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TNF Receptor-Associated Factor-2 Is Involved in Both IL-1ß and TNF- Signaling Cascades Leading to NF-B Activation and IL-8 Expression in Human Intestinal Epithelial Cells¹

Christian Jobin^2,*,, Lisa Holt^*, Cynthia A. Bradham^*, Konrad Streetz^*, David A. Brenner^*, and R. Balfour Sartor^*,

^* Departments of Medicine, Microbiology, and Immunology and Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, NC 27599

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytokine signaling involves the participation of many adaptor proteins, including the docking protein TNF receptor-associated factor-2 (TRAF-2), which is believed to transmit the TNF- signal through both the IB/NF-B and c-Jun N-terminal kinase (JNK)/stress-related protein kinase (SAPK) pathways. The physiological role of TRAF proteins in cytokine signaling in intestinal epithelial cells (IEC) is unknown. We characterized the effect of a dominant-negative TRAF-2 delivered by an adenoviral vector (Ad5dnTRAF-2) on the cytokine signaling cascade in several IEC and also investigated whether inhibiting the TRAF-2-transmitting signal blocked TNF--induced NF-B and IL-8 gene expression. A high efficacy and level of Ad5dnTRAF-2 gene transfer were obtained in IEC using a multiplicity of infection of 50. Ad5dnTRAF-2 expression prevented TNF--induced, but not IL-1ß-induced, IB degradation and NF-B activation in NIH-3T3 and IEC-6 cells. TNF--induced JNK activation was also inhibited in Ad5dnTRAF-2-infected HT-29 cells. Induction of IL-8 gene expression by TNF- was partially inhibited in Ad5dnTRAF-2-transfected HT-29, but not in control Ad5LacZ-infected, cells. Surprisingly, IL-1ß-mediated IL-8 gene expression was also inhibited in HT-29 cells as measured by Northern blot and ELISA. We concluded that TRAF-2 is partially involved in TNF--mediated signaling through IB/NF-B in IEC. In addition, our data suggest that TRAF-2 is involved in IL-1ß signaling in HT-29 cells. Manipulation of cytokine signaling pathways represents a new approach for inhibiting proinflammatory gene expression in IEC.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intestinal epithelial cells (IEC)³ form a single layer that isolates the host from the toxic gut luminal environment. Aside from their classical absorptive and physical barrier roles, IEC are now viewed as immunological sentinels of the gut (1). IEC respond to a wide array of agents commonly found in the normal gut, including bacterial products and invasive bacteria, by secreting chemokines and expressing adhesion molecules that promote the influx of inflammatory leukocytes. In addition, IEC respond to inflammatory stimuli such as cytokines released by activated lamina propria cells (1, 2). However, the mechanisms by which IEC regulate coordinated expression and suppression of proinflammatory genes are incompletely understood. Moreover, the intracellular signaling pathways associated with IEC proinflammatory gene expression are virtually unknown. Most of the molecules involved in immune and inflammatory responses are controlled at the transcriptional level by a coordinated group of transcription factors. We have shown that the IB/NF-B system is critical in mediating cytokine-induced ICAM-1, IL-1ß, IL-8, inducible nitric oxide synthase, and COX-2 gene expression in IEC (2, 3, 4, 5). NF-B is an inducible transcription factor comprised of dimers of c-Rel, RelA, RelB, p50, or p52 (synthesized as p105 and p100 precursors, respectively) (6, 7, 8). The NF-B prototype in IEC is composed of RelA (p65) and NF-B1 (p50) heterodimer subunits (2, 4) and is the most potent gene trans-activator in the NF-B family (9, 10).

NF-B activation is efficiently regulated by its endogenous cytoplasmic inhibitor, IB, which sequesters NF-B silently in the cytoplasm. Numerous stimuli, including IL-1ß and TNF- (11, 12), ubiquinate a kinase that phosphorylates IB at serine residues 32 and 36 on the N-terminus of the molecule (13). Phosphorylated IB is then selectively ubiquinated and rapidly degraded via a nonlysosomal, ATP-dependent, 26S proteolytic complex composed of a 700-kDa proteasome (14, 15).

