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Originally published online as doi:10.1189/jlb.0204115 on March 22, 2005

Published online before print March 22, 2005
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(Journal of Leukocyte Biology. 2005;77:975-983.)
© 2005 by Society for Leukocyte Biology

Inhibition of IKK down-regulates antigen + IgE-induced TNF production by mast cells: a role for the IKK-I{kappa}B-NF-{kappa}B pathway in IgE-dependent mast cell activation

Yongde Peng*,1, Melanie R. Power*, Bo Li* and Tong-Jun Lin*,{dagger},2

* Departments of Microbiology and Immunology and
{dagger} Pediatrics, Dalhousie University, Isaac Walton Killam Health Centre, Halifax, Nova Scotia, Canada

2 Correspondence: IWK Health Centre, Department of Pediatrics, 5850 University Ave., Halifax, NS, Canada, B3K 6R8. E-mail: tong-jun.lin{at}dal.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells (MC) are major effector cells for allergic diseases. Cross-linking of immunoglobulin E (IgE) and its high-affinity receptor, Fc{varepsilon}RI, by antigen initiates a cascade of signaling events leading to nuclear factor (NF)-{kappa}B activation and tumor necrosis factor (TNF) production. Here, we demonstrated that inhibition of inhibitor of {kappa}B (I{kappa}B) kinase (IKK) by a peptide IKK inhibitor or by four individual chemical IKK inhibitors including 15-deoxy-prostaglandin J2, BMS-345541, SC-514, or sulindac significantly blocked IgE + trinitrophenyl (TNP)-induced TNF production by mouse bone marrow-derived MC (BMMC). Moreover, IgE + TNP induced a rapid phosphorylation of IKK{alpha} but not IKKß in BMMC. IgE + TNP-induced phosphorylation of IKK{alpha} was accompanied with phosphorylation and degradation of I{kappa}B{alpha}, subsequent NF-{kappa}B activation, and TNF production. Inhibition of IKK by sulindac decreased IKK{alpha} phosphorylation, I{kappa}B{alpha} phosphorylation and degradation, NF-{kappa}B activation, and TNF production by BMMC. It is interesting that IgE + TNP stimulation also induced a prominent synthesis of IKK{alpha} and I{kappa}B{alpha}. Inhibition of NF-{kappa}B activity by pyrrolidine dithiocarbomate (PDTC) blocked IgE + TNP-induced I{kappa}B{alpha} synthesis. NF-{kappa}B activity and TNF production were also inhibited when PDTC was used even after IgE + TNP stimulation, suggesting a potential role for the newly synthesized I{kappa}B{alpha} in MC activation. In addition, IgE + TNP-induced IKK{alpha} and I{kappa}B{alpha} phosphorylation was inhibited by a protein kinase C (PKC) inhibitor Ro 31-8220. Taken together, our results support a role for the IKK-I{kappa}B-NF-{kappa}B pathway, which likely involves PKC in IgE-dependent TNF production by MC. Thus, IKK may serve as a new target for the regulation of MC function in allergy.

Key Words: Fc receptors • inflammation • allergy • protein kinases • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immunoglobulin E (IgE)-dependent mast cell (MC) activation plays an important role in the development of allergy. The cross-linking of allergen-specific IgE bound to its high-affinity receptor, Fc{varepsilon}RI, results in a series of molecular events leading to nuclear factor (NF)-{kappa}B activation [1 , 2 ] and subsequent cytokine production. Many MC-derived cytokines, such as tumor necrosis factor (TNF), are regulated by transcription factor NF-{kappa}B [3 ]. Five mammalian NF-{kappa}B proteins have been identified including NF-{kappa}B1 (p50 and its precursor p105), NF-{kappa}B2 (p52 and its precursor p100), RelA/p65, RelB, and cRel [3 ]. The predominant form of NF-{kappa}B in many cell types is a p65:p50 heterodimer. A family of inhibitory proteins, including inhibitor of {kappa}B (I{kappa}B){alpha}, I{kappa}Bß, and I{kappa}B{varepsilon}, which bind to NF-{kappa}B and mask its nuclear localization signal, causing its localization in the cytoplasm, controls the activity of NF-{kappa}B [3 ]. Phosphorylation and degradation of I{kappa}B allow NF-{kappa}B to translocate to the nucleus and to bind DNA to initiate gene expression. Thus, I{kappa}B plays an essential role in the control of NF-{kappa}B activity. Diverse signaling proteins have been shown to be involved directly in I{kappa}B phosphorylation and degradation. I{kappa}B kinase (IKK) is activated and responsible for cytokine interleukin (IL)-1 and TNF-induced I{kappa}B phosphorylation [3 , 4 ]. In macrophages, NF-{kappa}B activation is also mediated by c-Src tyrosine phosphorylation of I{kappa}B{alpha} [5 ]. Additional proteins, which interact directly with I{kappa}B and regulate its activity, include c-Abl [6 ], phosphoinositide-3 kinase (PI-3K) [7 ], m-calpain [8 ], protein kinase C (PKC) [9 , 10 ], and many others [3 ]. It is likely that a specific protein involved in the regulation of I{kappa}B activity is dependent on the specific stimuli and cell types. Although the biological relevance of IgE-dependent NF-{kappa}B activation in allergy is obvious, the way in which antigen (Ag) + IgE induces NF-{kappa}B activation in MC is still unclear.

IKK is a complex composed of three subunits: IKK{alpha} (IKK1), IKKß (IKK2), and IKK{gamma} [NF-{kappa}B essential modulator (NEMO)]. IKK{alpha} and IKKß are the catalytic subunits, and IKK{gamma} is the regulatory subunit [3 , 4 ]. NF-{kappa}B activation by TNF, IL-1, or lipopolysaccharide is blocked completely in the absence of IKK{gamma} and strongly reduced in the absence of IKKß [11 ]. IKK{alpha} is dispensable for cytokine-induced I{kappa}B degradation [11 ] but is critical for cytokine-induced transcription of NF-{kappa}B target genes by inducing histone phosphorylation [12 , 13 ]. In MC, a role for IKK in Ag + IgE-induced NF-{kappa}B activation and gene expression remains to be determined.

