Introduction

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Cells lining the respiratory tract are often exposed to reactive oxygen intermediates (ROI) resulting from inhalation of environmental pollutants (1), bacterial or viral infections (2, 3), or changes in oxygen tension (4). The responses induced by exposure of lung cells to oxidative stress vary from necrotic and/or apoptotic cell death to inflammation (5, 6), depending on the cell type and oxidant exposure. Many of these responses are orchestrated by changes in gene expression. Consequently, considerable effort has been directed toward examining the role of transcription factors, such as nuclear factor (NF) B, in oxidant-induced gene expression changes in airway cells.

In most unstimulated cell types, NF-B is sequestered as an inactive trimer, consisting of the transcriptionally active NF-B dimer, which is bound to an inhibitory subunit, IB. There are five known members of the NF-B family: p50, p65 (RelA), p52, c-Rel, and RelB, of which p65, RelB, and c-Rel are transcriptionally active (7). There are also several forms of IB, including IB , ,  , and Bcl-3 (7), which contain ankyrin-like repeat domains and regulate the DNA binding and subcellular localization of NF-B by masking their nuclear localization domain. Most work has focused on the predominant form of NF-B, the p65/p50 heterodimer, and its association with IB. Information regarding the NF-B activation cascade has been gathered mainly from cells activated with pro-inflammatory cytokines, such as interleukin (IL)-1 and tumor necrosis factor (TNF)-. However, many other stimuli, such as viral and bacterial products, T- and B-cell mitogens, phorbol esters, okadaic acid, and ionizing radiation have also been shown to be potent activators of NF-B (8). Although the upstream signal transduction cascades may vary with different stimuli, it is likely that most NF-B activation cascades converge at or near the point of activation of the IB kinases (IKKs), large multisubunit complexes that catalyze the phosphorylation of two N-terminal serine residues of IB (8). N-terminal phosphorylation of IB leads to the immediate recognition of phospho-IB by the ubiquitin- ligase complex, culminating in the polyubiquitination of IB, which in turn targets IB for rapid degradation by the 26S proteasome. Phosphorylation and polyubiquitination of IB are not sufficient to dissociate IB from NF-B and release the transcription factor to translocate into the nucleus because inhibitors of the 26S proteasome block nuclear translocation of NF-B.

It has been suggested that oxidative stress is a common intermediate in the activation of NF-B by diverse agents (9). This idea was supported by the observation that soluble antioxidants as well as overexpression of antioxidant enzymes modulate the activation of NF-B by some agents (10). In addition, increasing the levels of ROI, either by direct addition of H₂O₂ or by adding stimulants that also increase intracellular ROI levels, has been demonstrated to activate NF-B in some cell lines (11, 12). However, recently this model of oxidative stress as a universal mediator of NF-B activation has been challenged (13, 14). Whereas HeLa cells and a subclone of Jurkat T cells have been shown to activate NF-B after stimulation with H₂O₂, a number of other cell types, including KB epidermal cells (15), monocytic cells (16), and astrocytoma cells (17), appear to be insensitive to stimulation with H₂O₂. Furthermore, although there are numerous studies demonstrating activation of NF-B in vascular endothelial cell culture models stimulated with H₂O₂, some publications report the opposite effect. For example, high concentrations of H₂O₂ (1 mM) increased NF-B DNA binding in human umbilical vein endothelial cells (HUVECs) (18), whereas lower concentrations (50 to 200 µM) were unable to increase NF-B activity in the HUVEC culture model (19, 20). Furthermore, stimulation of a transformed human dermal microvessel endothelial cell line (HMEC-1) with H₂O₂ increased NF-B activity in one study (21), whereas it had no effect on NF-B activity in another study (22). Taken together, these observations indicate that stimulation with H₂O₂ can have opposite effects on NF-B activity in the same cell type.

In addition, the NF-B activation cascades induced by stimulation with either TNF- or IL-1 have been partially characterized from the receptor to the nucleus and the current model does not indicate any requirement for oxidative stress (23). We have recently demonstrated that stimulation with TNF- does not induce a detectable oxidative stress in bronchial epithelial cells and that TNF-- induced activation of NF-B is not affected by overexpression of the antioxidant enzymes catalase or copper zinc superoxide dismutase (24). In the same study, we showed that stimulation with vanadyl sulfate does induce an oxidative stress and that overexpression of catalase blocks vanadyl-induced activation of NF-B. These data suggest the existence of oxidant-sensitive and oxidant-insensitive pathways leading to activation of NF-B in the same cell. Whether both of these pathways act through the same IKK complex remains to be determined.

