, and TNF-{alpha} (Fig. 5Go). Thus, IL-17-mediated signal transduction shares the utilization of MAP3K NIK and the I{kappa}B{alpha} kinase IKK-{alpha} with IL-1ß and TNF-{alpha} in activation of the CINC promoter in IEC-6 cells.



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  		FIGURE 5. CINC promoter activation by IL-17 is dependent on NIK and IKK-{alpha} in IEC-6 cells. IEC-6 cells were transiently transfected with a control vector, kinase-negative NIK (NIKKK429–430AA), or kinase-negative IKK-{alpha} (CHUCKK44A) together with the CINC promoter luciferase reporter construct and the ß-galactosidase reporter. Twenty-four hours later IEC-6 cells were stimulated with 100 ng/ml TNF-{alpha}, 10 ng/ml IL-1ß, or 100 ng/ml IL-17, and luciferase activity was determined. All experiments were normalized for transfection efficiency. One representative experiment of three conducted is shown (¶, p < 0.001; #, p < 0.001; *, p < 0.01).

 
IL-17-induced nuclear translocation of NF-{kappa}B subunit p65 is NIK dependent

As demonstrated in Fig. 6Go, the inhibition of IL-17-mediated CINC promoter activation by the expression of kinase-negative NIKKK429–430AA mutant correlates with the inhibition of NF-{kappa}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-{kappa}B subunit p65 was determined in nuclear extracts without or after stimulation for 30 min with 100 ng/ml TNF-{alpha}, 10 ng/ml IL-1ß, or 100 ng/ml IL-17 by Western blot analysis (Fig. 6GoA). Densitometric quantification revealed that the expression of the kinase-negative NIK mutant reduced the nuclear translocation of p65 protein induced by TNF-{alpha}, IL-1ß, and IL-17 by 39.4, 38.4, and 45.4%, respectively (Fig. 6GoB). These experiments suggest NIK as a common mediator in IL-17 as well as IL-1ß and TNF-{alpha} signal transduction pathways leading to NF-{kappa}B activation in IEC-6 cells.



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  		FIGURE 6. Nuclear translocation of p65 by IL-17 is dependent on functional NIK in IEC-6 cells. A, IEC-6 cells were transfected with empty mock control vector (lanes 1–4) or with dominant-negative NIK (NIKKK429–430AA; lanes 5 and 6), and the expression of the NF-{kappa}B subunit p65 was determined by Western blot analysis in nuclear extracts without (lanes 1 and 5) or after stimulation for 30 min with 100 ng/ml TNF-{alpha} (lanes 2 and 6), 10 ng/ml IL-1ß (lanes 3 and 7), or 100 ng/ml IL-17 (lanes 4 and 8). The p65 protein concentrations in A were quantified with an densitometer (Molecular Dynamics, Sunnyvale, CA) and NIH Image 1.61 (National Institutes of Health, Bethesda, MD) and were expressed as the median density per area (B).

 
IL-17 induces MAPK activation in IEC-6 cells

Signal transduction events induced by TNF-{alpha} 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. 7GoA). 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. 7GoA). 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. 7GoB). IL-17 activated the JNK-mediated phosphorylation of c-Jun in intestinal epithelial cells (Fig. 7GoC). 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. 7GoC). 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. 8Go, 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. 8Go).



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  		FIGURE 7. IL-17 regulates ERK and JNK MAP kinase activities in intestinal epithelial cells. IEC-6 cells were stimulated with 100 ng/ml IL-17 for the indicated period of time. ERK1 (A), ERK2 (B), or JNK-1 (C) was immunoprecipitated with specific Abs, and activation of MAP kinases was determined by in vitro kinase assays as described in Materials and Methods.

 


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  		FIGURE 8. IL-17 induces phosphorylation of p38 in IEC-6 cells. IEC-6 cells were stimulated with 100 ng/ml IL-17 or 10 ng/ml IL-1ß for the indicated period of time. The p38 was immunoprecipitated with specific Abs and resolved on a 4–12% SDS-polyacrylamide gel, and activation of p38 was assayed by Western blotting with an Ab specifically recognizing p38 phosphorylated at Tyr182 (P-p38). To confirm equal loading, the blot was stripped and reprobed with Abs directed against p38 (p38).

 
IL-17 stimulates Ras-dependent ELK-1 trans-activation in IEC-6 cells

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. 9Go, 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. 9Go). This inhibition was specific, as the dominant negative RasN17 did not alter the baseline activity of the reporter construct in IEC-6 cells (Fig. 9Go). These results are consistent with an IL-17-dependent ERK MAP kinase activation in IEC-6 cells.



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  		FIGURE 9. IL-17 stimulates Ras-dependent trans-activation of ELK-1 in IEC-6 cells. IEC-6 cells were transiently transfected with 1 µg/ml of GAL4-Elk-1 and 5xGAL-luc constructs with either dominant-negative Ras (dn ras) construct or empty vector (Mock). After 24 h transfected IEC-6 cells were stimulated with or without 100 ng/ml IL-17 or 10 ng/ml IL-1ß, and luciferase activity was determined after 24 h and normalized for transfection efficiency. One representative experiment of four conducted is shown (*, p < 0.001; #, p < 0.001).