We demonstrated that a cell-permeable peptide targeting IKK and four individual chemical IKK inhibitors [15 deoxy-prostaglandin J2 (15dPGJ2), BMS-345541, SC-514, and sulindac] significantly inhibited Ag + IgE-induced TNF production by MC. IKK{alpha} is rapidly phosphorylated in MC after Ag + IgE stimulation. Inhibition of IgE-dependent IKK phosphorylation by sulindac correlates with decreased NF-{kappa}B activation and TNF production. Thus, IKK is likely involved in Fc{varepsilon}RI-induced TNF production by MC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
Antibodies to IKK{alpha}, phosphor (p)-IKK{alpha} (Ser180)/IKKß (Ser181), I{kappa}B{alpha}, and p-I{kappa}B{alpha} (Ser-32) were purchased from Cell Signaling Technology (Beverly, MA). Anti-IKKß (clone 10AG2) was obtained from Upstate (Charlottesville, VA). Anti-ß-actin (H-196) and antibodies to NF-{kappa}B p65, NF-{kappa}B p50 (H-119), I{kappa}Bß (S-20), and IKKß (H-470, partially cross-reactive with IKK{alpha}) were purchased from Santa Cruz Biotechnology (CA).

MC and stimulation
Murine bone marrow-derived MC (BMMC) were obtained by culturing bone marrow cells with 10% WEHI-3B-conditioned medium as a source of IL-3 [14 ]. After 4–6 weeks, MC purity of >99% was achieved, as assessed by toluidine blue staining of fixed cytocentrifuge preparations. For IgE-dependent activation studies, BMMC were sensitized overnight in fresh complete medium supplemented with T1B141-conditioned medium containing IgE directed against trinitrophenyl (TNP) at a ratio of 3:1. After washing, cells were stimulated with TNP-bovine serum albumin (BSA; 10 ng/ml, Biosearch Technologies, Inc., Novato, CA) for indicated times. In experiments with inhibitors, BMMC were treated with these inhibitors before stimulation with TNP-BSA, including pyrrolidine dithiocarbomate {PDTC; Sigma-Aldrich (St. Louis, MO), a NF-{kappa}B inhibitor [15 ]}, SC-514 {EMD Biosciences Inc. (San Diego, CA), an IKKß inhibitor [16 ]}, 15dPGJ2 (EMD Biosciences, an inhibitor for IKK{alpha} and IKKß [17 ]), a cell-permeable, NEMO-binding, domain-binding peptide (EMD Biosciences, an inhibitor for IKK{alpha} and IKKß, according to the manufacturer), BMS-345541 (a kind gift from Dr. James R. Burke, Bristol-Myers Squibb, New York, NY, an inhibitor for IKK{alpha} and IKKß [18 ]) for 1 h or indicated times, or sulindac (Sigma-Aldrich, an IKKß inhibitor [19 ]) for 1 or 2 h before Ag stimulation.

Preparation of nuclear extracts
Nuclear protein extracts were obtained using a nuclear extract kit (ActiveMotif, Carlsbad, CA), according to the manufacturer’s protocol. Cytoplasmic fractions were also saved. All preparation procedures were carried out at 4°C. Total protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Electrophoretic mobility shift assays (EMSA)
A consensus double-stranded NF-{kappa}B oligonucleotide (Promega, Madison, WI, 5'-AGT TGA GGG GAC TTT CCC AGG C-3') was used for EMSA. Probe-labeling was accomplished by treatment with T4 kinase (Promega) in the presence of 32P-adenosine 5'-triphosphate (Amersham Biosciences, Piscataway, NJ). Labeled oligonucleotides were purified on a Sephadex G-25 M column (Amersham Biosciences). Nuclear protein (10 µg) was added to 10 µl vol binding reaction with 1 µg poly(dI-dC; Amersham Biosciences) and incubated at room temperature for 15 min. Labeled, double-stranded NF-{kappa}B oligonucleotide was added to each reaction mixture, incubated at room temperature for 30 min, and separated by electrophoresis on a 6% polyacrylamide gel in 0.5 x Tris-boric acids–EDTA buffer. Gels were vacuum-dried and subjected to autoradiography. Cold competition was done by adding 1 µl (1.75 µM) specific unlabeled, double-stranded probe to the reaction mixture. Unlabeled, double-stranded oligonucleotide (1 µl, 1.75 µM), which does not bind NF-{kappa}B, was used for nonspecific competition. Polyclonal antibodies to p50 and p65 (1 µg/10 µl, Santa Cruz Biotechnology) were used for supershift assays for NF-{kappa}B proteins.

Immunoblotting
Denatured proteins were separated by electrophoresis on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) along with molecular weight markers. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories), which were blocked with 5% nonfat dried milk in Tris-buffered saline that contained 0.1% Tween-20. The membranes were probed with the specific primary antibody and an appropriate secondary horseradish peroxidase-conjugated antibody (anti-rabbit IgG, Cedarlane, Hornby, ON, Canada; or anti-goat IgG, Santa Cruz Biotechnology) and visualized by enhanced chemiluminescence.

TNF assay
TNF protein levels in supernatants were measured using antibodies from R&D Systems (Minneapolis, MN) by enzyme-linked immunosorbent assay (ELISA) as described previously [14 , 20 ]. The detection limit was 10 pg/ml. TNF mRNA was determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis using Assays-on-DemandTM reagents containing 6-carboxy fluorescein dye-labeled TaqMan® minor groove-binding probes (Applied Biosystems), according to the manufacturer’s protocol [20 ]. Glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous reference. Data were analyzed using the relative standard curve method according to the manufacturer’s protocol, and values from untreated MC were used as the calibrator. Thus, data are expressed as the fold-increase relative to untreated MC.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IKK inhibitors, a NEMO-binding peptide, 15dPGJ2, BMS-345541, SC-514, and sulindac blocked IgE + Ag-induced TNF production by mouse BMMC (mBMMC)
To determine if IKK plays a role in IgE-dependent TNF production by MC, various IKK inhibitors were used. These include a cell-permeable peptide targeting the regulatory subunit of the IKK complex, the NEMO-binding, domain-binding peptide, and four chemical compounds that have inhibitory effects on IKK{alpha} or IKKß, including sulindac, SC-514, 15dPGJ2, and BMS-345541. mBMMC, after IgE sensitization, were pretreated with these inhibitors for 1 h and stimulated with TNP-BSA (10 ng/ml) for 4 h. Cell-free supernatants were used to determine TNF production by ELISA. Treatment with the NEMO-binding peptide (150 µM) but not the control peptide (150 µM) inhibited TNP-induced TNF production (Fig. 1a ). Similarly, all four chemical IKK inhibitors, 15dPGJ2, BMS-345541, SC-514, and sulindac, strongly blocked IgE + TNP-induced TNF production (Fig. 1b 1c 1d 1e) , suggesting a role for IKK in IgE-dependent TNF production. These inhibitors by themselves had little effects on MC TNF production (Fig. 1a 1b 1c 1d 1e) and did not affect MC viability as tested by trypan blue assay (data not shown). In addition, the dimethyl sulfoxide (diluents for SC-514) had little effects on MC TNF production (Fig. 1d) .