In this study, we examined the effects of an oxidative stress imposed by addition of exogenous H₂O₂ on NF-B activation in human bronchial epithelial cells. Our data show that although treatment with H₂O₂ increases IKK activity and serine 32 phosphorylation of endogenous IB in a dose-dependent manner, there is no detectable IB breakdown or nuclear translocation of NF-B induced by H₂O₂. In addition, H₂O₂ inhibits the TNF--induced NF-B nuclear translocation and B-dependent transcription in bronchial epithelial cells. Treatment with H₂O₂ enhances the level of ubiquitinated proteins in these cells, suggesting that H₂O₂ interferes with proteasomal degradation of polyubiquitinated proteins and thus with the NF-B activation cascade.

	Materials and Methods

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Cell Culture and In Vitro Exposure

Primary normal human bronchial epithelial (NHBE) cells and the human BEAS-2B bronchoepithelial cell line were cultured as described previously (25, 26). H₂O₂ (Mallinckrodt Baker, Inc., Paris, KY) or TNF- (Peprotech Inc., Rocky Hill, NJ) were diluted in bronchial epithelial growth medium (NHBE) or keratinocyte growth medium (BEAS-2B) (both from Clonetics, San Diego, CA) before addition to the cell culture. In some experiments, 20 µM Z-Leu-Leu-Leu-CHO (MG 132; Calbiochem, San Diego, CA) or the respective dimethyl sulfoxide vehicle control were added 30 min before H₂O₂ or TNF- challenge. Effects of H₂O₂ or TNF- treatment on cell viability were measured with a fluorescence LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR) as per the supplier's instructions.

IKK Activity Assay

A GST-IB(1-54) fusion protein was prepared as follows. A double-stranded complementry DNA (cDNA) encoding the first 54 amino acids of IB flanked by a 5' BamHI restriction site and a 3' stop codon and a SalI restriction enzyme site was synthesized by polymerase chain reaction using the following oligonucleotide primers: 5'-ggatCCATGTTCCAGGCGGCCGAGC-3' and 5'-gtcgactaGAGGCGGATCTCCTGCAGCTCC-3. Lowercase bases indicate added flanking sequence not present in IB. The amplification products were subcloned into pCR4BLUNT-TOPO (Invitrogen, San Diego, CA) and the BamHI-SalI insert of a single clone with the correct sequence was ligated into BamHI and SalI-digested pGEX-6P-1 (AP Biotech, Arlington Heights, IN). The GST-IB fusion protein was expressed in BL21-CodonPlus-RIL cells (Stratagene, San Diego, CA) and isolated by affinity chromatography on glutathione agarose (Sigma, St. Louis, MO). One OD280 unit of GST-IB(1-54) (approximately 1.5 µg) was used per kinase assay. Cells were lysed in a buffer containing 0.1% Nonidet P-40 (NP-40), 250 mM NaCl, 50 mM Hepes, pH 7.8, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 20 mM -glycerophosphate, 20 mM NaF, 1 mM Na₃VO₄, 5.4 mM p-nitrophenyl phosphate, and protease inhibitors (1 mM AEBSF, 0.8 µM aprotinin, 50 µM bestatin, 15 µM E-64, 20 µM leupeptin, 10 µM pepstatin A; all part of Protease Inhibitor Cocktail Set III, Calbiochem). One milligram of the lysates was immunoprecipitated using 0.5 µg of anti-IKK/  antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4°C for 1 h, followed by addition of protein G agarose beads and further incubation for 2 h. After washing with kinase buffer (20 mM Hepes, pH 7.7, 20 mM -glycerophosphate, 1 mM MnCl₂, 5 mM MgCl₂, 2 mM NaF, 300 µM Na₃VO₄, 1 mM DTT), the immunoprecipitate was divided into two equal fractions. One fraction of the immunoprecipitate was resuspended in Laemmli buffer and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 9% Tris-glycine gels, followed by immunoblotting with antibodies against IKK/ (Santa Cruz Biotechnology). The other fraction of the immunoprecipitate was resuspended in 20 µl kinase buffer and mixed with GST-IB(1-54) substrate and 5 µCi [-³²P]adenosine triphosphate (ATP). In selected experiments, 500 µM H₂O₂ was added to the kinase reaction mixture before starting the kinase assay. The mixture was incubated at 30°C for 30 min and the samples were analyzed on a 12% SDS-PAGE. Gels were dried and analyzed by phosphorimaging (Molecular Dynamics, Sunnyvale, CA). IKK activities were normalized to IKK levels in the immunoprecipitates and expressed as fold induction over media controls.

