Differential requirement for NF-kappa B-inducing kinase in the induction of NF-kappa B by IL-1beta , TNF-alpha , and Fas

Maria P. Russo1, Brydon L. Bennett2, Anthony M. Manning2, David A. Brenner1,3, and Christian Jobin1,3

1 Department of Medicine and Center for Gastrointestinal Biology and Disease and 3 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7080; and 2 Signal Pharmaceuticals, Inc., San Diego, California 92121


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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In this study, we examined the role of the nuclear factor-kappa B (NF-kappa B)-inducing kinase (NIK) in distinct signaling pathways leading to NF-kappa B activation. We show that a dominant-negative form of NIK (dnNIK) delivered by adenoviral (Ad5dnNIK) vector inhibits Fas-induced Ikappa Balpha phosphorylation and NF-kappa B-dependent gene expression in HT-29 and HeLa cells. Interleukin (IL)-1beta - and tumor necrosis factor-alpha (TNF-alpha )-induced NF-kappa B activation and kappa B-dependent gene expression are inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells. Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF-alpha -induced apoptosis at an early time point. However, cytokine- and Fas-induced signals to NF-kappa B are finally integrated by the Ikappa B kinase (IKK) complex, since Ikappa Balpha phosphorylation, NF-kappa B DNA binding activity, and IL-8 gene expression were strongly inhibited in HT-29 and HeLa cells overexpressing dominant-negative IKKbeta (Ad5dnIKKbeta ). Our findings support the concept that cytokine signaling to NF-kappa B is redundant at the level of NIK. In addition, this study demonstrates for the first time the critical role of NIK and IKKbeta in Fas-induced NF-kappa B signaling cascade.

interleukin-8; inflammation; intestinal epithelial cells; signal transduction


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT HAS BEEN REPORTED that the inhibitor of kappa B (Ikappa B)/nuclear factor-kappa B (NF-kappa B) transcriptional system is implicated in multiple aspects of cell physiology, such as immune and inflammatory processes, cell growth, development, proliferation, and survival (6, 9). Activation of NF-kappa B is preceded by rapid serine-specific phosphorylation and degradation of its cytoplasmic inhibitory proteins of the Ikappa B family (23). Cytokine-induced serine phosphorylation of Ikappa Balpha has been shown to require the participation of a 700-kDa multisubunit protein complex, known as the Ikappa B kinase (IKK signalsome) (23). This complex contains at least two catalytic subunits, IKKalpha and IKKbeta , and the regulatory molecule IKKgamma /NEMO (56, 63). Overexpression of IKKalpha or IKKbeta triggers Ikappa Balpha serine phosphorylation and NF-kappa B activation; conversely, dominant-negative forms of both molecules impair interleukin (IL)-1beta - and tumor necrosis factor-alpha (TNF-alpha )-induced NF-kappa B-dependent reporter gene expression (10, 35, 48, 60, 64). However, gene deletion studies reveal that cytokine-induced Ikappa Balpha phosphorylation and NF-kappa B activation are predominantly accomplished by the catalytic subunit IKKbeta , and not by IKKalpha , and that the master regulator of both kinases is the IKK gamma -subunit (31, 53). Although these data established the IKK complex as the Ikappa B kinase, the events leading to its activation remain largely unknown.

The mitogen-activated protein 3-kinase (MAPKKK) NF-kappa B-inducing kinase (NIK) has been proposed to act as proximal inducer of the IKK catalytic subunit (9, 24), based on evidence that ectopic expression of the protein induced IKK kinase activity that triggers Ikappa Balpha serine phosphorylation and NF-kappa B-dependent gene transcription, a cascade of events blocked by dominant-negative NIK (dnNIK) (28, 29, 32, 38, 40, 46, 60).

Fas ligand, IL-1beta , and TNF-alpha utilize various and distinct adapter proteins and kinases to signal to downstream effector targets (5, 36, 42). It has been shown that Fas-, IL-1beta -, and TNF-alpha -induced kappa B-dependent transcription of a reporter gene is blocked by transient transfection of a dnNIK molecule (4, 28, 32, 41, 44, 51). These data positioned NIK at the intersection point of cytokines signaling to the NF-kappa B pathway.

The autosomal recessive mutation aly (alymphoplasia) causes lack of lymph nodes and Peyer's patches as well as disorganized splenic and thymic structures in aly/aly mice (33, 37). Interestingly, this phenotype is caused by a point mutation in the COOH-terminal region of NIK (54). Surprisingly, lymphotoxin (LT)-beta receptor (LTbeta R)-mediated, but not TNF-alpha -mediated, NF-kappa B activation is impaired in cells isolated from aly/aly mice (13, 34, 54). An analysis of CD40 signaling to NF-kappa B in the same mice (13) demonstrated that NIK is a critical mediator of NF-kappa B activation by CD40 in B cells, but not in dendritic cells. Moreover, embryonic fibroblasts isolated from NIK-deficient mice display a functional TNF-alpha -, IL-1beta -, and LTbeta R-induced NF-kappa B DNA binding activity (61). Overall, these data argue against a universal role for NIK in NF-kappa B signaling pathways and suggest eventually a cell-type and signal-specific relevance of this molecule.

Biochemical, pharmacological, and genetic data suggest that the control of NF-kappa B activation constitutes a relevant target for the treatment of inflammatory diseases, including inflammatory bowel disease (22). Therefore, the identification and functional characterization of the various kinases involved in the regulatory mechanisms of NF-kappa B activation are likely to help design new therapeutic targets. In this study, we evaluated the role of NIK and IKKbeta in Fas-, IL-1beta -, and TNF-alpha -induced NF-kappa B activation in HT-29 cells, an intestinal epithelial cell (IEC) line, and HeLa cells using adenoviral vectors encoding dominant-negative forms of these molecules. We show a strict requirement of NIK in Fas, but not in IL-1beta and TNF-alpha , signaling to NF-kappa B in IEC. However, cytokines and Fas signaling to NF-kappa B are inhibited in cells infected with an adenoviral vector encoding for dominant-negative IKKbeta (Ad5dnIKKbeta ). In this report, we have demonstrated for the first time the critical role of NIK and IKKbeta in Fas signaling to the NF-kappa B pathway.


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INTRODUCTION
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Cell culture. The human HT-29 colonic epithelial cells (HTB 38, American Type Culture Collection) were grown as described previously (18). HeLa cells were grown in Eagle's minimum essential medium with 10% FBS, 1× MEM nonessential amino acids, and antibiotics. The human HT-29 cell line was used, because IL-1beta , TNF-alpha , and Fas ligation stimulate NF-kappa B activation and IL-8 gene expression in these cells (2, 3, 19, 50). Cells were stimulated with human recombinant IL-1beta (5 ng/ml), TNF-alpha (5 ng/ml; both from Intergen, Purchase, NY), LT-alpha 1beta 2 (100 ng/ml; R & D Systems), or a Fas agonistic antibody (CH-11, 100 ng/ml; USB, Lake Placid, NY).