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  		Figure 1. Inhibition of IKK blocks IgE + TNP-induced TNF production. mBMMC were sensitized with anti-TNP IgE overnight. IgE-sensitized BMMC were treated with medium alone (Med) or with various IKK inhibitors including a cell-permeable, NEMO-binding, domain-binding peptide (NEMO Peptide, 150 µM) or its control peptide (150 µM; Fig. 1a), 15dPGJ2 (2.5, 5, 10 µg/ml; Fig. 1b), BMS-345541 (1, 10, 100 µM; Fig. 1c), SC-514 (1, 10, 100 µM; Fig. 1d), or sulindac (0.1, 1, 3, 5 mM; Fig. 1e) for 1 h. BMMC were then stimulated with TNP-BSA (10 ng/ml; as "TNP" in figure) for 4 h or left unstimulated (as "No TNP" in figure). Cell-free supernatants were collected to determine TNF production by ELISA. Results are the means ± SE of five to eight experiments, with each condition assayed in duplicate [*, P<0.05, compared with TNP-treated cells without inhibitor treatment (TNP in Med group)].

 
To determine effects of sulindac on TNF mRNA expression, BMMC, with or without sulindac (5 mM, 1 h pretreatment) treatment, were stimulated with TNP-BSA and subjected to real-time RT-PCR analysis for TNF expression. Using values from the untreated, resting MC as a calibrator, TNP-BSA stimulation induced a 43-fold increase of TNF mRNA relative to the untreated MC. Sulindac inhibited TNP-induced TNF mRNA to the level of 6.8-fold, relative to the untreated MC.

To examine if IKK inhibitors affect IgE-dependent MC degranulation, IgE-sensitized mBMMC were pretreated with various concentrations of BMS-345541 (1, 10, or 100 µg/ml) or sulindac (1 or 5 mM) for 60 min and stimulated with TNP-BSA (10 ng/ml). Histamine release was determined. We found that BMS-345541 had little effect on MC degranulation, and sulindac, at the concentration of 5 mM, blocked histamine release (data not shown). These results support the concept that MC degranulation and cytokine TNF production are two separate events. Additional studies are needed to define if IKK is involved in IgE-dependent MC degranulation.

Ag + IgE stimulation induces activation of the IKK{alpha}-I{kappa}B{alpha}-NF-{kappa}B pathway and I{kappa}B{alpha} but not IKBß regeneration
To determine if IgE + Ag stimulation induces IKK activation, IgE-sensitized mBMMC were stimulated with TNP-BSA for 40 min, and cytoplasmic proteins were used to examine IKK{alpha} and IKKß phosphorylation and total protein levels. Initially, protein samples in PVDF membranes were blotted with an antibody, which sees phosphorylated forms of IKK{alpha} and IKKß [p-IKK{alpha} (Ser180)/IKKß (Ser181), Cell Signaling Technology]. It is interesting that phosphorylation of IKK{alpha} but not IKKß was detected in cells following TNP-BSA stimulation (Fig. 2a , p-IKK{alpha}/IKKß panel). To verify the expression of IKK{alpha} and IKKß expression in MC, the same PVDF membrane was sequentially stripped and blotted using two additional antibodies. One specifically recognizes IKK{alpha} (Cell Signaling Technology), and the other recognizes IKKß with partial reaction with IKK{alpha}, according to the manufacturer (Santa Cruz Biotechnology). MC express IKK{alpha} (Fig. 2a , Total IKK{alpha} panel) and IKKß (Fig. 2a , Total IKK{alpha}/IKKß panel), and little or no TNP-induced IKKß phosphorylation was detected (Fig. 2a , p-IKK{alpha}/IKKß panel).



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  		Figure 2. IgE + TNP stimulation induces IKK{alpha} phosphorylation and I{kappa}B{alpha}-NF-{kappa}B pathway activation. (a) mBMMC were sensitized with anti-TNP IgE overnight and stimulated with TNP-BSA (10 ng/ml) for 40 min. The cytoplasmic proteins were analyzed by Western blotting. Initially, proteins in the PVDF membrane were blotted with antibodies (Abs) to p-IKK{alpha} (Ser180)/IKKß (Ser181). Then, the membrane was stripped and blotted with an antibody specific for total IKK{alpha}. Subsequently, the same membrane was again stripped and blotted with an antibody, which sees IKKß with partial reaction with IKK{alpha}. NT, Unstimulated. (b) Anti-TNP, IgE-sensitized mBMMC were stimulated for 5, 20, and 40 min and 1, 2, 3, or 6 h with TNP-BSA (10 ng/ml). The cytoplasmic proteins were separated in different SDS-PAGE gels and analyzed by Western blotting with antibodies to p-IKKß/IKK{alpha}, IKKß, IKK{alpha}, p-I{kappa}B{alpha}, I{kappa}B{alpha}, I{kappa}Bß, or NF-{kappa}B families (p65, p105, and p50). ß-Actin was used as a control.

 
To determine IgE-dependent activation of the IKK-I{kappa}B-NF-{kappa}B pathway, phosphorylation and total protein levels of members of the IKK-I{kappa}B-NF-{kappa}B family were determined following IgE + TNP stimulation. Cytoplasmic proteins from BMMC, with or without treatment with TNP-BSA (10 ng/ml), were separated by SDS-PAGE and analyzed with various antibodies in different blots. TNP stimulation induced a significant and rapid (5 min) phosphorylation of IKK{alpha}, which lasted up to 3 h (Fig. 2b , top panel). It is interesting that no IKKß phosphorylation was observed, as this antibody sees p-IKK{alpha} (Ser180) and p-IKKß (Ser181).