Separation of Cytoplasmic and Nuclear Fractions

After washing NHBE cells with ice-cold phosphate-buffered saline, 200 µl of cold cytoplasmic extraction buffer (CEB) (10 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM DTT) with protease inhibitors (as described previously) was added to each well. Using a rubber policeman, cells were scraped up and transferred into a microcentrifuge tube. The cells were allowed to swell on ice for 15 min, then NP-40 (Sigma) was added to a final concentration of 0.1% and the tube was vortexed for 10 s. Nuclei were pelleted by centrifugation at 15,000 × g for 30 s. The supernatant, containing the cytoplasmic fraction, was mixed with one-quarter volume of 4× loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.7 M -mercaptoethanol, 0.05% bromophenol blue), denatured at 95°C for 10 min, and stored at 70°C for immunoblot analysis. Protein content of a small aliquot of the cytoplasmic fraction was determined using the DC Bradford assay (BioRad, Richmond, CA). The nuclei were washed with CEB/ protease inhibitors (PI) and centrifuged again at 15,000 × g for 30 s. The supernatant was aspirated and the nuclei were incubated for 10 min on ice in nuclear extraction buffer (20 mM Tris-HCl, pH 8.0, 400 mM NaCl, 1.5 mM Mg Cl₂, 1.5 mM EDTA, 1 mM DTT, 25% glycerol) with PI. After brief centrifugation, the supernatants, containing the nuclear fraction, were either stored at 80°C until analysis by electrophoretic mobility shift assay or denatured and stored for immunoblot analysis as described previously.

Electrophoretic Mobility Shift Assay

Oligonucleotide probes containing the NF-B enhancer sequence from the major histocompatibility complex (MHC) class II gene (GGCTGGGGATTCCCCATCT) were synthesized on an Applied Biosystems model 391 DNA synthesizer (Perkin-Elmer, Norwalk, CT). The probes were labeled by incubating 15 U T4 polynucleotide kinase (New England Biolabs, Beverly, MA), 100 ng double-stranded probe, and 100 µCi adenosine 5'-[-³²P]triphosphate (ICN, Irvine, CA) at 37°C for 30 min. Unincorporated ³²P was removed using a desalting column (Nuc Trap; Stratagene). DNA protein binding reactions were performed for 10 min at room temperature in a mixture containing 4 µg of nuclear extract, 1 µl labeled probe, 10 µl running buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM EDTA, 1 mM DTT, 5% glycerol), and 2 µg poly dI/dC (Boehringer Mannheim, Indianapolis, IN). Samples were separated by electrophoresis through 4.5% nondenaturing polyacrylamide gels containing 0.5× tris borate EDTA. Gels were dried and analyzed by phosphorimaging (Molecular Dynamics).

Promoter-Reporter Constructs, Transfection, and Promoter-Reporter Assay

A B-dependent promoter-reporter construct, pNF-B-luc (Stratagene), was used. It was composed of a 5× tandem repeat of the NF-B response element of the mouse Ig gene intronic enhancer cloned upstream of a TATA box and a firefly luciferase cDNA. A constitutively active SV-40 promoter--galactosidase construct, pSV--galactosidase (Promega, Madison, WI), was used to adjust for well-to-well variation in cell number and transfection efficiency.

BEAS cells grown to 60 to 80% confluence in 24-well tissue culture dishes were cotransfected with 250 ng of the pNF-B-luc and 25 ng of pSV--galactosidase using 1.5 µg of DOTAP transfection reagent (Boehringer Mannheim). Forty-eight hours after transfection, cultures were treated for 8 h with 500 µM H₂O₂, 10 ng/ml TNF-, or H₂O₂ + TNF-. Luciferase and -galactosidase activity was determined using the Dual Light reporter gene assay system (Perkin-Elmer) and an AutoLumat LB953 luminometer (Berthold Analytical Instruments, Nashua, NH). Promoter activity was estimated as specific luciferase activity (luciferase counts per unit -galactosidase counts) and expressed as fold induction over the respective media control.

Immunoprecipitation and Immunoblot Analysis

Protein samples (50 µg of the cytoplasmic or nuclear protein fractions) were separated by SDS-PAGE on 12% Tris-glycine gels, followed by immunoblotting using specific antibodies to p65, IB, ubiquitin (all at 1:1,000; Santa Cruz Biotechnology) or ser-32 phospho-specific IB (1:1,000, New England Biolabs, Beverly, MA). Antigen-antibody complexes were stained with horseradish peroxidase-conjugated antibody (1:2,000, Santa Cruz Biotechnology) and SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and enhanced chemiluminescence film was used (Eastman Kodak Company, Rochester, NY). For immunoprecipitations, 500 µg of cytoplasmic protein fractions were incubated with 1 µg of antibodies against IB (Santa Cruz Biotechnology) for 1 h at 4°C, followed by addition of protein G plus A agarose beads (Calbiochem) and further incubation for 16 h at 4°C. Immunoprecipitates were washed, resuspended in Laemmli buffer, and separated by SDS-PAGE on 9% Tris-glycine gels. IB immunoprecipitates were immunoblotted with anti-ubiquitin antibody as described previously. Immunoblot films were digitized using the Kodak 1D Image Analysis Software (Eastman Kodak Company).