Ad5dnNIK and Ad5dnIKKbeta construction. The dnNIK consists of a truncated protein where the kinase domain and TNF receptor-associated factor (TRAF)-2-interacting domain (aa 1-623) were deleted (39). The adenovirus dnNIK (Ad5dnNIK) was constructed using the Cre-lox recombination method, as described previously (16). The subcloned gene contained an extra 27 bp of DNA nucleotides coding for a peptide derived from the hemagglutinin (HA) gene (YPYDVPDYA). The dnIKKbeta construct cloned in adenoviral vector carried a point mutation in the kinase domain (K44A), as described previously (35), and contained an extra 24 bp of DNA nucleotides coding for the FLAG peptide (DYLDDDDL). The Ad5Ikappa BAA virus has been characterized and described previously (20). Ad5LUC virus containing the luciferase gene and/or Ad5GFP virus containing the green fluorescent protein were used as control virus throughout the study.

IEC infection. After cells were cultured to 80% confluence, they were infected with the various adenoviral vectors in serum-free medium (Opti-MEM, GIBCO, Grand Island, NY) for 16 h. Different multiplicities of infection (0, 1:10, 1:50, and 1:100 IEC/virus particles) were tested to establish experimental conditions for high expression of the transgene with no cytotoxic effects. Cell viability estimated by trypan blue exclusion was always >97%. The adenovirus was then washed off, fresh medium containing serum was added, and cells were treated at the various time points with human recombinant IL-1beta , TNF-alpha , Fas agonistic antibody, or LT-alpha 1beta 2.

RNA extraction and RT-PCR analysis. RNA was isolated using TRIzol (GIBCO), reverse transcribed, and amplified as described elsewhere (18). The PCR products (5 µl) were subjected to electrophoresis on 2% agarose gels containing Gel Star fluorescent dye (FMC, Philadelphia, PA). Fluorescent staining was captured using an AlphaImager 2000 (AlphaInnotech, San Leandro, CA). Negative controls included amplifications with no nucleic acid or no reverse transcriptase. The IL-8 and beta -actin primers have been previously described (18). The NIK primers used were (5')5-CTGGCCTGTGTAGACAGCCAGA-3 (position 1091, NIK-A) and (3')3-TAATCCACTGGCTTGAGTTTCTCA-5 (position 1381, NIK-B). The length of the amplified product was 314 bp. To confirm the specificity and identity of the amplified product, the DNA was sequenced at the University of North Carolina, Chapel Hill, Automated Sequencing Facility on a model 377 DNA sequencer (Applied Biosystems Division, Perkin Elmer) using the ABI PRISM dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase FS (Applied Biosystems Division, Perkin Elmer).

Western blot analysis. Uninfected or Ad5dnNIK- or Ad5dnIKKbeta -infected cells were stimulated with IL-1beta or TNF-alpha (both at 5 ng/ml) for 0-60 min. The cells were then lysed in 1× Laemmli buffer (18), and 20 µg of protein were subjected to electrophoresis on 10% SDS-polyacrylamide gels. Antiphosphoserine Ikappa Balpha (New England Biolabs, Beverly, MA), anti-Ikappa Balpha (Santa Cruz Biotechnology, Santa Cruz, CA), anti-HA (Boehringer Mannheim, Indianapolis, IN), anti-NIK (Santa Cruz Biotechnology), and anti-FLAG M2 (Eastman Kodak, New Haven, CT) antibodies were all used at 1:1,000 dilution. The specific immunoreactive proteins were detected using the enhanced chemiluminescence kit (ECL; Amersham), as described previously (18).

Immunofluorescence analysis. HT-29 and HeLa cells were infected with Ad5dnNIK or left uninfected for 16 h. Cells were fixed with 100% ice-cold methanol, and dnNIK was detected using a mouse anti-HA antibody followed by incubation with an anti-mouse rhodamine-conjugated antibody, as described previously (20).

Nuclear extraction and electrophoretic mobility shift assay. Uninfected or adenovirus-infected cells were stimulated with IL-1beta or TNF-alpha (both at 5 ng/ml) for 30 min or with Fas agonistic antibody for 6 h (100 ng/ml), and nuclear extracts were prepared as described previously (18). Extracts (5 µg) were incubated with radiolabeled double-stranded oligonucleotides specifying the consensus sequence for class I major histocompatibility complex kappa B sites (GGCTGGGGATTCCCCATCT). Protein-DNA complexes were then separated by nondenaturating electrophoresis and visualized by autoradiography, as described previously (18).

Cell death assay. HT-29 cells (1 × 106) were grown in six-well plates and at 80% of confluence were infected with Ad5Ikappa BAA or Ad5dnNIK for 16 h and then exposed to TNF-alpha (5 ng/ml) for 6 h. Cell death was determined by counting the number of floating cells per well with a hemocytometer, a technique commonly used to quantify cell death in HT-29 cells as well as other cell lines of epithelial origin (14, 17, 43). In addition, cell death was evaluated by DNA fragmentation analysis on agarose gels. After treatment, cells were washed twice with PBS and then lysed in a hypotonic lysis buffer (10 mM Tris, 1 mM EDTA, and 0.2% Triton X-100, pH 7.5). Proteinase K digestion was performed for 16 h at 37°C. The DNA was then extracted with phenol-chloroform-isoamyl alcohol (25:24:1), precipitated in ethanol, and resuspended in TE buffer (10 mM Tris · HCl-1 mM EDTA). DNA was separated by electrophoresis on a 1.5% agarose gel.

Luciferase assay. HT-29 and HeLa cells were infected with a kappa B-luciferase adenoviral vector (Ad5kappa BLUC) alone or coinfected with Ad5dnNIK or control virus (Ad5GFP) for 16 h. After infection, cells were washed in PBS, and fresh medium containing serum was added before stimulation. Cells were stimulated with TNF-alpha (0.05 ng/ml), IL-1beta (0.5 ng/ml), and Fas antibody (100 ng/ml). After stimulation with TNF-alpha and IL-1beta for 8 h and with Fas antibody for 16 h, cells were harvested and lysed as previously described (18). kappa B-dependent luciferase activity was evaluated on a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Values are expressed as means ± SE derived by triplicates of each condition tested.

Statistical analysis. Statistical significance was evaluated by the two-tailed Student's t-test for paired data. P < 0.05 was considered statistically significant.