IgE + TNP induced rapid (5 min) phosphorylation of I{kappa}B{alpha}, which maintained over 6 h (Fig. 2b , p-I{kappa}B{alpha} panel). IgE + TNP-induced degradation of constitutive I{kappa}B{alpha} can be seen readily after 20 min stimulation, which was followed by a prominent regeneration of I{kappa}B{alpha} (Fig. 2b , Total I{kappa}B{alpha} panel). There was a decrease of I{kappa}B{alpha} phosphorylation level at 20 min, which is likely a result of the decrease of total I{kappa}B proteins at this time-point because of the degradation of I{kappa}B{alpha} after initial phosphorylation (5 min). It is noticeable that phosphorylation of I{kappa}B{alpha} was evident after TNP stimulation for 40 min–6 h (Fig. 2b , p-I{kappa}B{alpha} panel), suggesting that newly generated I{kappa}B{alpha} was again phophorylated after TNP stimulation. Total I{kappa}Bß protein levels remained constant before or after IgE + TNP stimulation (Fig. 2b , Total I{kappa}Bß panel). An increase of NF-{kappa}B p65 protein level was observed after IgE + TNP stimulation (Fig. 2b , NF-{kappa}B, p65 panel), suggesting a regeneration of this protein after TNP stimulation.

No effect of histamine on IKK{alpha} phosphorylation was observed when BMMC were treated with histamine (10 µg/ml) for 5 min (data not shown). This excludes the possibility that IgE + TNP-induced IKK{alpha} phosphorylation is a secondary effect induced by histamine following degranulation.

Sulindac blocked IgE + TNP-induced IKK-I{kappa}B-NF-{kappa}B pathway activation and TNF production by mBMMC
Sulindac is a widely used, nonsteroidal, anti-inflammatory agent, which has inhibitory effects for IKK [19 , 21 ]. To determine if sulindac inhibits IgE-dependent TNF production through IKK-related mechanisms, IgE-sensitized BMMC were pretreated with sulindac (5 mM) for 2 h and then were stimulated with TNP-BSA (10 ng/ml) for 5 or 40 min. As shown in Figure 3 (p-IKK{alpha} panel), sulindac inhibited IgE + TNP-induced IKK{alpha} phosphorylation. IgE + TNP-induced I{kappa}B{alpha} phosphorylation at 5 min and 40 min was also inhibited by sulindac treatment (Fig. 3 , p-I{kappa}B{alpha} panel). TNP-induced I{kappa}B{alpha} degradation at 5 min was blocked by sulindac (Fig. 3 , Total I{kappa}B{alpha} panel). It is noteworthy that at 40 min after TNP stimulation, a decreased level of total I{kappa}B{alpha} was observed in sulindac-treated cells (Fig. 3 , Total I{kappa}B{alpha} panel). This is likely a result of the combination of a low level of I{kappa}B{alpha} degradation and decreased I{kappa}B{alpha} regeneration in these cells following sulindac and TNP-BSA cotreatment.



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  		Figure 3. Sulindac inhibits IgE-dependent IKK{alpha} phosphorylation and I{kappa}B{alpha} activation. After sensitization with anti-TNP IgE, mBMMC were treated with an IKK inhibitor sulindac (5 mM) for 2 h or were sham-treated with medium and then stimulated with TNP-BSA (10 ng/ml) for 5 or 40 min. The cytoplasmic proteins were analyzed by Western blotting with antibodies to p-IKK{alpha}, total IKK{alpha}, p-I{kappa}B{alpha}, total I{kappa}B{alpha}, or ß-actin. NT, Unstimulated.

 
To determine the effect of sulindac on IgE-dependent NF-{kappa}B activation, IgE-sensitized BMMC were treated with sulindac for 2 h before TNP stimulation. NF-{kappa}B activation was determined by EMSA. Sulindac treatment inhibited IgE + TNP-induced NF-{kappa}B activation (Fig. 4a ). To verify the specific binding of NF-{kappa}B with 32P-labeled NF-{kappa}B oligonucleotide, nuclear proteins from TNP-activated MC were incubated with anti-p50 or anti-p65 antibodies. The anti-p50 antibody induced a complete shift of the NF-{kappa}B-binding band. In addition, 100x concentrated, unlabeled NF-{kappa}B oligonucleotide (cold probe) was used to compete with 32P-labeled NF-{kappa}B oligonucleotide, and 100x concentrated, unlabeled AP-1 oligonucleotide was used as a nonspecific control probe. The specific inhibition of NF-{kappa}B binding by the cold NF-{kappa}B probe but not by the AP-1 probe further supports the specificity of NF-{kappa}B binding in our EMSA experiments (Fig. 4b) .



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  		Figure 4. Sulindac inhibits IgE-dependent NF-{kappa}B activation and TNF production. Anti-TNP, IgE-sensitized BMMC were treated with an IKK inhibitor sulindac (5 mM) for 2 h or sham-treated with medium and then stimulated with TNP-BSA (10 ng/ml) for 5, 20, and 40 min and 1, 3, or 6 h or were unstimulated (NT). (a) Nuclear extracts were isolated and analyzed by EMSA using 32P-labeled NF-{kappa}B oligonucleotide. (b) To verify the specificity binding of NF-{kappa}B with oligonucleotide probe, TNP-activated MC nuclear proteins were incubated with anti-p50 or anti-p65 antibodies (Ab). Furthermore, 100x concentrated, unlabeled NF-{kappa}B oligonucleotide was used to compete with 32P-labeled NF-{kappa}B oligonucleotide, and 100x concentrated, unlabeled activator protein-1 (AP-1) oligonucleotide was used as a nonspecific control probe. (c) Cell-free supernatants were used to determine TNF levels by ELISA.

 
To examine the correlations between the effects of sulindac on IgE-dependent NF-{kappa}B activation and TNF production, cell-free supernatants collected from sulindac-treated cells as described in Figure 4a were used to determine TNF by ELISA. Consistent with the inhibitory effects of sulindac on IKK, I{kappa}B{alpha}, and NF-{kappa}B activation, treatment with sulindac completely blocked IgE + TNP-induced cytokine TNF production (Fig. 4c) .

To determine a concentration-dependent effect of sulindac on IgE-dependent MC activation, IgE-sensitized BMMC were pretreated with sulindac at the concentrations of 0.1, 1, and 5 mM for 2 h and stimulated with TNP for 6 h. As shown in Figure 5a 5b 5c , TNP-induced phosphorylation and synthesis of IKK{alpha} and I{kappa}B{alpha} and activation of NF-{kappa}B were blocked by sulindac in a concentration-dependent manner.