Real-Time Reverse Transcriptase/Polymerase Chain Reaction

Extraction of RNA and first-strand cDNA synthesis were performed as described previously (26). Real-time reverse transcriptase/ polymerase chain reaction (RT-PCR) using quantitative fluorogenic amplification of first strand cDNAs were performed using the ABI Prism 7700 Sequence Detector System (PE Biosystems, Foster City, CA), TaqMan Universal PCR Master Mix (PE Biosystems), and primers and fluorophore-labeled probes listed subsequently. Real-time fluorescence measurements were used to determine the threshold cycle (CT) for each amplification by calculating the number of cycles required to reach a fluorescence intensity 10 baseline standard deviations greater than the baseline fluorescence intensity (27, 28). A standard curve relating CT to a serial dilution of a standard pool of first strand cDNAs prepared from human bronchial epithelial cells was used to compute a relative abundance for IL-8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) in each sample. The relative abundance of GAPDH mRNA in each sample was used to normalize the IL-8 mRNA levels.

IL-8: probe, 5'-FAM-CCTTGGCAAAACTGCACCTTCACACA-TAMRA-3'; sense, 5'-TTGGCAGCCTTCCTGATTTC-3'; antisense, 5'-TATGCACTGACATCTAAGTTCTTTAGCA-3'; GAPDH: probe, 5'-JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA-3'; sense, 5'-GAAGGTGAAGGTCGGAGTC-3'; antisense, 5'-GAAGATGGTGATGGGATTTC-3'.

Statistical Analysis

Data are presented as mean ± standard error of the mean SEM of at least three separate experiments. Data comparisons were carried out using a one-way analysis of variance followed by Newman-Keul's post hoc test for multigroup analysis.

	Results

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Treatment with H₂O₂ Increases IKK Activity and Phosphorylation of IB

Although several reports have suggested that oxidative stress can induce NF-B activity, it is still unclear whether ROI affect the activity of IKK. In this study, we analyzed IKK activities in lysates from cells treated with H₂O₂. Preliminary studies conducted in our laboratory indicated that H₂O₂ enhances IKK activity within 15 min, where stimulation with TNF-, a known activator of IKK (29), caused a more rapid activation of IKK within 5 min (data not shown). It is shown in Figures 1A and 1C that treatment with H₂O₂ enhances IKK activity in a dose-dependent manner. To assess whether H₂O₂ has a direct effect on the activity of IKK in vitro, we added H₂O₂ to IKK immunoprecipitates of either control or TNF--stimulated cells before starting the kinase assay. It is shown in Figure 1B that stimulation with TNF- increases IKK activity, which was unaffected by the presence of H₂O₂ (compare lanes 2 and 4). More importantly, addition of H₂O₂ to IKK immunoprecipitates of control cells did not enhance IKK activity (Figure 1B, compare lanes 1 and 3), suggesting that activation of IKK in H₂O₂-treated NHBE cells results from the activation of proximal signaling step(s) in the IKK activation cascade rather than from the activation of IKK directly. Next, we examined whether the increased IKK activity measured with in vitro kinase assays corresponds to phosphorylation of endogenous IB. NHBE cells were pretreated with the proteasome inhibitor MG132, which prevents IB degradation via the 26S proteasome and thereby permits the accumulation of phosphorylated IB in stimulated cells (30). Cells were stimulated with either TNF- or H₂O₂, and the cytoplasmic protein fractions were separated by SDS-PAGE and immunoblotted with a phospho-specific antibody against the Ser-32-phosphorylated form of IB. As shown in Figure 1D, stimulation with TNF- increased phosphorylation of endogenous IB. Similarly, treatment with 100 and 500 µM H₂O₂ increased the levels of phosphorylated IB in NHBE cells.

View larger version (17K): [in this window] [in a new window]   Figure 1.   H2O2 increases IKK activity and phosphorylation of IB. (A) NHBE cells were treated with 100 or 500 µM H2O2 for 15 min. Whole-cell lysates were immunoprecipitated with anti-IKK/ antibody and in vitro kinase assays were performed with the immunoprecipitates using GST- IB(1-54) as substrates. (B) NHBE cells were treated with or without 10 ng/ml TNF- for 5 min. Immunoprecipitates generated with anti-IKK/ antibody were divided into two equal portions and in vitro kinase assays were performed in the presence or absence of 500 µM H2O2. (C) Densitometric analysis of IKK activity normalized to IKK/ immunoblots of three separate experiments. (D) NHBE cells were pretreated with 20 µM MG132 or the vehicle control for 30 min and subsequently stimulated with 100 or 500 µM H2O2, or 10 ng/ml TNF- for 30 min. Cytoplasmic extracts were separated by SDS-PAGE and immunoblotted with an antibody against the Ser-32 phosphorylated form of IB (p-IB).