The mechanisms by which cytokines transduce their signals through the IB/NF-B system have only recently been described. The cloning and characterization of the kinase responsible for inducible IB phosphorylation (16, 17) and recent findings in cytokine signaling have provided a better understanding of cytokine-mediated NF-B activation. Following TNF- stimulation, TNF receptor-associated factor-2 (TRAF-2) is recruited to the cytoplasmic portion of the TNF-R1 via the intermediate action of TNF receptor 1-associated death domain (18). In contrast, IL-1ß signals through the action of IL-1R-associated kinase (IRAK), which associates with IL-1R1 and activates TRAF-6 (19, 20). Activated TRAF-2 and TRAF-6 are then able to associate/activate the NF-B-inducing kinase (NIK) (21), a kinase dedicated to the NF-B pathway. NIK, in turn, associates/activates the IB kinase (IKK) complex (22, 23), which is composed of the IKK-1 and IKK-2 subunits, both critical in mediating cytokine-induced IB phosphorylation (24, 25, 26). Activation of the IKK complex leads to specific IB phosphorylation/degradation and subsequent release of NF-B, which migrates to the nucleus and activates transcription of B-specific genes. TRAF-2 has been shown to play an important role in transducing the TNF- signal through the IB/NF-B axis (27, 28, 29, 30, 31, 32). However, most of these data were obtained by reporter gene assay systems using transient overexpression of TRAF-2 in Jurkat T cells, so the specificity of the endogenous TRAF-2 pathway has not been explored.

We constructed an adenoviral vector bearing a dominant negative form of the adaptor protein TRAF-2 (Ad5dnTRAF-2) to study endogenous TNF- signaling and gene expression in transformed IEC cell lines. We report an efficient transduction of dnTRAF2 into transformed IEC and demonstrate that the dnTRAF-2 molecule partially blocked TNF--induced IB degradation and NF-B activation in several cell lines. IL-8 gene expression induced by both TNF- and IL-1ß was also inhibited by dnTRAF-2 in HT-29 cells, suggesting that TRAF2 may also be involved in IL-1ß signaling in this IEC line, whose pattern of IB phosphorylation and degradation most closely resembles that of native colonic epithelial cells. Our data indicate that TRAF2 plays a key role in TNF- signaling in IEC and also suggest that signaling through this adaptor protein is not uniquely linked to TNF- signal transduction in these cells.

	Materials and Methods

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Cell culture

Transformed human HT-29 colonic epithelial cells (American Type Culture Collection HTB 38, Manassas, VA) were used between passages 10 and 25, and Caco-2 epithelial cells (American Type Culture Collection HTB 37) were used between passages 29 and 40. HT-29 cells were grown in DMEM with high glucose (Life Technologies, Grand Island, NY). The rat nontransformed intestinal epithelial cell line IEC-6 (American Type Culture Collection CRL 1592) was used between passages 3 and 15 and was grown in high glucose DMEM (Life Technologies) with 5% heat-inactivated FBS (Life Technologies), 2 mM L-glutamine, 1 U/ml insulin, and antibiotics (Pen/Strep/fungizone, 1 x, Life Technologies). Mouse embryo NIH-3T3 (American Type Culture Collection CRL 1658) were grown in DMEM with high glucose (Life Technologies) in the presence of 5% heat-inactivated newborn calf serum (Life Technologies). Cells were cultured in a water-saturated atmosphere of 95% air and 5% CO₂ at 37°C.

Ad5dnTRAF2 construction

The recombinant replicative-deficient adenovirus was constructed by the method of Graham et al. (33) as previously described (3). The dominant negative TRAF-2 consisted of a truncated variant of TRAF-2 in which nucleotides 87–501 corresponding to the N-terminal region of the ring finger domain were deleted (27). This deletion prevents interaction between downstream interacting effecter molecules (27). The Ad5dnTRAF2 gene contained extra 24-bp DNA nucleotides coding for the FLAG peptide (DYLDDDDL). Ad5LacZ, which contains the Escherichia coli ß-galactosidase gene (3), was grown and purified as described above and was used as a control virus throughout the study.

IEC infection

IEC cell lines were cultured to postconfluence, after which they were infected with Ad5dnTRAF2 or Ad5LacZ in serum-free medium (Opti-MEM, Life Technologies) at different multiplicities of infection (moi; 0, 1/10, 1/50, and 1/100 IEC/viral particles) for 12 h. The adenovirus was then washed off, fresh medium-containing serum was added to the transfection medium, and cells were treated at various time points with human recombinant IL-1ß (5 ng/ml) or TNF- (10 ng/ml; both from Intergen, Purchase, NY).