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  		Figure 5. Concentration-dependent inhibition of sulindac on IKK{alpha} phosphorylation and I{kappa}B-NF-{kappa}B pathway activation. mBMMC, after sensitization with IgE, were pretreated with sulindac at the concentrations of 0.1, 1.0, or 5.0 mM for 2 h and then stimulated with TNP-BSA (10 ng/ml) for 40 min (a), or 3 h (b and c). Cytoplasmic proteins were analyzed by Western blotting with antibodies to p-IKK{alpha}, total IKK{alpha}, or ß-actin (a) or antibodies to p-I{kappa}B{alpha}, I{kappa}B{alpha}, I{kappa}Bß, NF-{kappa}B, p50 p65, or ß-actin (b). Nuclear extracts were isolated and analyzed by EMSA using 32P-labeled NF-{kappa}B oligonucleotide (c).

 
PDTC blocks Fc{varepsilon}RI-dependent cytokine production and synthesis of I{kappa}B and NF-{kappa}B
In addition to NF-{kappa}B, several transcription factors such as AP-1 and NF of activated T cells have been implicated in IgE-dependent MC cytokine production [22 ]. To examine the importance of the IKK-I{kappa}B-NF-{kappa}B pathway in IgE-dependent TNF production, BMMC were pretreated with a NF-{kappa}B inhibitor PDTC before TNP stimulation. PDTC inhibited IgE + TNP-induced TNF production in a dose-dependent manner (Fig. 6a ), suggesting a role for NF-{kappa}B in IgE-dependent MC TNF production.



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  		Figure 6. PDTC blocks IgE-dependent I{kappa}B and NF-{kappa}B synthesis. (a–c) BMMC, after sensitization with IgE, were pretreated with various concentrations of PDTC for 1 h and then were stimulated with TNP-BSA (10 ng/ml) for 3 h. (a) TNF levels in cell-free supernatants were determined by ELISA. (b) The cytoplasmic proteins were analyzed by Western blotting with antibodies to IKK{alpha}, p-I{kappa}B{alpha}, I{kappa}B{alpha}, I{kappa}Bß, or ß-actin. (c and d) PDTC (50 µM) was added to BMMC before (–30 min or –1 h), at the same time (0), or after (+20 min or +40 min) TNP-BSA (10 ng/ml) stimulation. Nuclear extracts were isolated and analyzed for NF-{kappa}B activation by EMSA (c), and cell-free supernatants were used to determine TNF production by ELISA (d).

 
IgE + TNP-induced stimulation also induces resynthesis of I{kappa}B{alpha} and an increase of protein levels of IKK{alpha}, as well as several NF-{kappa}B family members (Fig. 2b) , suggesting a dynamic involvement of the IKK-I{kappa}B-NF-{kappa}B pathway in IgE-induced MC activation. To examine a role for NF-{kappa}B in IgE-induced IKK and I{kappa}B synthesis, BMMC were treated with PDTC (50, 100, 500 µM) for 1 h before TNP-BSA stimulation. PDTC treatment had little effect on IgE + TNP-induced IKK{alpha} synthesis, even at the high concentration of 500 µM (Fig. 6b , Total IKK{alpha} panel) but strongly inhibited I{kappa}B{alpha} regeneration (Fig. 6b , Total I{kappa}B{alpha} panel), suggesting that synthesis of I{kappa}B{alpha} but not IKK{alpha} is regulated by NF-{kappa}B. It is interesting that although total I{kappa}B{alpha} levels were low in PDTC-treated cells, the phosphorylation level of this protein remained high in these cells (Fig. 6b , p-I{kappa}B{alpha} panel), a piece of evidence that deserves further study. In addition, I{kappa}Bß protein level was not affected by PDTC treatment (Fig. 6b , Total I{kappa}Bß panel). Basal I{kappa}B{alpha} levels appear to be unaffected by PDTC treatment (50 µM, 4 h; data not shown).

Stimulation with IgE + TNP induces degradation of I{kappa}B{alpha} (20 min), which is followed with regeneration of I{kappa}B{alpha} (Fig. 2b , Total I{kappa}B{alpha} panel). It is interesting that phosphorylation of I{kappa}B{alpha} lasts hours, suggesting that newly synthesized I{kappa}B{alpha} becomes phosphorylated (Fig. 2b , p-I{kappa}B{alpha} panel). Thus, IgE + TNP-induced MC cytokine production likely involves constitutively expressed and newly synthesized I{kappa}B{alpha}. To examine if constitutively expressed I{kappa}B{alpha} and other NF-{kappa}B family members are sufficient in mediating IgE + TNP-induced NF-{kappa}B activation and cytokine production, PDTC was added to BMMC before and after TNP-BSA stimulation. NF-{kappa}B activation was significantly inhibited when PDTC was added to BMMC before (1 h or 30 min), together with, or after (20 min) TNP-BSA stimulation (Fig. 6c and 6d) . When PDTC was added to BMMC 40 min after TNP stimulation, evident NF-{kappa}B activities were observed (Fig. 6c) . It is interesting that PDTC only partially inhibited IgE + TNP-induced TNF production when it was added after TNP stimulation (Fig. 6d) , suggesting constitutively expressed and newly synthesized I{kappa}B and NF-{kappa}B members are involved in IgE-dependent MC cytokine production.

IgE-dependent IKK{alpha} phosphorylation was inhibited by Ro 31-8220
To examine the potential mechanisms involved in IgE-dependent IKK{alpha} phosphorylation, BMMC, after IgE sensitization, were treated for 2 h with several protein kinase inhibitors including PP2 (Src family protein tyrosine kinase inhibitor, 10 µM), SB 203580 [p38 mitogen-activated protein kinase (MAPK) inhibitor, 10 µM], PD 98059 [MAPK and MAPK kinase (MEK) inhibitor, 50 µM], wortmannin (PI-3K inhibitor, 100 nM), and Ro 31-8220 (PKC inhibitor, 5 µM). It is interesting that Ro 31-8220 but not other kinase inhibitors blocked IgE + TNP-induced IKK{alpha} and I{kappa}B{alpha} phosphorylation (Fig. 7 , p-IKK{alpha} and p-I{kappa}B{alpha} panels), suggesting a role for PKC in IgE-dependent IKK-I{kappa}B-NF-{kappa}B activation.