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ABSTRACT
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Selective role of NIK in Fas-, TNF-alpha -, and IL-1beta -mediated kappa B-dependent transcriptional activity. To determine whether NIK has a differential role in Fas, TNF-alpha and IL-1beta signaling to NF-kappa B in epithelial cells, we constructed adenoviral vectors encoding for a dnNIK (Ad5dnNIK). Adenoviral vectors are not only suitable for gene therapy but are also a useful tool to dissect endogenous signaling cascades in cells refractory to regular lipid-based transfection (21). Immunofluorescence analysis using an anti-HA antibody demonstrated that HA-tagged dnNIK was expressed by >90% of Ad5dnNIK-infected HT-29 and HeLa cells (Fig. 1). We next investigated the role of NIK in cytokine- and Fas death receptor-induced NF-kappa B activation in the two different epithelial cells, HeLa and HT-29. Cells were infected with an adenoviral vector encoding for the kappa B-luciferase gene (Ad5kappa BLUC) alone or with Ad5dnNIK or the control virus and then were stimulated with IL-1beta , TNF-alpha , or Fas antibody. TNF-alpha , IL-1beta , and Fas antibody induced NF-kappa B-dependent transcriptional activity in both cell lines, although Fas ligation induced lower activation than cytokines (data not shown). To compare the ability of dnNIK to block signal-induced NF-kappa B transcriptional activity, we established doses of IL-1beta , Fas antibody, and TNF-alpha that give similar levels of NF-kappa B induction (data not shown). Figure 2 shows that IL-1- and TNF-induced NF-kappa B transcriptional activity is significantly inhibited in Ad5dnNIK-infected HeLa cells but not in Ad5dnNIK-infected HT-29 cells, while Fas-induced NF-kappa B transcriptional activity is strongly blocked in both cell lines.


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Fig. 1.   Expression of dominant-negative (dn) nuclear factor-kappa B (NF-kappa B)-inducing kinase (dnNIK) in HT-29 (A) and HeLa (B) cells infected with an adenoviral (Ad) vector encoding for a dominant-negative form of NIK (Ad5dnNIK). Cells were infected with Ad5dnNIK (multiplicity of infection = 50) for 16 h and then double stained with Hoechst dye for detecting nuclear DNA and a rhodamine-conjugated antibody (left panel) specific for the antihemagglutinin (HA) antibody used to detect dnNIK tag expression. Results are representative of 4 different experiments.



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Fig. 2.   Differential requirement of NIK in interleukin (IL)-1beta -, tumor necrosis factor-alpha (TNF-alpha )-, and Fas-induced NF-kappa B activation. HT-29 (A) and HeLa (B) cells were infected with an adenovirus containing the luciferase gene (Ad5kappa BLUC) for 16 h and then stimulated with TNF-alpha (0.05 ng/ml) or IL-1beta (0.5 ng/ml) for 8 h or Fas antibody (Fas Ab, 100 ng/ml) for 16 h. Cells were then lysed, and the luciferase assay was performed. Results (means ± SE) were normalized for extract protein concentration and expressed as fold induction over control. * P < 0.05. Results are representative of 2 independent experiments.

Differential involvement of NIK in regulating phosphorylation of Ikappa Balpha induced by cytokines and Fas death receptor in HT-29 and HeLa cells. NIK has been positioned as a proximal kinase involved in IKK activation; therefore, a dominant-negative molecule should inhibit cytokine-induced Ikappa Balpha phosphorylation. To test this hypothesis, HeLa and HT-29 cells were infected with Ad5dnNIK and stimulated with TNF-alpha , IL-1beta , or Fas antibody for various times, then phosphorylation at serine-32 of Ikappa Balpha was analyzed by Western blotting. Because phosphorylated Ikappa Balpha is an unstable intermediate because of its rapid proteasome-mediated degradation, cells were preincubated with the proteasome inhibitor MG-132 to allow accumulation of phosphorylated Ikappa Balpha . As shown in Fig. 3, TNF-alpha , IL-1beta , and Fas antibody induced Ikappa Balpha phosphorylation in both HeLa and HT-29 cells (compare lanes 2-4 with lane 1), although the kinetics of phosphorylation were slower (>30 min) in Fas- than in cytokine-stimulated cells (<10 min). In accordance with data presented in Fig. 2, Ad5dnNIK strongly reduced IL-1beta - and TNF-alpha -induced Ikappa Balpha phosphorylation in HeLa cells, but only marginally in HT-29 cells (Fig. 3), whereas Fas-induced phosphorylation of Ikappa Balpha was blocked in both cell lines. Overall, this suggests that NIK has a critical role in regulating phosphorylation of Ikappa Balpha in Fas signaling to NF-kappa B in both epithelial cell lines.


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Fig. 3.   Differential effect of Ad5dnNIK on TNF-alpha -, IL-1beta -, and Fas antibody-mediated phosphorylation of Ikappa Balpha in HeLa (A) and HT-29 (B) cells. Cells were infected with Ad5dnNIK for 16 h or left untreated and then pretreated with the proteasome inhibitor MG-132 (20 µM for 30 min) and stimulated with IL-1beta , TNF-alpha (both at 5 ng/ml), or Fas antibody (100 ng/ml) for the indicated times. Total proteins were extracted, and 25 µg of protein were subjected to SDS-PAGE followed by immunoblotting with antiphosphoserine-32 Ikappa Balpha antibody. Phosphorylated Ikappa Balpha (P-Ikappa Balpha ) was detected using the enhanced chemiluminescence technique. A representative blot showing actin and HA-dnNIK expression for each cell line is also shown. Results are representative of 3 different experiments.

Differential ability of NIK in regulating cytokine- and Fas-induced NF-kappa B DNA binding activity and gene expression in HT-29 and HeLa cells. We next used HT-29 and HeLa cells to further characterize the role of NIK in cytokine- and Fas-induced NF-kappa B-dependent gene expression and DNA binding activity. As shown in Fig. 4A, all three stimuli, TNF-alpha , IL-1beta , and Fas antibody, induced NF-kappa B DNA binding activity, although Fas ligation induced only a modest increase. In agreement with Fig. 2A, Ad5dnNIK blocked Fas antibody-induced NF-kappa B DNA binding activity but only weakly blocked IL-1beta - or TNF-alpha -induced NF-kappa B DNA binding activity in HT-29 cells (Fig. 4A). In contrast, TNF-alpha -induced NF-kappa B DNA binding activity was strongly inhibited in Ad5dnNIK-infected HeLa cells (Fig. 4B). As in HT-29 cells, Fas-induced NF-kappa B DNA binding activity was also blocked in Ad5dnNIK-infected HeLa cells (Fig. 4B). To rule out the possibility that dnNIK failed to overcome the strong activation of NF-kappa B by cytokines in HT-29 cells but was efficient in blocking the weaker Fas signal, we next performed a TNF dose response in HT-29 cells. As shown in Fig. 4C, Ad5dnNIK failed to prevent NF-kappa B DNA binding activity even at low TNF concentrations, suggesting that the lack of inhibition is not related to the extent of NF-kappa B activation. This is in agreement with a recent report showing that TNF-alpha - and IL-1beta -induced NF-kappa B DNA binding activity is NIK independent in mouse embryonic fibroblasts (MEF) isolated from NIK-/- mice (61). Western blot analysis revealed equal expression of dnNIK in infected cells (Fig. 4C, bottom). LT-alpha 1beta 2 has been shown to induce NF-kappa B activation in HT-29 cells (30). Figure 4D shows that Fas-induced NF-kappa B DNA binding activity is inhibited by Ad5dnNIK, whereas the LTbeta R signal is minimally affected. Interestingly LTbeta R-induced NF-kappa B DNA binding activity is not blocked in cells isolated from NIK-/- mice, further confirming the validity of our in vitro approach through delivery of a dominant-negative molecule.