 
IL-17-induced activation of JNK-1 is linked to the NF-{kappa}B signal transduction pathway

TNF-{alpha} activation of the JNK/stress-activated protein kinase (SAPK) pathway bifurcates from the NF-{kappa}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. 10Go, A and B, expression of dominant TRAF6289–522 (Fig. 10Go, lanes 6–10) inhibited the IL-17-mediated activation of JNK-1 after 15 min (Fig. 10Go, lane 9) by 52.6% compared with that of mock-transfected IEC-6 cells (Fig. 10Go, lane 19). In contrast, the expression of either TRAF287–501 (Fig. 10Go, lanes 1–5) or kinase-deficient NIK (NIKKK429–430AA; Fig. 10Go, 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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  		FIGURE 10. JNK-1 activation by IL-17 in IEC-6 cells is inhibited by dominant-negative TRAF6 and is NIK independent. In A, IEC-6 cells were transiently transfected to express Flag epitope-tagged dominant-negative TRAF287–501 (dn TRAF2; lanes 1–5, filled bars) or dominant-negative TRAF6289–522 (dn TRAF6; lanes 6–10, open bars), kinase-deficient NIKKK429–430AA (dn NIK; lanes 11–15, hatched bars), and empty control vector (Mock; lanes 16–20, cross-hatched bars) and stimulated with 100 ng/ml IL-17 for the indicated period of time. JNK1 was immunoprecipitated with anti-JNK-1 Abs conjugated to agarose (Santa Cruz Biotechnology), and in vitro kinase assays were conducted with c-Jun as the substrate as described in Materials and Methods. B, The densitometric quantification of the IL-17-induced JNK-1-dependent phosphorylation of c-Jun in A.

 
IL-17 inhibits the proliferation of synchronized IEC-6 cells

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. 11Go). As shown in Fig. 11Go, 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. 9Go). 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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  		FIGURE 11. IL-17 inhibits the proliferation of synchronized IEC-6 cells. IEC-6 cells were grown in 96-well plates at a density of 5000 cells/well and were cultured with the indicated amount of rIL-17 for 48 h in the presence (open bars) or the absence of 5% FCS (filled bars). Triplicate samples for each concentration were measured by MTS assay as described in Materials and Methods (*, p < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-17 is a potent inducer of NF-{kappa}B and subsequent chemokine expression in intestinal epithelial cell line IEC-6. NF-{kappa}B is a ubiquitously expressed transcription factor with particular importance in immune and inflammatory responses (22). The DNA-binding NF-{kappa}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-{kappa}B1), c-Rel, p52 (NF-{kappa}B2), and RelB (I-Rel). These different forms of NF-{kappa}B probably activate different sets of target genes as demonstrated by the different phenotypes of knockout mice for the different NF-{kappa}B subunits (23). In unstimulated cells, NF-{kappa}B is bound in the cytoplasm to the family of I{kappa}B proteins, which prevents NF-{kappa}B from entering the nuclei. Upon stimulation, I{kappa}B proteins are phosphorylated by I{kappa}B kinases and rapidly degraded (24, 25), followed by the translocation of NF-{kappa}B into the nucleus. Although it had been demonstrated that IL-17 induced NF-{kappa}B consensus sequence binding complexes in fibroblasts, the induced NF-{kappa}B proteins had not been identified (5). IL-17 induced NF-{kappa}B heterodimers were supershifted by Abs directed against p65 or p50, whereas the NF-{kappa}B consensus binding activity induced by IL-1ß and TNF-{alpha} in IEC-6 cells may encompass additional NF-{kappa}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-{alpha}, GRO-ß, and GRO-{gamma} 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-{kappa}B activation and subsequent chemokine expression (36, 37, 38, 39).

NF-{kappa}B-regulated gene transcription by IL-17 has been demonstrated in conjunction with TNF-{alpha} and IFN-{gamma} (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-{kappa}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-{alpha} are among the best characterized inducers of NF-{kappa}B activation (41, 42). Although IL-1ß and TNF-{alpha} initiate signaling cascades leading to NF-{kappa}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-{alpha}-induced NF-{kappa}B activation requires TRAF2 (8). In contrast, TRAF6 participates in NF-{kappa}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-{kappa}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-{alpha} events downstream of TRAF6. In IEC-6 cells, dominant-negative mutants of NIK and IKK-{alpha} both inhibited CINC promoter activation by IL-17 as well as the activation induced by IL-1ß and TNF-{alpha}. Downstream of TRAF family members, IL-1ß and TNF-{alpha} NF-{kappa}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 I{kappa}B{alpha}-kinase (11), but recently IKK-{alpha} has been identified as I{kappa}B{alpha} 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-{kappa}B through IKK-{alpha}.

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-{kappa}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-{kappa}B activation by TNF-{alpha} (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-{kappa}B site by TNF-{alpha} 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-{kappa}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-{kappa}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-{kappa}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-{alpha} in the activation of the NF-{kappa}B-dependent CINC promoter. Our data support the concept that NIK is a central upstream mediator of NF-{kappa}B activation by extracellular signals.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant DK51003 and a research grant from the Crohn’s and Colitis Foundation of America (to H.-C.R.). This work was presented in part at the annual meeting of the American Gastroenterology Association in Washington, 1997 and 1998. Back

2 M.A. and P.G.A. contributed equally to this work. Back

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: Back

4 Abbreviations used in this paper: ERK, extracellular regulated kinase; JNK, c-Jun NH2-terminal kinase; TRAF, TNF receptor-associated factor; NIK, NF-{kappa}B-inducing kinase; IKK, I{kappa}B kinase; SAPK, stress-activated protein kinase; CHUCK, conserved helix loop helix ubiquitous kinase; MCP-1, monocyte chemoattractant protein-1. Back

Received for publication August 24, 1998. Accepted for publication February 8, 1999.


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