RNA extraction and Northern blot analysis

Uninfected, Ad5dnTRAF2-infected, or Ad5LacZ-infected cells were stimulated with IL-1ß (5 ng/ml) or TNF- (10 ng/ml) for 3 h. RNA was isolated using the Trizol method (Life Technologies). Total RNA (10 µg) was electrophoresed on 1.5% denaturing gels as previously described (34). The RNA was blotted onto Hybond-N paper (Amersham, Arlington Heights, IL) overnight followed by UV fixation. The integrity of RNA was checked by methylene blue staining as previously described (34). Membranes were prehybridized for 60 min, then hybridized for 120 min in RapidHyb buffer (Amersham) containing [³²P]dCTP-labeled cDNA probe (1 x 10⁶ cpm/ml) encoding human IL-8. The blot was washed for 30 min in 2x SSPE/0.1% SDS at room temperature followed by 30 min in 0.1x SSPE/0.1% SDS at room temperature, and finally 30 min in 0.1x SSPE/0.1% SDS at 65°C. The blot was exposed as described previously (35).

RT-PCR analysis

RNA was isolated by the Trizol method as described above (Life Technologies) and was reverse transcribed using 1 µg total RNA, 15 U RNA Guard (Pharmacia, Piscataway, NJ), 1x first-strand buffer (Life Technologies), 12.5 mM dNTP (Pharmacia), 125 pmol of random hexamer primers (Pharmacia), and 125 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies) in a final volume of 25 µl. The reaction was conducted for 1 h at 39°C followed by 7 min at 93°C and 1 min at 24°C, and then slowly cooled to 4°C for 20 min. PCR was conducted in a volume of 50 µl containing 2 µl of the RT mixture, 1x Taq buffer (Applied Biosystem, Foster City, CA), 5 pmol of each primer, 2.5 mM dNTPs and 1 U of Thermo aquaticus polymerase (Applied Biosystem). PCR was conducted in a 9600 Perkin-Elmer cycler (Applied Biosystem) set for various cycles (18–30 cycles) to monitor the linearity of the amplification. The PCR temperatures used were 94°C for 45 s (denaturing), 56°C for 45 s (annealing), and 72°C for 2 min (polymerization) followed by an extension of 5 min at 72°C. The PCR products (5 µl) were electrophoresed on a 2% agarose gel containing GelStar fluorescent dye (FMC, Philadelphia, PA). The gel was captured using an AlphaImager 2000 (Alpha Innotech, San Leandro, CA). Negative controls consisted of tubes with no nucleic acid or RNA only. The oligonucleotide TRAF-2 primers used were TRAF-2 5' (5'-CGGACACCAGCTATCTTCTC-3'; position 1161) and 3' (5'-TA GAGTCCTGTTAGGTCCACA-3; position 1552). The length of the amplified products was 391. To confirm the specificity and identity of the amplified product, the DNA was sequenced at the UNC-CH Automated sequencing facility on a model 377 DNA sequencer (Perkin-Elmer, Applied Biosystems Division) using the ABI PRISM dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase, FS (Perkin-Elmer, Applied Biosystems Division).

Western blot analysis

Uninfected or Ad5dnTRAF2- or Ad5LacZ-infected cells were stimulated with IL-1ß (5 ng/ml) or TNF- (10 ng/ml) for 0–60 min. The cells were lysed in 1x Laemmli buffer, and 20 µg of proteins were electrophoresed on 10% SDS-polyacrylamide gels. Anti-FLAG M2 (Eastman Kodak, New Haven, CT) or anti-IB Ab (Santa Cruz Biotechnology, Santa Cruz, CA) were used to detect immunoreactive FLAG-dnTRAF2 or IB, respectively, using the enhanced chemiluminescence light-detecting kit (ECL, Amersham) as described previously (2). For endogenous TRAF-2 protein analysis, the blots were incubated for 45 min with an anti-TRAF-2 Ab (C-20, Santa Cruz Biotechnology) diluted 1/1000, and immunoreactive complexes were detected as described above.