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  		Figure 7. Effects of kinase inhibitors on IgE-dependent IKK{alpha} phosphorylation and I{kappa}B{alpha} phosphorylation and degradation. BMMC, after sensitization with IgE, were pretreated for 2 h with the following protein kinase inhibitors: PP2 (10 µM), SB 203580 (10 µM), PD 98059 (50 µM), wortmannin (100 nM), and Ro 31-8220 (PKC inhibitor, 5 µM). Treated or untreated (NT) BMMC were stimulated with TNP-BSA for 10 min, and cytoplasmic proteins were analyzed by Western blotting using antibodies to p-IKK{alpha}, IKK{alpha}, p-I{kappa}B{alpha}, I{kappa}B{alpha}, I{kappa}Bß, or ß-actin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although Fc{varepsilon}RI-induced NF-{kappa}B activation and I{kappa}B phosphorylation have been reported [1 , 2 ], little is known about the mechanisms directly involved in Fc{varepsilon}RI-induced I{kappa}B-NF-{kappa}B activation in MC. Depending on the nature of specific stimuli-receptor involved, a number of signaling molecules (>12), including IKK [3 , 4 ], c-Src [5 ], pp90rsk [23 ], m-calpain [8 ], and others, have been shown to interact directly with I{kappa}B in different cell types. We examined if IKK is required for IgE-dependent cytokine production by MC. Five IKK inhibitors were used including 15dPGJ2, BMS-345541, and a peptide that inhibits IKK{alpha} and IKKß activities and SC-514 and sulindac, which inhibit IKKß. All five IKK inhibitors strongly blocked IgE + TNP-induced TNF production by MC. Moreover, activation of MC by IgE + TNP-BSA induced a rapid IKK{alpha} phosphorylation (5 min), which was accompanied with a rapid I{kappa}B{alpha} phosphorylation (5 min) and subsequent I{kappa}B{alpha} degradation, a sustained NF-{kappa}B activation (>6 h), and production of TNF. These data support a role for IKK in the initiation of an IKK-I{kappa}B-NF-{kappa}B cascade following Fc{varepsilon}RI-cross-linking in MC. The rapid phosphorylation of IKK and I{kappa}B suggests that this effect is most likely directly induced by Fc{varepsilon}RI cross-linking and excludes the possibility of the secondary effect induced by MC-secreted mediators such as TNF [24 ].

It is noteworthy that no IKKß phosphorylation was observed in activated BMMC, although in other cell types, IKKß is considered mainly responsible for I{kappa}B phosphorylation [25 , 26 ], and IKK{alpha} is required for processing of NF-{kappa}B2 [27 ]. However, this cannot exclude a potential role for IKKß in IgE-dependent TNF production, as inhibitors that block IKKß activity, such as SC-514 [16 ] or sulindac [19 ], inhibited IgE + TNP-induced TNF production. In addition, a recent report showed IKKß phosphorylation in a rat MC line, BRL 2H3 cells [28 ]. Thus, further study is required to determine the specific contribution of IKK{alpha} versus IKKß in IgE-dependent I{kappa}B-NF-{kappa}B pathway activation and subsequent TNF production by MC. Considering the significant role of MC in allergy, our finding of IKK in IgE + TNP-induced signaling in MC, together with a recent report of IKK activation in bronchiolar epithelium of allergic airway [29 ], suggests that IKK may have an important role in the pathogenesis of allergic diseases.

Our results also showed that Fc{varepsilon}RI cross-linking on MC induces a new synthesis of several signaling molecules including I{kappa}B{alpha} and NF-{kappa}B p65. It is interesting that an IgE-induced increase of I{kappa}B{alpha} synthesis is accompanied with a sustained I{kappa}B{alpha} phosphorylation without evident I{kappa}B{alpha} decrease (degradation; from 40 min to 6 h). This may likely be a result of the fact that IgE-induced synthesis of I{kappa}B{alpha} exceeds its degradation. To differentiate the role of the constitutively existed I{kappa}B and the newly synthesized I{kappa}B in IgE-dependent MC activation, PDTC was used to block NF-{kappa}B activity, as I{kappa}B synthesis is NF-{kappa}B-dependent. PDTC was added at various times before or after TNP stimulation. When BMMC were initially activated by IgE + TNP, which allows the constitutively existed I{kappa}B{alpha} phosphorylation and degradation, and then followed by treatment with PDTC to block the new synthesis of I{kappa}B, IgE + TNP-induced cytokine production was inhibited. These data support a role for the newly synthesized I{kappa}B{alpha} and NF-{kappa}B p65 in IgE-dependent NF-{kappa}B activation and cytokine production. The prolonged phosphorylation of IKK, sustained I{kappa}B{alpha} phosphorylation, and NF-{kappa}B activation and significant synthesis of IKK and NF-{kappa}B families suggest that Fc{varepsilon}RI aggregation induces a dynamic IKK-I{kappa}B-NF-{kappa}B activation cycle that leads to cytokine production.

Regulation of IgE-dependent IKK activation in MC has not been reported previously. In other cell types, IKK is associated with and regulated by several signaling molecules such as c-Src [30 ], PKC{theta} [31 ], protein phosphatase 2A [32 ], or steroid receptor coactivator [33 ]. In MC, we were unable to demonstrate physical association between IKK and PP2A (data not shown), although such association was shown in MT4 cells [32 ]. In other cells, IKK{alpha} is preferentially phosphorylated by NF-{kappa}B-inducing kinase [34 ], and IKKß was preferentially phosphorylated by MEK kinase 1 [35 ]. We attempted to examine mechanisms involved in IgE-dependent IKK activation by using several inhibitors including inhibitors for PKC (Ro 31-8220), Src tyrosine kinase (PP2), PI-3K (wortmannin), MAPK p38 (SB 203580), and MEK (PD 98059). It is interesting that only the PKC inhibitor demonstrated an inhibitory effect on IgE + TNP-induced IKK{alpha} phosphorylation, suggesting that PKC may serve as an up-stream molecule in IgE-induced MC activation. Consistent with this notion, PKCß controls NF-{kappa}B activation in B cells through selective regulation of the IKK{alpha} [36 ].

Based on these findings, we propose a working model for the regulatory role of IKK in IgE-dependent TNF production (Fig. 8 ). Ag-induced cross-linking of IgE-Fc{varepsilon}RI mediates PKC activation and subsequent IKK activation, leading to I{kappa}B{alpha} phosphorylation and degradation. This allows NF-{kappa}B to translocate into the nucleus and initiates TNF production. Activation of NF-{kappa}B also induces new synthesis of I{kappa}B{alpha} and NF-{kappa}B p65. As newly synthesized I{kappa}B{alpha} is phosphorylated rapidly during Ag stimulation, which is accompanied with prolonged phosphorylation of IKK{alpha} and sustained NF-{kappa}B activation, Fc{varepsilon}RI aggregation induces a dynamic IKK{alpha}-I{kappa}B{alpha}-NF-{kappa}B activation cycle that leads to TNF production. Blockade of IKK{alpha}-I{kappa}B{alpha}-NF-{kappa}B pathway activation and subsequent TNF production by IKK inhibitors or by NF-{kappa}B inhibitor PDTC suggests an essential role of this pathway in IgE-dependent MC TNF production.