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Fig. 4.   Ad5dnNIK blocks Fas-mediated, but not IL-1beta -, TNF-alpha -, or lymphotoxin (LT)-alpha beta -mediated NF-kappa B DNA binding activity. A: HT-29 cells were infected with Ad5dnNIK for 16 h and then stimulated with IL-1beta (5 ng/ml) or TNF-alpha (5 ng/ml) for 30 min or Fas antibody (100 ng/ml) for 6 h. Nuclear extracts were tested for kappa B binding activity by electrophoretic mobility shift assay (EMSA). Results are representative of 5 different experiments. B: HeLa cells were infected with Ad5dnNIK for 16 h and then stimulated with TNF-alpha (5 ng/ml) for 30 min or Fas antibody (100 ng/ml) for 6 h. Nuclear extracts were tested for kappa B binding activity by EMSA. Results are representative of 3 different experiments. C: cells were infected with Ad5dnNIK or left untreated for 16 h and then stimulated with increasing doses of TNF-alpha (0.1-10 µg/ml). Nuclear extracts were tested for kappa B binding activity by EMSA. Results are representative of 3 different experiments. D: cells were infected with Ad5dnNIK or left untreated for 16 h and then stimulated with LT-alpha beta (100 ng/ml for 1 h) or Fas antibody (100 ng/ml for 6 h). Nuclear extracts were tested for kappa B binding activity by EMSA. Results are representative of 3 different experiments.

To validate the role of NIK in Fas signaling to NF-kappa B, we next investigated the effect of Ad5dnNIK on IL-8 gene expression. We have shown that cytokine-induced IL-8 gene expression is dependent on NF-kappa B activation in IEC (19, 20). Uninfected and Ad5dnNIK-infected HT-29 cells were stimulated with the agonistic Fas antibody for 8 h or with IL-1beta and TNF-alpha for 3 h, and then IL-8 mRNA accumulation was analyzed by RT-PCR. All three stimuli induced IL-8 mRNA accumulation in HT-29 cells (Fig. 5, compare lane 1 with lanes 2-4), in agreement with previous findings (3, 18, 20, 50). Fas antibody-induced, but not IL-1beta - and TNF-alpha -induced, IL-8 mRNA was inhibited in Ad5dnNIK-infected cells (Fig. 5, compare lane 8 with 4 and lanes 6 and 7 with 2 and 3). This suggests that NIK is essential for Fas-induced NF-kappa B-dependent IL-8 gene expression in HT-29 cells.


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Fig. 5.   Ad5dnNIK blocks Fas-induced, but not IL-1beta - or TNF-alpha -induced, IL-8 gene expression in HT-29 cells. Cells were infected with Ad5dnNIK for 16 h and then stimulated with IL-1beta (5 ng/ml) or TNF-alpha (5 ng/ml) for 30 min or Fas antibody (100 ng/ml) for 6 h. Total RNA was extracted, reverse transcribed, and amplified using specific IL-8 primers. PCR products were run on a 2% agarose gel stained with Gel Star. Results are representative of 3 different experiments.

It might be argued that the lack of total inhibition of NF-kappa B activation by dnNIK is due to high expression of endogenous wild-type NIK molecule. We therefore compared the level of expression of NIK in uninfected and Ad5dnNIK-infected HT-29 cells. With the use of a primer set common to both the wild-type and the truncated NIK molecule, a high level of dnNIK mRNA in Ad5dnNIK-infected cells was found, while wild-type endogenous NIK was detected only on further amplification cycles (Fig. 6A). In addition, similar levels of NIK mRNA and protein were detected in HT-29 and HeLa cells (Fig. 6B, top and middle, respectively), suggesting that the differential role of NIK in cytokine signaling to NF-kappa B is not related to the level of expression of the endogenous NIK molecule.


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Fig. 6.   Expression of NIK and dnNIK in Ad5dnNIK-infected cells. A: total RNA was extracted from uninfected HT-29 cells or HT-29 cells infected with Ad5dnNIK for 16 h. RNA was then reverse transcribed and amplified using specific NIK primers. PCR products were run on a 2% agarose gel stained with Gel Star. B: NIK mRNA and protein levels in HT-29 and HeLa cells. Top: total RNA from HT-29 and HeLa cells was extracted, reverse transcribed, and amplified using specific NIK primers. PCR products were run on a 2% agarose gel stained with Gel Star. Middle and bottom: total proteins were extracted, and 25 µg of protein were subjected to SDS-PAGE followed by immunoblotting of NIK and actin. NIK standard used as a control contains an HA tag and migrates slower than the endogenous NIK. Results are representative of 3 different experiments.

Increased apoptosis in Ad5Ikappa BAA- but not Ad5dnNIK-infected HT-29 cells. Blockade of NF-kappa B has been shown to sensitize cells to TNF-alpha -induced apoptosis (58). Therefore, we compared the effect of Ad5Ikappa BAA delivering a superrepressor of NF-kappa B (20) and Ad5dnNIK on TNF-alpha -stimulated HT-29 cells. Interestingly, cell detachment increased in Ad5Ikappa BAA-infected TNF-alpha -stimulated HT-29 cells as early as 6 h but not in Ad5dnNIK-infected cells, as seen by morphological analysis (Fig. 7A) and by counting floating cells (Fig. 7B), a widely used method for evaluation of cell death in cells of epithelial origin (14, 17, 43). Moreover, DNA laddering was increased in Ad5Ikappa BAA-infected, but not in Ad5dnNIK-infected, HT-29 cells stimulated with TNF-alpha for 6 h. This may suggest that the protective role of NF-kappa B is still present in conditions where NIK function is impaired.


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Fig. 7.   Ad5dnNIK failed to sensitize HT-29 cells to TNF-alpha -induced cell death. A: morphological analysis of TNF-alpha -induced cell detachment. HT-29 cells were infected with Ad5dnNIK or Ad5Ikappa BAA for 16 h and then stimulated with TNF-alpha (5 ng/ml) for 6 h. B: counting of detached cells. Cells were treated as described in A, and the number of dead cells per well was assayed. Values are means ± SE of 3 independent experiments. C: TNF-alpha -induced DNA laddering is increased in Ad5Ikappa BAA-infected, but not Ad5dnNIK-infected, HT-29 cells. Cells were treated as described in A, and DNA was extracted. Samples were run on 1.5% agarose gel stained with Gel Star. Results are representative of 3 different experiments.