Immunofluorescence study

Uninfected, Ad5dnTRAF2-infected, or Ad5LacZ-infected cells were stimulated for 30 min with TNF- (10 ng/ml) or IL-1ß (5 ng/ml), after which they were fixed with 100% ice-cold methanol. Blocking was performed using 10% nonimmune goat serum (NGS; Sigma, St. Louis, MO) for 30 min. After blocking, rabbit anti-RelA Ab (Rockland, Gilberville, PA; diluted 1/200) or mouse anti-FLAG M2 Ab (Eastman Kodak; diluted 1/1000), both in 10% NGS, was added for 30 min, after which rhodamine isothiocyanate-conjugated goat anti-rabbit IgG Ab for RelA detection (Jackson ImmunoResearch Laboratories, West Grove, PA) or FITC goat anti-mouse IgG for dnTRAF-2, both diluted 1/100 in 10% NGS, was added for 30 min. RelA and FLAG-dnTRAF2 expression were visualized with a fluorescent light microscope.

Transfections

The IL-8-reporter luciferase construct consists of the native IL-8 promoter (36). IL-8-Luc (1 µg) or dnTRAF-2 (3 µg) was transfected into NIH-3T3 cells using lipofectamine reagent (Life Technologies) as described previously (2). The total amount of transfected DNA was adjusted using an empty vector. Transfected cells were incubated overnight at 37°C and 5% CO₂, after which the DNA/lipofectamine medium was replaced with the serum-containing medium, and the cells were incubated for an additional 24 h. Cells were treated with TNF- (10 ng/ml) or IL-1ß (2 ng/ml) or were left untreated for 8 h, after which extracts were prepared using enhanced luciferase assay reagents (Analytical Luminescence, San Diego, CA). Luciferase assays was performed on a Monolight 2010 luminometer (Analytical Luminescence) for 20 s, and results were normalized for the extract protein concentration measured with the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).

Whole cell extracts

Cells were plated (2 x 10⁶ cells) in 100-mm dishes and infected when approximately 80% confluence was obtained. The cells were washed with PBS, grown in under serum-starved conditions (0.5% FBS) for 48 h, and finally treated with TNF- (30 ng/ml) for 30 min. The cells were scraped with a rubber policeman, washed with ice-cold PBS, and then lysed in Dignam C buffer (420 mM NaCl, 1.5 mM MgCl₂, 20 mM HEPES (pH 7.0), 0.2 mM EDTA, 25% glycerol, and 0.5 mM DTT) containing protease and phosphatase inhibitors (0.5 mM PMSF, 0.1 mM p-nitrophenyl phosphate, 0.04 mM ß-glycerophosphate, 0.05 mM Na₃VO₄, 40 mg/ml bestatin, 2 mg/ml aprotinin, 0.54 mg/ml leupeptin, and 0.7 mg/ml pepstatin A). Lysates were rotated at 4°C for 30 min. Cell membranes were then pelleted by cold centrifugation at 14,000 rpm and discarded. The supernatant was aliquoted and stored at -80°C. The protein concentrations of whole cell extracts were determined using the Bio-Rad protein assay.

JNK assay

JNK activity was assessed in HT-29 cells using an in vitro kinase assay as described previously (4). Recombinant GST-c-Jun protein (amino acids 1–79) containing the activation domain of the c-Jun protein was used as substrate. Twenty-five micrograms of whole cell extracts were incubated with 5 µg of substrate protein linked to glutathione-Sepharose beads. After extensive washing of the complexes, the kinase reaction was performed with [-³²P]ATP (4500 Ci/mmol; ICN Biochemicals, Costa Mesa, CA). The proteins were fractionated using 12.5% SDS-PAGE and were visualized/quantitated by phosphorimager analysis. Coomassie staining was used to demonstrate equal protein loading. A mutated GST-c-Jun (Ser⁶³ and Ser⁷³ substitution to Ala;GST-c-JunAA) was not phosphorylated by colonic epithelial cells (37). Kinase assays were performed in duplicate using whole cell extracts from two independent experiments.

Nuclear extracts and electrophoretic mobility shift assay (EMSA)

Ad5dnTRAF-2- and Ad5Luc-infected cells were stimulated for 30 min with TNF- (10 ng/ml) or IL-1ß (5 ng/ml) and lysed, and nuclear protein extracts were prepared as previously described (2). Extracts (5 µg) were incubated with radiolabeled double-stranded class I MHC B sites (GGCT GGGGATTCCCCATCT), separated by nondenaturing electrophoresis, and analyzed by autoradiography as described previously (2). For Ab supershifting analysis, nuclear extracts were preincubated with 1 µl of RelA Ab directed against the C-terminus portion of the molecule (Rockland) or with 1 µl of p50 Ab directed against the NLS portion of the molecule (SC-144X, Santa Cruz Biotechnology) for 15 min at room temperature before addition of the binding buffer and probe.