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  		Figure 8. Working model for the regulatory role of IKK in IgE-dependent TNF production by MC. Ag-induced cross-linking of IgE-Fc{varepsilon}RI mediates PKC activation and subsequent IKK activation, leading to I{kappa}B{alpha} phosphorylation and degradation. This allows NF-{kappa}B to translocate into the nucleus and initiates TNF production. Activation of NF-{kappa}B also induces a new synthesis of I{kappa}B{alpha} and NF-{kappa}B p65. As newly synthesized I{kappa}B{alpha} is phosphorylated rapidly during Ag stimulation, which is accompanied with prolonged phosphorylation of IKK and sustained NF-{kappa}B activation, Fc{varepsilon}RI aggregation induces a dynamic IKK-I{kappa}B-NF-{kappa}B activation cycle that leads to TNF production. Inhibition of IKK by the IKK inhibitor blocks IgE + Ag-induced TNF production by MC.

 


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Canadian Institutes of Health Research (CIHR; ROP 44944 and MGC-57081), Canadian Cystic Fibrosis Foundation, Canadian Foundation for Innovation, and Isaac Walton Killam (IWK) Health Centre to T-J. L. M. R. P. is supported by a Studentship from IWK Health Centre. T-J. L. is the recipient of a New Investigator Award from CIHR and an Investigatorship Award from the IWK Health Centre.


   FOOTNOTES
 
1 Current address: Department of Endocrinology, Diabetes Research Laboratory, The First People’s Hospital, Shanghai Jiaotong University, 85 Wujin Road, Shanghai 200080, P. R. China Back