IKKbeta is critical for Fas- and cytokine-induced NF-kappa B activation and IL-8 gene expression in HT-29 and HeLa cells. We next investigated whether in both cell lines Fas and cytokine signaling to NF-kappa B proceed through the IKK complex. We first compared the level of Ikappa Balpha phosphorylation in cells infected with an adenoviral vector delivering a kinase-deficient mutant of IKKbeta (Ad5dnIKKbeta ). TNF-alpha , IL-1beta , and Fas antibody induced Ikappa Balpha phosphorylation in HeLa cells (Fig. 8A) and HT-29 cells (Fig. 8B), in accordance with data presented in Fig. 3. Interestingly, as opposed to Ad5dnNIK, a blockade at the level of IKKbeta by Ad5dnIKKbeta strongly reduced Ikappa Balpha phosphorylation in both cell lines and in all the conditions tested. Moreover, Ad5dnIKKbeta strongly inhibited Fas- and IL-1beta -induced NF-kappa B DNA binding activity in HT-29 (Fig. 9) and HeLa (data not shown) cells. In accordance with blockade of NF-kappa B activation, cytokine- and Fas antibody-induced IL-8 mRNA accumulation was blocked in Ad5dnIKKbeta -infected HT-29 cells (Fig. 10A). In addition, TNF-alpha - and Fas antibody-induced IL-8 mRNA accumulation was also blocked in Ad5dnIKKbeta -infected HeLa cells (Fig. 10B). Although IL-1beta induced kappa B-dependent transcriptional activity (Fig. 2B), it failed to induce significant IL-8 mRNA expression (unpublished observation). Together, these data demonstrate that NIK is an essential component of Fas signaling to NF-kappa B but is dispensable for IL-1beta - and TNF-alpha -induced NF-kappa B activation. On the other hand, cytokine- and Fas-initiated signals converge on the IKK complex and utilize the IKKbeta catalytic subunit to induce NF-kappa B activity.


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Fig. 8.   Ad5dnIKKbeta inhibits TNF-alpha -, IL-1beta -, and Fas antibody-mediated Ikappa Balpha phosphorylation in HeLa (A) and HT-29 (B) cells. Cells were infected with Ad5dnIKKbeta for 16 h or left untreated; then cells were pretreated with the proteasome inhibitor MG-132 (20 µM for 30 min) and stimulated with IL-1beta (5 ng/ml), TNF-alpha (5 ng/ml), or Fas antibody (100 ng/ml) for the indicated times. Total proteins were extracted, and 25 µg of proteins were subjected to SDS-PAGE followed by immunoblotting of phosphoserine Ikappa Balpha . A representative blot showing actin and FLAG-dnIKKbeta expression for each cell line is shown. Results are representative of 3 different experiments.



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Fig. 9.   Fas- and IL-1beta -mediated NF-kappa B DNA binding activity is inhibited in Ad5dnIKKbeta -infected intestinal epithelial cells. A: HT-29 cells were infected with Ad5dnIKKbeta for 16 h and then stimulated with IL-1beta (5 ng/ml) for 30 min or Fas antibody (100 ng/ml) for 6 h. Nuclear extracts were tested for kappa B binding activity by EMSA. Results are representative of 3 different experiments.



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Fig. 10.   Ad5dnIKKbeta inhibits cytokine- and Fas-induced IL-8 gene expression in HT-29 (A) and HeLa (B) cells. Cells were infected with Ad5dnIKKbeta for 16 h and then stimulated with IL-1beta or TNF-alpha (both at 5 ng/ml) or Fas antibody (100 ng/ml) for 3 and 6 h, respectively. Total RNA was extracted, reverse transcribed, and amplified using specific IL-8 and beta -actin primers. PCR products were run on a 2% agarose gel stained with Gel Star. Results are representative of 3 different experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Numerous unrelated signals such as IL-1beta , TNF, lipopolysaccharide, and the viral protein Tax induce NF-kappa B activity through IKK kinase (63). An important question is whether these various inducers utilize diverse or common transmitting pathways to relay the signal to IKK. NIK has been proposed as an upstream kinase activated by diverse stimuli and inducing the IKK complex, and, as such, it may represent a potent therapeutic target for disorders involving NF-kappa B dysregulation.

Our data indicate that NIK is obligatory for NF-kappa B activation induced by Fas ligation in HeLa and HT-29 cells, since a dominant-negative molecule blocks Ikappa Balpha phosphorylation, NF-kappa B DNA binding activity, kappa B-dependent gene expression, and IL-8 mRNA accumulation. Recently, it has been reported that LT-beta , but not TNF, utilizes NIK to activate the NF-kappa B pathway (34). Our data show that TNF-, IL-beta -, and LT-beta -induced NF-kappa B DNA binding activity is not blocked in Ad5dnNIK-infected HT-29 cells. This suggests that the role of NIK in the NF-kappa B pathway is restricted to the Fas signaling cascade, at least in HT-29 and HeLa cells. During the preparation/submission of our manuscript, a study has demonstrated that IL-1beta -, TNF-, and LT-beta -induced NF-kappa B DNA binding activity proceeds through a NIK-independent mechanism in MEF or B cells isolated from NIK-deficient mice (61). Therefore, our findings that cytokine-induced NF-kappa B activation is NIK independent in HT-29 cells are in line with this new study. In addition, we demonstrated for the first time that Fas signaling to the NF-kappa B pathway is NIK dependent.

Although Fas ligation has been shown to induce kappa B-dependent transcription and IL-8 gene expression (3, 8, 11, 15, 16, 32, 45, 50), the signaling cascade responsible for NF-kappa B activity has not been established yet. Our study shows that the Fas signaling cascade to NF-kappa B activation is blocked by Ad5dnNIK and Ad5dnIKKbeta . From these data, an emerging pathway for Fas signaling to NF-kappa B includes the obligatory role of NIK and IKKbeta . It is still unclear how Fas signals to NIK and IKK proteins. The Fas cytoplasmic tail is able to recruit Fas-associated death domain and the receptor-interacting protein (RIP) (57). Recently, it has been shown that TNF receptor 1 utilizes RIP to mediate IKK activation (12). Therefore, similar to TNF type1 receptor, Fas may utilize RIP to activate IKK. Although it remains to be demonstrated whether RIP is necessary for Fas-mediated NF-kappa B activation, a potential pathway could involve RIP, NIK, and IKKbeta . It remains to be seen whether cells isolated from NIK-deficient mice display an impaired Fas signaling to NF-kappa B.