IL-8 ELISA

A human IL-8 ELISA of cell culture supernatants from noninfected, Ad5LacZ-infected, and Ad5dnTRAF-2-infected IEC was performed in duplicate according to the manufacturer’s specifications (R&D Systems, Minneapolis, MN).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immunofluorescence using an anti-Flag Ab revealed an increased expression of dnTRAF-2 with increasing Ad5dnTRAF-2 moi (Fig. 1A). At an moi of 50, approximately 80% of the cell population expressed the dnTRAF-2 compared with the same cell field viewed under light microscopy (compare panel 5 Fig. 1A to panel 2 in Fig. 1B). Although most HT-29 cells were infected, levels of dnTRAF2 protein expression varied considerably among cells (Fig. 1A, panel 5). Western blot analysis of HT-29 cell extract infected with increased Ad5dnTRAF2 moi revealed a high level of dnTRAF2 expression (Fig. 1C). Interestingly, endogenous wild-type TRAF-2 mRNA expression was very low in HT-29 by RT-PCR compared with the mutant TRAF-2, which was clearly detectable in infected cells in a dose-dependent fashion (Fig. 2A). The endogenous TRAF-2 mRNA was detected only at higher PCR cycles, at which dnTRAF-2 mRNA plateaued (Fig. 2A, lower panel). In addition, endogenous TRAF-2 protein levels were significantly lower than dnTRAF-2 concentrations in infected HT-29 cells (Fig. 2B). Interestingly, endogenous TRAF-2 protein was undetectable in the TNF--unresponsive Caco-2 cells. Note that the dnTRAF-2 protein migrates faster than endogenous TRAF-2 due to deletion in the ring finger domain. Therefore, although the level of dnTRAF-2 varied considerably among infected cells (Fig. 1A), it remained much higher than that of the endogenous wild-type TRAF-2 (Fig. 2). These data indicate that HT-29 cells express much higher levels of dnTRAF-2 than endogenous TRAF-2 following Ad5dnTRAF-2 infection.

View larger version (84K): [in this window] [in a new window]   FIGURE 1. Expression of dnTRAF-2 in HT-29 cells following various Ad5dnTRAF-2 moi (0–100 cells/virus). A, HT-29 cells were infected with different Ad5dnTRAF-2 moi (panels 1–6 represent moi of 0, 1, 10, 25, 50, and 100, respectively), and dnTRAF-2 was visualized 12 h postinfection using an anti-FLAG Ab followed by a rhodamine-conjugated Ab. B, The same cell field as in A viewed under light microscopy. C, HT-29 cells were infected with different Ad5dnTRAF-2 moi, and 12 h postinfection, total protein was extracted, and 20 µg of protein was subjected to SDS-PAGE followed by immunoblotting using an anti-FLAG Ab and an ECL technique as described in Materials and Methods. These results are representative of three different experiments.

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Expression of endogenous and exogenous TRAF-2 in HT-29 cells. A, HT-29 cells were infected with different Ad5dnTRAF-2 moi (0–100 cells/virus), and total RNA was extracted, reverse transcribed, and amplified for the indicated cycle using a specific TRAF-2 primer. PCR product was run on a 2% agarose gel and stained with GelStar. B, Proteins from Caco-2 and uninfected or Ad5dnTRAF-2-infected HT-29 cells (moi of 50) were extracted, and 20 µg was subjected to SDS-PAGE followed by immunoblotting using a C-terminus TRAF-2 Ab as described in Materials and Methods. These results are representative of two different experiments.

View larger version (110K): [in this window] [in a new window]   FIGURE 3. Ad5dnTRAF-2 selectively inhibits TNF--stimulated RelA nuclear migration in IEC-6 cells. A, IEC-6 cells were infected with either Ad5dnTRAF-2 (panels 3 and 5) or Ad5LacZ (panels 2 and 4) or were left untreated (panel 1) for 12 h, after which cells were stimulated with IL-1ß (5 ng/ml), TNF- (10 ng/ml), or medium alone. RelA localization was visualized using an anti-RelA Ab followed by a rhodamine-conjugated Ab as described in Materials and Methods. B, IEC-6 cells were infected with either Ad5dnTRAF-2 or Ad5LacZ or were left untreated for 12 h, after which cells were stimulated with TNF- (10 ng/ml) or medium alone. RelA localization and dnTRAF-2 expression were visualized (x100) using an anti-RelA or anti-FLAG Ab followed by a rhodamine-conjugated or fluorescein Ab, respectively, as described in Materials and Methods. These results are representative of three different experiments.