Received February 26, 2004; revised February 25, 2005; accepted February 27, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kalesnikoff, J., Baur, N., Leitges, M., Hughes, M. R., Damen, J. E., Huber, M., Krystal, G. (2002) SHIP negatively regulates IgE + antigen-induced IL-6 production in mast cells by inhibiting NF-{kappa} B activity J. Immunol. 168,4737-4746[Abstract/Free Full Text]
  2. Marquardt, D. L., Walker, L. L. (2000) Dependence of mast cell IgE-mediated cytokine production on nuclear factor-{kappa}B activity J. Allergy Clin. Immunol. 105,500-505[CrossRef][Medline]
  3. Karin, M., Ben-Neriah, Y. (2000) Phosphorylation meets ubiquitination: the control of NF-[{kappa}]B activity Annu. Rev. Immunol. 18,621-663[CrossRef][Medline]
  4. Ghosh, S., Karin, M. (2002) Missing pieces in the NF-{kappa}B puzzle Cell 109(Suppl),S81-S96
  5. Abu-Amer, Y., Ross, F. P., McHugh, K. P., Livolsi, A., Peyron, J. F., Teitelbaum, S. L. (1998) Tumor necrosis factor-{alpha} activation of nuclear transcription factor-{kappa}B in marrow macrophages is mediated by c-Src tyrosine phosphorylation of I{kappa} B{alpha} J. Biol. Chem. 273,29417-29423[Abstract/Free Full Text]
  6. Kawai, H., Nie, L., Yuan, Z. M. (2002) Inactivation of NF-{kappa}B-dependent cell survival, a novel mechanism for the proapoptotic function of c-Abl Mol. Cell. Biol. 22,6079-6088[Abstract/Free Full Text]
  7. Beraud, C., Henzel, W. J., Baeuerle, P. A. (1999) Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-{kappa}B activation Proc. Natl. Acad. Sci. USA 96,429-434[Abstract/Free Full Text]
  8. Han, Y., Weinman, S., Boldogh, I., Walker, R. K., Brasier, A. R. (1999) Tumor necrosis factor-{alpha}-inducible I{kappa}B{alpha} proteolysis mediated by cytosolic m-calpain. A mechanism parallel to the ubiquitin-proteasome pathway for nuclear factor-{kappa}b activation J. Biol. Chem. 274,787-794[Abstract/Free Full Text]
  9. Han, Y., Meng, T., Murray, N. R., Fields, A. P., Brasier, A. R. (1999) Interleukin-1-induced nuclear factor-{kappa}B-I{kappa}B{alpha} autoregulatory feedback loop in hepatocytes. A role for protein kinase c{alpha} in post-transcriptional regulation of i{kappa}b{alpha} resynthesis J. Biol. Chem. 274,939-947[Abstract/Free Full Text]
  10. Lallena, M. J., Diaz-Meco, M. T., Bren, G., Paya, C. V., Moscat, J. (1999) Activation of I{kappa}B kinase ß by protein kinase C isoforms Mol. Cell. Biol. 19,2180-2188[Abstract/Free Full Text]
  11. Israel, A. (2000) The IKK complex: an integrator of all signals that activate NF-{kappa}B? Trends Cell Biol. 10,129-133[CrossRef][Medline]
  12. Anest, V., Hanson, J. L., Cogswell, P. C., Steinbrecher, K. A., Strahl, B. D., Baldwin, A. S. (2003) A nucleosomal function for I{kappa}B kinase-{alpha} in NF-{kappa}B-dependent gene expression Nature 423,659-663[CrossRef][Medline]
  13. Yamamoto, Y., Verma, U. N., Prajapati, S., Kwak, Y. T., Gaynor, R. B. (2003) Histone H3 phosphorylation by IKK-{alpha} is critical for cytokine-induced gene expression Nature 423,655-659[CrossRef][Medline]
  14. Boudreau, R. T., Garduno, R., Lin, T. J. (2002) Protein phosphatase 2A and protein kinase C{alpha} are physically associated and are involved in Pseudomonas aeruginosa-induced interleukin 6 production by mast cells J. Biol. Chem. 277,5322-5329[Abstract/Free Full Text]
  15. Liu, S. F., Ye, X., Malik, A. B. (1999) Inhibition of NF-{kappa}B activation by pyrrolidine dithiocarbamate prevents in vivo expression of proinflammatory genes Circulation 100,1330-1337[Abstract/Free Full Text]
  16. Kishore, N., Sommers, C., Mathialagan, S., Guzova, J., Yao, M., Hauser, S., Huynh, K., Bonar, S., Mielke, C., Albee, L., Weier, R., Graneto, M., Hanau, C., Perry, T., Tripp, C. S. (2003) A selective IKK-2 inhibitor blocks NF-{kappa} B-dependent gene expression in interleukin-1 ß-stimulated synovial fibroblasts J. Biol. Chem. 278,32861-32871[Abstract/Free Full Text]
  17. Rossi, A., Kapahi, P., Natoli, G., Takahashi, T., Chen, Y., Karin, M., Santoro, M. G. (2000) Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of I{kappa}B kinase Nature 403,103-108[CrossRef][Medline]
  18. Burke, J. R., Pattoli, M. A., Gregor, K. R., Brassil, P. J., MacMaster, J. F., McIntyre, K. W., Yang, X., Iotzova, V. S., Clarke, W., Strnad, J., Qiu, Y., Zusi, F. C. (2003) BMS-345541 is a highly selective inhibitor of I {kappa} B kinase that binds at an allosteric site of the enzyme and blocks NF-{kappa} B-dependent transcription in mice J. Biol. Chem. 278,1450-1456[Abstract/Free Full Text]
  19. Yamamoto, Y., Yin, M. J., Lin, K. M., Gaynor, R. B. (1999) Sulindac inhibits activation of the NF-{kappa}B pathway J. Biol. Chem. 274,27307-27314[Abstract/Free Full Text]
  20. Power, M. R., Peng, Y., Maydanski, E., Marshall, J. S., Lin, T. J. (2004) The development of early host response to Pseudomonas aeruginosa lung infection is critically dependent on myeloid differentiation factor 88 in mice J. Biol. Chem. 279,49315-49322[Abstract/Free Full Text]
  21. Kwak, Y. T., Guo, J., Shen, J., Gaynor, R. B. (2000) Analysis of domains in the IKK{alpha} and IKKß proteins that regulate their kinase activity J. Biol. Chem. 275,14752-14759[Abstract/Free Full Text]
  22. Lorentz, A., Klopp, I., Gebhardt, T., Manns, M. P., Bischoff, S. C. (2003) Role of activator protein 1, nuclear factor-{kappa}B, and nuclear factor of activated T cells in IgE receptor-mediated cytokine expression in mature human mast cells J. Allergy Clin. Immunol. 111,1062-1068[CrossRef][Medline]
  23. Ghoda, L., Lin, X., Greene, W. C. (1997) The 90-kDa ribosomal S6 kinase (pp90rsk) phosphorylates the N-terminal regulatory domain of I{kappa}B{alpha} and stimulates its degradation in vitro J. Biol. Chem. 272,21281-21288[Abstract/Free Full Text]
  24. Coward, W. R., Okayama, Y., Sagara, H., Wilson, S. J., Holgate, S. T., Church, M. K. (2002) NF-{kappa} B and TNF-{alpha}: a positive autocrine loop in human lung mast cells? J. Immunol. 169,5287-5293[Abstract/Free Full Text]
  25. Delhase, M., Hayakawa, M., Chen, Y., Karin, M. (1999) Positive and negative regulation of I{kappa}B kinase activity through IKKß subunit phosphorylation Science 284,309-313[Abstract/Free Full Text]
  26. Senftleben, U., Karin, M. (2002) The IKK/NF-{kappa}B pathway Crit. Care Med. 30,S18-S26[CrossRef][Medline]
  27. Senftleben, U., Cao, Y., Xiao, G., Greten, F. R., Krahn, G., Bonizzi, G., Chen, Y., Hu, Y., Fong, A., Sun, S. C., Karin, M. (2001) Activation by IKK{alpha} of a second, evolutionary conserved, NF-{kappa} B signaling pathway Science 293,1495-1499[Abstract/Free Full Text]
  28. Qu, X., Sada, K., Kyo, S., Maeno, K., Miah, S. M., Yamamura, H. (2004) Negative regulation of Fc{varepsilon}RI-mediated mast cell activation by a ubiquitin-protein ligase Cbl-b Blood 103,1779-1786[Abstract/Free Full Text]
  29. Poynter, M. E., Irvin, C. G., Janssen-Heininger, Y. M. (2002) Rapid activation of nuclear factor-{kappa}B in airway epithelium in a murine model of allergic airway inflammation Am. J. Pathol. 160,1325-1334[Abstract/Free Full Text]
  30. Huang, W. C., Chen, J. J., Chen, C. C. (2003) c-Src-dependent tyrosine phosphorylation of IKKß is involved in tumor necrosis factor-{alpha}-induced intercellular adhesion molecule-1 expression J. Biol. Chem. 278,9944-9952[Abstract/Free Full Text]
  31. Khoshnan, A., Bae, D., Tindell, C. A., Nel, A. E. (2000) The physical association of protein kinase C {theta} with a lipid raft-associated inhibitor of {kappa} B factor kinase (IKK) complex plays a role in the activation of the NF-{kappa} B cascade by TCR and CD28 J. Immunol. 165,6933-6940[Abstract/Free Full Text]
  32. Fu, D. X., Kuo, Y. L., Liu, B. Y., Jeang, K. T., Giam, C. Z. (2003) Human T-lymphotropic virus type I tax activates I-{kappa} B kinase by inhibiting I-{kappa} B kinase-associated serine/threonine protein phosphatase 2A J. Biol. Chem. 278,1487-1493[Abstract/Free Full Text]
  33. Wu, R. C., Qin, J., Hashimoto, Y., Wong, J., Xu, J., Tsai, S. Y., Tsai, M. J., O’Malley, B. W. (2002) Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) coactivator activity by I {kappa} B kinase Mol. Cell. Biol. 22,3549-3561[Abstract/Free Full Text]
  34. Ling, L., Cao, Z., Goeddel, D. V. (1998) NF-{kappa}B-inducing kinase activates IKK-{alpha} by phosphorylation of Ser-176 Proc. Natl. Acad. Sci. USA 95,3792-3797[Abstract/Free Full Text]
  35. Nakano, H., Shindo, M., Sakon, S., Nishinaka, S., Mihara, M., Yagita, H., Okumura, K. (1998) Differential regulation of I{kappa}B kinase {alpha} and ß 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[Abstract/Free Full Text]
  36. Saijo, K., Mecklenbrauker, I., Santana, A., Leitger, M., Schmedt, C., Tarakhovsky, A. (2002) Protein kinase C ß controls nuclear factor {kappa}B activation in B cells through selective regulation of the I{kappa}B kinase {alpha} J. Exp. Med. 195,1647-1652[Abstract/Free Full Text]



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