The role of Fas in intestinal homeostasis is not well understood. However, this pathway may participate in mucosal injury by reducing intestinal barrier function through induction of cell apoptosis and/or by impairing epithelial barrier function (1). Therefore, our finding that NIK is selectively involved in Fas signal transduction in IEC provides an interesting checkpoint to modulate this pathway.

The failure of Ad5dnNIK to completely block IL-1beta - and TNF-alpha -induced NF-kappa B activation in HT-29 cells strongly suggests the existence of an alternate route of activation. Mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) kinase-1 (MEKK1) has been postulated to be a strong inducer of IKKbeta kinase activity (26, 27, 38, 40). Recently, it was reported that cells isolated from MEKK1-/- mice display a functional NF-kappa B activation after IL-1beta and TNF-alpha stimulation (62). In addition, significant IKK and NF-kappa B activity was detected in cells where both MEKK1 and NIK signals were blocked (49). This suggests the existence of a signaling pathway to NF-kappa B utilized by IL-1beta and TNF-alpha that is independent of NIK and MEKK1. Potential candidate kinases include p38 kinase, phosphoinositide 3-kinase, protein kinase B (Akt), MEKK2, MEKK3, and the atypical protein kinase C, which are all capable of NF-kappa B activation (7, 25, 44, 47, 52, 55, 65). Interestingly, phosphoinositide 3-kinase inhibition by pharmacological blockade does not prevent TNF-alpha -induced NF-kappa B activation (59). From our data, we suggest that different stimuli target various intermediate kinases to activate NF-kappa B and that NIK is part of a multiple network of kinases but is not a critical component of IKK activation. It is still unclear why NIK is obligatory for Fas signaling to NF-kappa B but dispensable for TNF-alpha or IL-1beta . The differences may relate to the pleiotrophic nature of IL-1 and TNF signaling, which involve a multitude of kinases as opposed to the more linear Fas signaling. However, regardless of the intermediate kinases used by IL-1beta and TNF-alpha , a blockade imposed by Ad5dnIKKbeta totally prevented NF-kappa B signaling and IL-8 gene expression. Thus, although the signal from the cell surface receptor may lead to redundant and alternate routes, these different pathways converge on IKKbeta to activate NF-kappa B. This indicates that IKKbeta , but not NIK, represents a potential target for therapeutic intervention targeting the NF-kappa B pathway in many cell types, including IEC.

In summary, we have demonstrated that NIK is an essential signaling molecule for Fas-mediated NF-kappa B activity. However, the role of NIK in IL-1beta - or TNF-alpha -induced NF-kappa B activity appears to be signal and cell type specific. These findings may help design new therapeutic targets for inflammatory disorders.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grant ROI-DK-47700 to C. Jobin.


    FOOTNOTES

Address for reprint requests and other correspondence: C. Jobin, Div. of Digestive Diseases and Nutrition, CB# 7038, Glaxo Bldg., University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7038 (E-mail: Job{at}med.unc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 27, 2002;10.1152/ajpcell.00166.2001

Received 2 April 2001; accepted in final form 21 February 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abreu, MT, Palladino AA, Arnold ET, Kwon RS, and McRoberts JA. Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells. Gastroenterology 119: 1524-1536, 2000[ISI][Medline].

2.   Abreu-Martin, MT, Palladino AA, Faris M, Carramanzana NM, Nel AE, and Targan SR. Fas activates the JNK pathway in human colonic epithelial cells: lack of a direct role in apoptosis. Am J Physiol Gastrointest Liver Physiol 276: G599-G605, 1999[Abstract/Free Full Text].

3.   Abreu-Martin, MT, Vidrich A, Lynch DH, and Targan SR. Divergent induction of apoptosis and IL-8 secretion in HT-29 cells in response to TNF-alpha and ligation of Fas antigen. J Immunol 155: 4147-4154, 1995[Abstract].

4.   Awane, M, Andres PG, Li DJ, and Reinecker HC. NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha -, and IL-1beta -induced chemokine promoter activation in intestinal epithelial cells. J Immunol 162: 5337-5344, 1999[Abstract/Free Full Text].

5.   Baker, SJ, and Reddy EP. Modulation of life and death by the TNF receptor superfamily. Oncogene 17: 3261-3270, 1998[ISI][Medline].

6.   Barnes, PJ, and Karin M. Nuclear factor-kappa B, a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336: 1066-1071, 1997[Free Full Text].

7.   Berghe, W, Plaisance S, De Brosscher K, Schmitz ML, Fiers W, and Haegeman G. p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappa B p65 transactivation mediated by tumor necrosis factor. J Biol Chem 273: 3285-3290, 1998[Abstract/Free Full Text].

8.   Cheema, ZF, Wade SB, Sata M, Walsh K, Sohrabja F, and Miranda RC. Fas/Apo (apoptosis)-1 and associated proteins in the differentiating cerebral cortex: induction of caspase-dependent cell death and activation of NF-kappa B. J Neurosci 19: 1754-1770, 1999[Abstract/Free Full Text].

9.   Chen, F, Castranova V, Shi X, and Demers LM. New insights into the role of nuclear factor-kappa B, a ubiquitous transcription factor in the initiation of diseases. Clin Chem 45: 7-17, 1999[Abstract/Free Full Text].

10.   Cohen, L, Henzel WJ, and Baeuerle PA. IKAP is a scaffold protein of the Ikappa B kinase complex. Nature 395: 292-296, 1998[ISI][Medline].

11.   Depraetere, V, and Golstein P. Fas and other cell death signaling pathways. Semin Immunol 9: 93-107, 1997[Medline].

12.   Devin, A, Cook A, Lin Y, Rodriguez Y, Kelliher M, and Liu ZG. The distinct roles of TRAF2 and RIP in IKK activation by TNF-R1: TRAF-2 recruits IKK to TNF-R1 while RIP mediates IKK activation. Immunity 12: 419-429, 2000[ISI][Medline].

13.   Garceau, N, Kosaka Y, Masters S, Hambor J, Shinkura R, Honjo T, and Noelle RJ. Lineage-restricted function of nuclear factor-kappa B-inducing kinase (NIK) in transducing signals via CD40. J Exp Med 191: 381-386, 2000[Abstract/Free Full Text].

14.   Giardina, C, Boulares H, and Inan MS. NSAID and butyrate sensitize a human colorectal cancer cell line to TNF-alpha and Fas ligation: the role of reactive oxygen species. Biochim Biophys Acta 1448: 425-438, 1999[ISI][Medline].

15.   Hagimoto, N, Kuwano K, Kawasaki M, Yoshimi M, Kaneko Y, Kunitake R, Maeyama T, Tanaka T, and Hara N. Induction of interleukin-8 secretion and apoptosis in bronchiolar epithelial cells by fas ligation. Am J Respir Cell Mol Biol 21: 436-445, 1999[Abstract/Free Full Text].