View larger version (57K): [in this window] [in a new window]   FIGURE 4. Inhibition of NF-B binding activity in TNF-stimulated Ad5dnTRAF-2-infected HT-29 cells. HT-29 cells were infected with either Ad5dnTRAF-2 or Ad5LacZ for 12 h, after which cells were stimulated with TNF- (10 ng/ml). Nuclear extracts were tested for B binding activity by EMSA 30 min after TNF- stimulation. Ab supershifting is indicated by arrows. These results are representative of three different experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 5. dnTRAF-2 inhibits TNF--induced IB degradation in IEC-6 cells and NIH-3T3 fibroblasts. A, IEC-6 cells were infected with Ad5LacZ or Ad5dnTRAF-2 or were left untreated for 12 h, after which cells were stimulated with IL-1ß (5 ng/ml), TNF- (10 ng/ml), or medium alone for 20 min. Total protein was extracted after cytokine stimulation, and 20 µg of protein was subjected to SDS-PAGE followed by IB immunoblotting using the ECL technique as described in Materials and Methods. B, NIH-3T3 cells were infected with Ad5LacZ or Ad5dnTRAF-2 or were left untreated for 12 h, after which cells were stimulated with TNF- (10 ng/ml) or medium alone for 0–60 min. Total protein was extracted after stimulation, and 20 µg of protein was subjected to SDS-PAGE followed by IB immunoblotting using the ECL technique as described above. These results are representative of two different experiments.

View larger version (11K): [in this window] [in a new window]   FIGURE 6. The dnTRAF-2 specifically inhibits TNF- induced transcription of an IL-8-luciferase reporter gene transfected in NIH-3T3 cells. Cells were transfected with 1 µg of IL-8 reporter luciferase plasmid alone or cotransfected with 3 µg of dnTRAF-2 plasmids as described in Materials and Methods. Cells were stimulated with IL-1ß (2 ng/ml), TNF- (10 ng/ml), or medium alone for 8 h, after which extracts were prepared for determination of luciferase activity. Results from one representative experiment performed in duplicate are shown.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Ad5dnTRAF-2 blocks TNF--induced JNK kinase activity in HT-29 cells. HT-29 cells were infected with either Ad5dnTRAF-2 or Ad5LUC for 12 h, after which cells were stimulated with TNF- (30 ng/ml) or medium alone for 30 min. Phosphorylated GST-c-Jun was visualized after protein fractionation using 12.5% SDS-PAGE and was quantitated using phosphorimager analysis. Coomassie staining documented equal protein loading. Shown is a representative result of three independent experiments.

View larger version (33K): [in this window] [in a new window]   FIGURE 8. Ad5dnTRAF-2 inhibits both IL-1ß- and TNF--induced IL-8 expression in HT-29 cells. A, IEC were infected with either Ad5dnTRAF-2 or Ad5LacZ for 12 h, after which cells were stimulated with IL-1ß (5 ng/ml), TNF- (10 ng/ml), or medium alone for 4 h. Total RNA was extracted, and an IL-8 Northern blot was performed as described in Materials and Methods. These results are representative of three different experiments. B, HT-29 cells were treated as described in A and incubated with medium or IL-1ß (5 ng/ml), TNF- (10 ng/ml), or medium alone for 12 h, after which immunoreactive IL-8 concentrations were measured in cell supernatants using an ELISA technique. These results are representative of two different experiments performed in duplicate.