Treatment with H₂O₂ Does Not Induce IB Breakdown, p65 Nuclear Translocation, or NF-B DNA Binding

Phosphorylation of IB at Ser-32 and Ser-36 marks it for subsequent ubiquitination and degradation by the 26S proteasome (30), which enables NF-B to translocate into the nucleus. As expected, treatment with TNF- rapidly induces the breakdown of IB in the cytoplasmic protein fractions of NHBE cells, followed by an apparent resynthesis of IB after 60 min (Figure 2A). However, treatment with either 100 or 500 µM H₂O₂ induced no apparent breakdown of IB in NHBE cells at any of those timepoints. Densitometric analysis of IB levels in NHBE cells treated with either 100 or 500 µM H₂O₂ or TNF- for 30 min shown in Figure 2B indicates that neither concentration of H₂O₂ caused significant degradation of IB. Similarly, comparison of p65 levels in the cytoplasmic and nuclear fractions of TNF- and H₂O₂-treated cells shows that TNF- induced nuclear translocation of the NF-B subunit p65, whereas treatment with H₂O₂ did not increase nuclear p65 levels (Figure 2C). Moreover, treatment with either 100 or 500 µM H₂O₂ did not enhance NF-B DNA binding activity, however, treatment with TNF- increased the DNA-binding activity to a radiolabeled oligonucleotide containing the sequence of the MHC class II NF-B response element (Figure 2D). The failure to induce IB breakdown and NF-B DNA binding was not caused by cytotoxicity of the H₂O₂ treatment because stimulation with either 100 or 500 µM H₂O₂ or TNF- for 24 h had no effect on cell viability (data not shown). These data suggest that although treatment with H₂O₂ increased IKK activity and levels of phosphorylated IB, H₂O₂ did not induce breakdown of IB, nuclear translocation of p65, or NF-B DNA binding.

View larger version (30K): [in this window] [in a new window]   Figure 2.   TNF-, but not H2O2, induced IB breakdown and NF-B nuclear translocation. (A) NHBE cells were treated with 100 or 500 µM H2O2, or 10 ng/ml TNF- for the indicated times. Cytoplasmic extracts were separated by SDS-PAGE and immunoblotted with anti-IB antibodies. (B) Densitometric analysis of three separate experiments. *Significantly different from control; P < 0.05. (C) Cytoplasmic and nuclear extracts from NHBE cells treated with TNF- or H2O2 for 30 min were separated by SDS-PAGE and immunoblotted with anti-p65 antibodies. (D) Nuclear extracts from NHBE cells treated with TNF- for 30 min or with 100 or 500 µM H2O2 for 30 or 60 min were analyzed for DNA binding activities to the MHC class II NF-B response element. The arrow indicates the NF-B p65/p50 heterodimeric binding complex, which was determined previously using supershift analysis (24).

Treatment with H₂O₂ Inhibits TNF--Induced IB Breakdown and NF-B DNA Binding

The lack of H₂O₂-induced IB breakdown despite enhanced IKK activity and phosphorylated IB levels suggested that the pathway culminating in IB breakdown was disrupted by treatment with H₂O₂. To evaluate whether the H₂O₂-induced disruption of the NF-B activation pathway occurs also in TNF--treated cells, we examined whether treatment with H₂O₂ could affect TNF--induced IB breakdown and NF-B DNA binding. It is shown in Figure 3A that H₂O₂ inhibits TNF--induced IB breakdown in a dose-dependent manner (compare lanes 4, 5, and 6). The densitometric analysis shown in Figure 3B illustrates that treatment with 100 or 500 µM H₂O₂ significantly inhibits TNF--induced IB breakdown in NHBE cells. Similarly, NF-B DNA binding activities in the nuclear protein fractions of these cells showed that H₂O₂ inhibited TNF--induced NF-B DNA binding (Figure 3C, compare lanes 3 and 4). Previous reports have suggested that H₂O₂ can modulate cellular responses to TNF- by increasing the shedding of soluble TNF receptors (31) or decreasing TNF receptor binding (20), thus reducing the ability of cells to respond to stimulation with TNF-. To test whether the inhibition of TNF--induced IB breakdown and NF-B DNA binding by H₂O₂ seen in Figures 3A, 3B, and 3C was caused by modulating the ability of NHBE cells to respond to TNF-, we examined the levels of phosphorylated IB. There was no difference in phosphorylated IB levels in cells treated with TNF- alone or in combination with H₂O₂ (Figure 3D, compare lanes 3 and 4). These data suggested that treatment with H₂O₂ exerts an inhibitory effect on the NF-B activation cascade downstream of IB phosphorylation and upstream of IB breakdown.