16.   Hardy, S, Kitamura M, Harris-Stansil T, Dal Y, and Phipps L. Construction of adenovirus vectors through Cre-lox recombination. J Virol 71: 1842-1849, 1997[Abstract].

17.   Heerdt, BG, Houston MA, and Augenlicht LH. Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines. Cancer Res 54: 3288-3293, 1994[Abstract].

18.   Jobin, C, Haskill S, Mayer L, Panja A, and Sartor RB. Evidence for an altered regulation of Ikappa Balpha degradation in human colonic epithelial cells. J Immunol 158: 226-234, 1997[Abstract].

19.   Jobin, C, Holt L, Bradham CA, Streetz K, Brenner DA, and Sartor RB. TRAF-2 is involved in both IL-1beta - and TNF-alpha -signaling cascade leading to NF-kappa B activation and IL-8 expression in human intestinal epithelial cells. J Immunol 162: 4447-4454, 1999[Abstract/Free Full Text].

20.   Jobin, C, Panja A, Hellerbrand C, Iimuro Y, Didonato J, Brenner DA, and Sartor RB. Inhibition of proinflammatory molecule production by adenovirus-mediated expression of an NF-kappa B super-repressor in human intestinal epithelial cells. J Immunol 160: 410-418, 1998[Abstract/Free Full Text].

21.   Jobin, C, and Sartor RB. The Ikappa B/NF-kappa B system: a key determinant of mucosal inflammation and protection. Am J Physiol Cell Physiol 278: C451-C462, 2000[Abstract/Free Full Text].

22.   Jobin, C, and Sartor RB. NF-kappa B signaling proteins as therapeutic targets for inflammatory bowel diseases. Inflamm Bowel Dis 6: 206-213, 2000[ISI][Medline].

23.   Karin, M. The beginning of the end: Ikappa B kinase (IKK) and NF-kappa B activation. J Biol Chem 274: 27399-27342, 1999[Abstract/Free Full Text].

24.   Karin, M, and Delhase M. JNK or IKK, AP-1 or NF-kappa B, which are the targets for MEK kinase 1 action? Proc Natl Acad Sci USA 95: 9067-9069, 1998[Free Full Text].

25.   Lallena, MJ, Diaz-Meco MT, Bren G, Paya CV, and Moscat J. Activation of Ikappa B kinase beta  by protein kinase C isoforms. Mol Cell Biol 19: 2180-2188, 1999[Abstract/Free Full Text].

26.   Lee, FS, Hagler J, Chen ZJ, and Maniatis T. Activation of the Ikappa Balpha kinase complex by MEKK1, a kinase of the JNK pathway. Cell 88: 213-222, 1997[ISI][Medline].

27.   Lee, FS, Peters RT, Dang LC, and Maniatis T. MEKK1 activates both Ikappa B kinase alpha  and Ikappa B kinase beta . Proc Natl Acad Sci USA 95: 9319-9324, 1998[Abstract/Free Full Text].

28.   Lin, X, Mu Y, Cunningham ET, Marcu KB, Geleziunas R, and Greene WC. Molecular determinants of NF-kappa B-inducing kinase action. Mol Cell Biol 18: 5899-5907, 1998[Abstract/Free Full Text].

29.   Ling, L, Cao Z, and Goeddel DV. NF-kappa B-inducing kinase activates IKK-alpha by phosphorylation of Ser-176. Proc Natl Acad Sci USA 95: 3792-3797, 1998[Abstract/Free Full Text].

30.   Mackay, F, Majeau GR, Hochman PS, and Browning JL. Lymphotoxin-beta receptor triggering induces activation of the nuclear factor kappa B transcription factor in some cell types. J Biol Chem 271: 24934-24938, 1996[Abstract/Free Full Text].

31.   Makris, C, Godfrey VL, Krahn-Senftleben G, Takahashi T, Roberts JL, Schwarz T, Feng L, Johnson RS, and Karin M. Female mice heterozygous for IKKgamma /NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Mol Cell 5: 969-979, 2000[ISI][Medline].

32.   Malinin, NL, Boldin MP, Kovalenko AV, and Wallach D. MAP3K-related kinase involved in NF-kappa B induction by TNF, CD95 and IL-1. Nature 385: 540-544, 1997[ISI][Medline].

33.   Matsumoto, M, Iwamasa K, Rennert PD, Yamada T, Suzuki R, Matsushima A, Okabe M, Fujita S, and Yokoyama M. Involvement of distinct cellular compartments in the abnormal lymphoid organogenesis in lymphotoxin-alpha -deficient mice and alymphoplasia (aly) mice defined by the chimeric analysis. J Immunol 163: 1584-1591, 1999[Abstract/Free Full Text].

34.   Matsushima, A, Kaisho T, Rennert PD, Nakano H, Kurosawa K, Uchida D, Takeda K, Akira S, and Matsumoto M. Essential role of nuclear factor (NF)-kappa B-inducing kinase and inhibitor of kappa B (Ikappa B) kinase alpha  in NF-kappa B activation through lymphotoxin beta  receptor, but not through tumor necrosis factor receptor I. J Exp Med 193: 631-636, 2001[Abstract/Free Full Text].

35.   Mercurio, F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li JW, Young DB, Barbosa M, Mann M, Manning A, and Rao A. IKK-1 and IKK-2: cytokine-activated Ikappa B kinases essential for NF-kappa B activation. Science 278: 860-866, 1997[Abstract/Free Full Text].

36.   Miagkov, AV, Kovalenko DV, Brown CE, Didsbury JR, Cogswell JP, Stimpson SA, Baldwin AS, and Makarov SS. NF-kappa B activation provides the potential link between inflammation and hyperplasia in the arthritic joint. Proc Natl Acad Sci USA 95: 13859-13864, 1998[Abstract/Free Full Text].

37.   Miyawaki, S, Nakamura Y, Suzuka H, Koba M, Yasumizu R, Ikehara S, and Shibata Y. A new mutation, aly, that induces a generalized lack of lymph nodes accompanied by immunodeficiency in mice. Eur J Immunol 24: 429-434, 1994[ISI][Medline].

38.   Nakano, H, Shindo M, Sakon S, Nishinaka S, Mihara M, Yagita H, and Okumura K. Differential regulation of Ikappa B kinase alpha  and beta  by two upstream kinases, NF-kappa B-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1. Proc Natl Acad Sci USA 95: 3537-3542, 1998[Abstract/Free Full Text].

39.   Natoli, G, Costanzo A, Moretti F, Fulco M, Balsano C, and Levrero M. Tumor necrosis factor (TNF) receptor 1 signaling downstream of TNF receptor-associated factor 2. J Biol Chem 272: 26079-26082, 1997[Abstract/Free Full Text].