View larger version (53K): [in this window] [in a new window]   FIGURE 9. Inhibition of NF-B binding activity in IL-1ß-stimulated Ad5dnTRAF-2-infected HT-29 cells. HT-29 cells were infected with either Ad5dnTRAF-2 or Ad5LacZ for 12 h, after which cells were stimulated with IL-1ß (5 ng/ml). Nuclear extracts were tested for B binding activity by EMSA 30 min after IL-1ß stimulation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The intermediate steps involved in TNF- signaling through the IB/NF-B system have not been previously investigated in IEC. We used a recombinant adenoviral vector encoding a dnTRAF-2 (Ad5dnTRAF-2) to study the TNF- signaling pathway in IEC. This technology allows the study of endogenous gene expression and therefore is a more accurate measurement of a cell’s physiological response than transient transfection using reporter gene assays, which is the method previously used in most studies of TRAF-2 function in other cell lines (27, 28, 29, 30, 31, 32). Our results indicate that TRAF-2 is an important adaptor protein in the TNF- signaling cascade in IEC. First, the signal leading to activation of the JNK/SAPK pathway was inhibited by expression of dnTRAF-2, as indicated by diminution of JNK kinase activity. Second, the signal resulting in IB degradation and subsequent NF-B nuclear translocation was partially inhibited by the dnTRAF-2 protein. Finally, IL-8, an NF-B-dependent gene, was partially inhibited by the dnTRAF-2 protein. Therefore, we conclude that TRAF-2 plays a role as a proximal transmitter of the TNF- signal through both the JNK and NF-B pathways in IEC, but is not the sole molecule involved in NF-B activation.

There are conflicting data regarding the role of TRAF-2 in TNF-mediated NF-B activation in other cells (39, 40). The data suggesting a role for TRAF-2 in TNF- signaling through the NF-B pathway were mostly generated using a reporter gene assay (27, 28, 29, 30, 31, 32). However, more recent physiological studies contradict these early findings. For example, lymphocytes from dnTRAF-2 transgenic mice displayed a functional NF-B pathway following TNF- stimulation (40), and TNF--stimulated lymphocytes isolated from TRAF-2-deficient mice also showed activation of NF-B (39). These data suggest that TRAF-2 alone is neither sufficient nor required to transduce TNF- signals through NF-B in lymphocytes and that TRAF-2 is an adapter protein dedicated to the JNK/SAPK pathway. Our data partially agree with these findings, in that TRAF-2 may not be the sole adapter protein involved in TNF- signaling since dnTRAF-2 only partially inhibited TNF- stimulation of both the NF-B and JNK/SAPK pathways in IEC. However, at least 50% inhibition of NF-B activation and IL-8 gene expression by dnTRAF-2 indicates that TRAF-2 does have a significant role in the TNF- signaling cascade leading to NF-B activation in IEC in contrast to that in murine lymphocytes. The critical role of TRAF-2 in conducting TNF- signals toward the JNK/SAPK pathway is also confirmed by our study. This may suggest a universal role of TRAF-2 in the JNK/SAPK pathway activation in multiple cell types. The discrepancy in TRAF-2 function between our findings and others (39, 40) may lie in the cell type used in each study. It remains to be seen whether IEC isolated from dnTRAF-2 transgenic mice behave like their lymphocyte counterparts or like the rat and human IEC lines.

Only partial IL-8 gene inhibition by dnTRAF-2 suggests that an attenuated signal is still going through the NF-B pathway in IEC when TRAF-2 is blocked. This may indicate that TRAF-2 is not the sole molecule transmitting the TNF- signal to the IB/NF-B system in these cells. This observation raises the possibility of a TRAF-2-independent route used by TNF- to activate NF-B, such as participation of other molecules that associate with TNF receptor 1-associated death domain or TRAF-2 and transmit a signal to NIK and downstream effector molecules. It is interesting to note that embryonic fibroblasts from receptor interacting protein (RIP)-deficient gene mouse failed to activate NF-B following TNF- stimulation (41). One could speculate that the RIP is partially responsible for the TNF- activation of NF-B and IL-8 gene expression in Ad5dnTRAF-2-infected cells. Another possible explanation of this incomplete blockade is that since only 80% of IEC were transfected, the remaining uninfected cells could contribute to the residual activation of NF-B and IL-8 in TNF--stimulated IEC. However, this seems to be less likely, since similar transfection efficacy using an NF-B super-repressor delivered by an adenoviral vector resulted in an almost total inhibition of IL-8 mRNA expression (3). Furthermore, the ability of dnTRAF-2 to suppress TNF--stimulated IL-8 mRNA (49–68%) was substantially less than the transfection efficiency (80%) and the reduction in IL-1ß-stimulated IL-8 mRNA (65–84%) with identical Ad5dnTRAF-2 infection (Fig. 8). In addition, the JNK activity was more strongly down-regulated than the NF-B activity, suggesting a predominant effect of TRAF-2 on the JNK pathway. These observations suggest that strategies aimed at effector molecules dedicated to the NF-B pathway (e.g., NIK and IKK) might be more efficient inhibitors than divergent effector molecules (e.g., TRAF and IRAK). Therefore, our data suggest that TRAF-2 may not be the target of choice to inhibit NF-B and cytokine gene expression.