View larger version (22K): [in this window] [in a new window]   Figure 3.   H2O2 inhibits TNF--induced IB breakdown and nuclear translocation of NF-B. NHBE cells were treated with 10 ng/ml TNF-, 500 µM H2O2, or TNF- and H2O2 for 30 min. (A) Cytoplasmic extracts were separated by SDS-PAGE and immunoblotted with anti-IB antibodies. (B) Densitometric analysis of three separate experiments. *Significantly different from control; # significantly different from TNF--treated cells; P < 0.05. (C) Nuclear extracts were analyzed for DNA binding activities to the MHC class II NF-B response element. (D) Cytoplasmic extracts from NHBE cells pretreated with 20 µM MG132 and subsequently treated with TNF-, H2O2, or TNF- and H2O2 were separated by SDS-PAGE and immunoblotted with an antibody against the Ser-32 phosphorylated form of IB (p-IB).

Treatment with H₂O₂ Increases IB-Specific and Total Ubiquitination

As indicated previously, under most circumstances phosphorylation of IB at Ser-32 and Ser-36 marks it for ubiquitination, which in turn initiates the proteolytic degradation of IB by the 26S proteasome (30). To investigate whether IB becomes ubiquitinated in response to treatment with H₂O₂ or TNF-, we immunoprecipitated IB and immunoblotted the immunoprecipitate with anti-ubiquitin antibodies. To prevent proteolytic breakdown of ubiquitinated proteins, we pretreated NHBE cells with MG132. Treatment with either H₂O₂ or TNF- increased the levels of ubiquitinated IB (Figure 4A). In addition, the combination treatment of H₂O₂ and TNF- also increased ubiquitinated IB levels. This suggested that the H₂O₂-induced phosphorylation of IB observed in Figure 1 is followed by ubiquitination of this protein. Moreover, treatment with H₂O₂ had no effect on TNF--induced ubiquitination of IB, suggesting that H₂O₂-induced inhibition of IB breakdown does not occur at the level of IB ubiquitination. To examine whether H₂O₂ inhibits proteolytic degradation of other polyubiquitinated proteins, we measured total levels of ubiquitinated proteins in H₂O₂-, TNF--, or H₂O₂/TNF--treated cells. As expected, pretreatment with the proteasome inhibitor MG132 increased the levels of polyubiquitinated proteins in all samples (Figure 4B, left part of the blot). Interestingly, treatment with H₂O₂ alone or in combination with TNF-, but not TNF- alone, also enhanced the levels of ubiquitinated proteins in NHBE cells. These data suggest that treatment with H₂O₂ does not interfere with the ubiquitination process but inhibits the proteolytic degradation of polyubiquitinated proteins, thus leading to the accumulation of ubiquitinated proteins in the cell.

View larger version (64K): [in this window] [in a new window]   Figure 4.   Treatment with H2O2 enhances ubiquitination of IB and other cytoplasmic proteins. (A) Cytoplasmic extracts from NHBE cells pretreated with 20 µM MG132 and subsequently stimulated with TNF-, H2O2, or TNF- and H2O2 were immunoprecipitated (IP) with anti-IB antibodies. The immunoprecipitates were separated by SDS-PAGE and immunoblotted (IB) with anti-ubiquitin antibodies. (B) Cytoplasmic extracts from NHBE cells pretreated with 20 µM MG132 or the respective vehicle control and subsequently stimulated with TNF-, H2O2, or TNF- and H2O2 were separated by SDS-PAGE and immunoblotted with anti-ubiquitin antibodies.

Treatment with H₂O₂ Inhibits TNF--Induced NF-B-Dependent Transcription

To determine whether the inhibitory effect of H₂O₂ on NF-B activity also occurs at the level of NF-B-dependent transcription, we conducted promoter-reporter assays using a 5× tandem repeat NF-B promoter-reporter construct. It is shown in Figure 5A that treatment with TNF- induced a significant increase in NF-B-dependent promoter-reporter activity, which was inhibited by H₂O₂. We have previously shown that IL-8 gene expression is NF-B-dependent in NHBE cells (26). Stimulation with TNF- increased the levels of IL-8 mRNA, which was significantly inhibited by H₂O₂ in a dose-dependent manner (Figure 5B). Neither NF-B-dependent promoter-reporter activity nor IL-8 mRNA levels were significantly increased by H₂O₂ alone. Taken together, these data indicate that treatment with H₂O₂ inhibits TNF--induced NF-B-dependent transcription in bronchial epithelial cells.