40.   Nemoto, S, DiDonato JA, and Lin A. Coordinate regulation of Ikappa B kinases by mitogen-activated protein kinase kinase kinase 1 and NF-kappa B-inducing kinase. Mol Cell Biol 18: 7336-7343, 1998[Abstract/Free Full Text].

41.   Ninomiya-Tsuji, J, Kishimoto K, Hiyama A, Inoue JI, Cao Z, and Matsumoto K. The kinase TAK1 can activate the NIK-Ikappa B as well as the MAP kinase cascade in the IL-1 signaling. Nature 398: 252-256, 1999[ISI][Medline].

42.   O'Neill, LAJ, and Greene C. Signal transduction pathways activated by IL-1 receptor family: ancient machinery in mammals, insects, and plants. J Leukoc Biol 63: 650-657, 1998[Abstract].

43.   Ossina, NK, Cannas A, Powers VC, Fitzpatrick PA, Knight JD, Gilbert JR, Shekhtman EM, Tomei LD, Umansky SR, and Kiefer MC. Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem 272: 16351-16357, 1997[Abstract/Free Full Text].

44.   Ozes, ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, and Donner DB. NF-kappa B activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401: 82-85, 1999[ISI][Medline].

45.   Ponton, A, Clement MV, and Stamenkovic I. The CD95 (APO/Fas) receptor activates NF-kappa B independently of its cytotoxic function. J Biol Chem 271: 8991-8995, 1996[Abstract/Free Full Text].

46.   Regnier, CH, Song HY, Gao X, Goeddel DV, Cao Z, and Rothe M. Identification and characterization of an Ikappa B kinase. Cell 90: 373-383, 1997[ISI][Medline].

47.   Romashkova, JA, and Makarov SS. NF-kappa B is a target of AKT in anti-apoptotic PDGF signalling. Nature 401: 86-90, 1999[ISI][Medline].

48.   Rothwarf, DM, Zandi E, Natoli G, and Karin M. IKK-gamma g is an essential regulatory subunit of the Ikappa B kinase complex. Nature 395: 297-300, 1998[ISI][Medline].

49.   Russo, MP, Bradham CA, Bennett BL, Manning AM, Brenner DA, and Jobin C. Ikappa B kinase beta  (IKKbeta ), but not NF-kappa B-inducing kinase (NIK) or IKKalpha , represents a therapeutic target for modulation of Fas, IL-1beta and TNFalpha -mediated NF-kappa B activation in intestinal epithelial cells (Abstract). Gastroenterology 118: A587, 2000.

50.   Russo, MP, Mehta NP, Keku TO, Sartor RB, and Jobin C. Increased susceptibility to Fas-mediated apoptosis in differentiated HT-29 cells independent of its effects on NF-kappa B activation and IL-8 secretion (Abstract). Gastroenterology 118: A820, 2000.

51.   Sakurai, H, Miyoshi H, Toriumi W, and Sugita T. Functional interactions of transforming growth factor beta -activated kinase 1 with Ikappa B kinases to stimulate NF-kappa B activation. J Biol Chem 274: 10641-10648, 1999[Abstract/Free Full Text].

52.   Sanz, L, Sanchez P, Lallena MJ, Diaz-Meco MT, and Moscat J. The interaction of p62 with RIP links the atypical PKCs to NF-kappa B activation. EMBO J 18: 3044-3053, 1999[Abstract/Free Full Text].

53.   Schmidt-Supprian, M, Bloch W, Courtois G, Addicks K, Israel A, Rajewsky K, and Pasparakis M. NEMO/IKKgamma -deficient mice model incontinentia pigmenti. Mol Cell 5: 981-992, 2000[ISI][Medline].

54.   Shinkura, R, Kitada K, Matsuda F, Tashiro K, Ikuta K, Suzuki M, Kogishi K, Serikawa T, and Honjo T. Alymphoplasia is caused by a point mutation in the mouse gene encoding NF-kappa B-inducing kinase. Nat Genet 22: 74-77, 1999[ISI][Medline].

55.   Sizemore, N, Leung S, and Stark GR. Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-kappa B p65/RelA subunit. Mol Cell Biol 19: 4798-4805, 1999[Abstract/Free Full Text].

56.   Tak, PP, and Firestein GS. NF-kappa B: a key role in inflammatory diseases. J Clin Invest 107: 7-11, 2001[Free Full Text].

57.   Wallach, D, Varfolomeev EE, Malinin NL, Goltsev YV, Kovalenko AV, and Boldin MP. Tumor necrosis factor receptor and Fas signaling mechanisms. Annu Rev Immunol 17: 331-367, 1999[ISI][Medline].

58.   Wang, CY, Mayo MW, and Baldwin AS, Jr. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappa B. Science 274: 784-787, 1996[Abstract/Free Full Text].

59.   Weaver, SA, Russo MP, Wright KL, Kolios G, Jobin C, Robertson DA, and Ward SG. Regulatory role of phosphatidylinositol 3-kinase on TNF-alpha -induced cyclooxygenase 2 expression in colonic epithelial cells. Gastroenterology 120: 1117-1127, 2001[ISI][Medline].

60.   Woronicz, JD, Gao X, Cao Z, Rothe M, and Goeddel DV. Ikappa B kinase-beta : NF-kappa B activation and complex formation with Ikappa B kinase-alpha and NIK. Science 278: 866-869, 1997[Abstract/Free Full Text].

61.   Yin, L, Wu L, Wesche H, Arthur CD, White JM, Goeddel DV, and Schreiber RD. Defective lymphotoxin-beta receptor-induced NF-kappa B transcriptional activity in NIK-deficient mice. Science 291: 2162-2165, 2001[Abstract/Free Full Text].

62.   Yujiri, T, Ware M, Widmann C, Oyer R, Russel D, Chan E, Zaitsu Y, Clarke P, Tyler K, Oka Y, Fanger GR, Henson P, and Johnson GL. MEK kinase 1 gene disruption alters cell migration and c-Jun NH2-terminal kinase regulation but does not cause a measurable defect in NF-kappa B activation. Proc Natl Acad Sci USA 97: 7272-7277, 2000[Abstract/Free Full Text].

63.   Zandi, E, and Karin M. Bridging the gap: composition, regulation, and physiological function of the Ikappa B kinase complex. Mol Cell Biol 19: 4547-4551, 1999[Free Full Text].

64.   Zandi, E, Rothwarf DM, Belhase M, Hayakama M, and Karin M. The Ikappa B kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta , necessary for Ikappa B phosphorylation and NF-kappa B activation. Cell 91: 243-252, 1997[ISI][Medline].

65.   Zhao, Q, and Lee FS. Mitogen-activated protein kinase/ERK kinase kinase 2 and 3 activate nuclear factor-kappa B through Ikappa B kinase-alpha and Ikappa B kinase-beta . J Biol Chem 274: 8355-8358, 1999[Abstract/Free Full Text].


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