An unexpected finding in our study is the critical role of TRAF-2 in IL-1ß-mediated IL-8 gene expression in HT-29 cells. IL-1ß signaling through NF-B in fibroblasts has been shown to involve the participation of the TRAF-6 adapter protein based on the observation that TRAF-6, but not the TRAF-2 protein, associates with IRAK following IL-1ß stimulation of embryonic kidney 293 cells overexpressing an exogenous IL-1 type 1 receptor (19). In contrast, we have measured an endogenous gene response following stimulation of the natural IL-1 cell receptor. Therefore, we believe that our data represent a more physiological situation than receptor overexpression and luciferase measurement. We are currently investigating the role of TRAF-6 in cytokine signaling pathways in IEC using an adenoviral vector encoding a dnTRAF-6.

The discrepancy between the involvement of TRAF-2 in NF-B activation by IL-1ß in IEC-6 and HT-29 cells is unknown at this point. We have previously shown that IEC-6 cells display a classical pattern of rapid and complete IB degradation (4) as opposed to the delayed, incomplete IB degradation found in HT-29 and primary IEC (2). Of interest, overexpression of IKK in HT-29 cells leads to complete IB degradation following cytokine stimulation (16). In addition, both IKK activity and IB serine 32 phosphorylation are strongly reduced in HT-29 cells compared with Caco-2 cells (C. Jobin, manuscript in preparation). These observations suggest that HT-29 cells have an aberrant cytokine signaling cascade in respect to their IB/NF-B axis. It remains to be seen whether the HT-29 cytokine signaling pathway is characteristic of primary human IEC or, rather, represents a unique phenotypic transformation. A careful dissection of the cytokine signaling pathway using dominant negative forms of various effector molecules cloned into adenoviral vectors, as performed in the present study, should shed light on the aberrant signal transduction in HT-29 cells, which more closely mimic the physiologic activation pathway in primary IEC than do Caco-2 and IEC-6 cells (2, 4).

The biological activity of proinflammatory molecules can be modulated using several strategies, including blockade of synthesis by antisense oligonucleotides (42), inhibition of activity by Ab neutralization (43), or inhibition of synthesis by immunosuppressive substances (e.g., IL-10, IL-4, etc.) (44). To this repertoire, targeted manipulation of the cytokine signaling cascade could prove to be a new, powerful, and selective therapeutic tool for blocking gene expression. The relevance of selective TNF signaling cascade blockade is exemplified by the discovery of two natural TRAF-2 inhibitory proteins (45, 46).

Collectively, these data suggest that TRAF-2 is partially involved in TNF- signaling through the IB/NF-B system in IEC, but is not a potent target for immunomodulation. Also, our data suggest a potential role of TRAF-2 in IL-1ß signaling in HT-29 cells.

	Acknowledgments

 
We thank Julie Vorobiov from the Immunoassay Core of the Center for Gastroenterology Biology and Disease for expert assistance with IL-8 quantification.

	Footnotes

 
¹ This work was supported by a Career Development Award from the Crohn’s and Colitis Foundation of America (to C.J.), National Institutes of Health Grant RO1-DK47700 (to R.B.S.), and National Institutes of Health Center Grant DK34087.

² Address correspondence and reprint requests to Dr. Christian Jobin, Division of Digestive Diseases and Nutrition, CB 7038, Glaxo Building, University of North Carolina, Chapel Hill, NC 27599-7080. E-mail address:

³ Abbreviations used in this paper: IEC, intestinal epithelial cells; IB, inhibitor B; TRAF-2, TNFR-associated factor-2; IRAK, IL-1 R-associated kinase; NIK, NF-B-inducing kinase; IKK, IB kinase; Ad5, adenovirus type 5; dn, dominant negative; moi, multiplicity of infection; NGS, nonimmune goat serum; JNK, c-Jun N-terminal kinase; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; SAPK, stress-related protein kinase.

Received for publication September 28, 1998. Accepted for publication January 21, 1999.

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