View larger version (19K): [in this window] [in a new window]   Figure 5.   H2O2 decreases TNF--induced NF-B-dependent transcription and IL-8 gene expression. (A) BEAS-2B cultures were transiently cotransfected with pNF-B-luc and pSV-galactosidase. Cultures were stimulated with TNF-, H2O2, or TNF- and H2O2 for 8 h, 24 h after transfection. Specific luciferase activity in culture lysates was determined using -galactosidase activity as a normalizing factor. The data are expressed as mean specific luciferase activity ± SEM. *Significantly different from media control; # significantly different from TNF--treated cells; P < 0.05. (B) NHBE cells were stimulated with or without TNF- in the presence or absence of 100 or 500 µM H2O2 for 1 h. Total RNA was analyzed for IL-8 and GAPDH mRNA levels by real-time RT-PCR. IL-8 mRNA levels were normalized to GAPDH mRNA levels and expressed as mean ± SEM. *Significantly different from media control; #significantly different from TNF--treated cells; P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recent studies have advanced our understanding of the signal transduction cascades mediating phosphorylation and subsequent degradation of IB in response to pro-inflammatory cytokines. The signaling steps from the receptor to activation of IKK, IB breakdown, and activation of NF-B have been well described for stimulation with pro-inflammatory cytokines (8). Although earlier studies have implicated oxidative stress as an important activator of NF-B, no studies have reported activation of IKK in response to oxidative stress. In this study, we examined the effects of H₂O₂ on IKK activity and subsequent activation of NF-B in airway epithelial cells. Our results demonstrate that treatment with H₂O₂ increases IKK activity, phosphorylation, and ubiquitination of IB but fails to induce IB breakdown and nuclear translocation of NF-B. In addition, although H₂O₂ had no effect on TNF-- induced phosphorylation and ubiquitination of IB, the presence of H₂O₂ inhibited TNF--induced IB breakdown, NF-B DNA binding, and NF-B-dependent transcription. These data suggest that in airway epithelial cells treatment with H₂O₂ affects the NF-B activation cascade at two different levels: (1) activation of IKK and (2) inhibition of the proteolytic degradation of phosphorylated and ubiquitinated IB.

IKK/-containing complexes were activated by H₂O₂ in NHBE cells, despite of the absence of NF-B activation. The mechanism and consequences of this activation remain to be investigated. Because addition of H₂O₂ had no direct effect on IKK activity in vitro (Figure 1B), the enhanced IKK activity in NHBE cells treated with H₂O₂ is probably caused by activation of signaling steps proximal to IKK. The activation of IKK and  is mediated by phosphorylation on serine residues within a short activation loop (T-loop) (32, 33), which has a canonical mitogen-activated protein kinase (MAPK) kinase activation loop motif (Ser-X-X-X-Ser). Accordingly, IKK/-containing complexes are activated in vivo by overexpression of a number of wild-type MAPK kinase kinase (MAPKKK) family kinases (34- 36) and activation is inhibited by overexpression of dominant interfering mutants of MAPKKKs. IKK or  or both are also activated by overexpression of certain isoforms of protein kinase C (37), by the serine-threonine kinase Akt (38, 39), and by NF-B-activating kinase (TBK1), a novel IKK-like kinase (40, 41). This diversity of IKK kinases is believed to mediate the convergence of signals initiated by a variety of environmental stimuli on IKK/ -containing signaling complexes. None of the kinases immediately upstream of IKK and  are known to be activated by oxidative stress. However, one of the MAPKKKs that activates IKK/-containing complexes, MEKK1 (42), mediates activation of the p38 MAPK, a MAPK that is activated by H₂O₂ in NHBE cells (Dr. Jaspers, unpublished observation). Thus, H₂O₂ may activate a kinase upstream of MEKK1.

Phosphorylation at serine residues 32 and 36 by the IKK complex marks IB for subsequent ubiquitination by the ubiquitin ligase complex (43). However, neither phosphorylation nor polyubiquitination of IB is sufficient for nuclear translocation of NF-B (44, 45). Polyubiquitination targets IB for rapid degradation by the 26S proteasome, which results in exposure of the nuclear localization sequence of NF-B and translocation of the transcription factor into the nucleus (46). Interestingly, the data presented here show that although treatment of airway epithelial cells with H₂O₂ increased phosphorylation and polyubiquitination of IB, these cells showed no enhanced degradation of IB or nuclear translocation of NF-B. In addition, treatment of airway epithelial cells with H₂O₂ induced the accumulation of other ubiquitinated proteins. Generally, steady-state levels of ubiquitinated proteins depend on the rate of ubiquitination and proteolytic activity of the 26S proteasome. Because H₂O₂ enhanced ubiquitination, but not degradation of IB, our results suggest that H₂O₂ inhibits proteolytic degradation of polyubiquitinated proteins by the 26S proteasome. This hypothesis is supported by previous studies that have demonstrated that H₂O₂ inhibits the proteolytic enzyme activities of the 26S proteasome in vitro as well as in K562 cells treated with H₂O₂ (47). Interestingly, this study also demonstrated that in H₂O₂-treated K562 cells the activity of the ATP- and ubiquitin-dependent 26S proteasome was several times more sensitive to inhibition by oxidants as compared with the activity of the ATP- and ubiquitin-independent 20S proteasome, which was slightly enhanced at low oxidant levels (47). In addition, 4-hydroxy-2-nonenal (HNE), which is generated by peroxidation of membrane lipids during oxidative stress, decreased proteolytic activity of proteasomal enzymes and increased accumulation of ubiquitinated proteins in the kidneys of mice exposed to oxidative stress (48), suggesting inhibition of the 26S proteasome. In vitro incubation of kidney homogenates with HNE decreased proteasomal enzyme activity, suggesting that interaction of HNE with the proteasomal enzymes decreases their catalytic activity. Thus, H₂O₂-induced formation of HNE or other lipid peroxidation products could interact with the 26S proteasome and inhibit proteasomal degradation of ubiquitinated proteins, including IB.

Although H₂O₂ does induce IB breakdown and NF-B nuclear translocation in certain cell lines (11, 19, 49), it is not known whether the phosphorylation/ubiquitination/ 26S proteasome pathway described previously mediated the proteolytic degradation of IB in these experiments. A recent study reported that in the mouse EL4 lymphoblastoid cell line, H₂O₂ induced IB breakdown and NF-B nuclear translocation by a mechanism that is independent of IKK/ and the 26S proteasome (50). Thus, H₂O₂- induced IB degradation does not preclude inhibition of the 26S proteasome. This alternative pathway leading to IB degradation is present in other cell lines as well (51, 52), although its sensitivity to H₂O₂ in these cell lines has not been examined. The generality of this alternative IB degradation mechanism in H₂O₂-induced NF-B nuclear translocation needs further investigation and it is possible that inhibition of the 26S proteasome by H₂O₂ in vivo is a general phenomenon.

In this study, we investigated whether H₂O₂ affects TNF-- induced breakdown of IB, activation of NF-B-dependent transcription, and expression of pro-inflammatory cytokines. Modulation of TNF--induced gene expression by costimulation with H₂O₂ would be physiologically relevant. For example, during acute inflammation, lung epithelial cells are potentially exposed to both TNF- and oxidative stress due to the release of TNF- and reactive oxygen species by infiltrating phagocytes that reside in close proximity to the lung epithelium. Airway epithelial cells, composing a physical barrier between the lung lumen and the underlying interstitium, are in a position to mediate recruitment of inflammatory phagocytes into the airways in response to pro-inflammatory stimuli. Stimulation with TNF- causes airway epithelial cells to synthesize and release chemokines that mediate the recruitment of phagocytes into the airways. At sites of acute inflammation, with increased release of both TNF- and reactive oxygen species by infiltrated phagocytes, H₂O₂-induced attenuation of NF-B-dependent pro-inflammatory gene expression in human airway epithelial cells may represent a mechanism that downregulates pro-inflammatory responses in the lung.

H₂O₂ activates B-dependent transcription in a spontaneously transformed rat lung epithelial (RLE) cell line, but this response is slow, requiring 8 h, and is independent of IB breakdown (53). We have not observed enhanced B-dependent transcription in human bronchial epithelial cell lines over a wide range of H₂O₂ exposure concentrations and duration (Dr. Jaspers, unpublished observation) (Figure 5). Taken together, these observations suggest that RLE cells have an H₂O₂-activated mechanism that enhances B-dependent transcription but does not mobilize NF-B, which is absent or suppressed in NHBE cells. Numerous signaling mechanisms that affect transactivation of NF-B without affecting nuclear translocation of NF-B have been described or proposed (24, 54). Whether the mechanism in RLE cells is a novel one or is due to a cell line-specific modification that confers H₂O₂ sensitivity on an identified mechanism remains to be determined. In any case, it is evident that the alternative mechanisms of IB degradation and NF-B transactivation responsible for the activation of NF-B by H₂O₂ in certain cell lines are absent or suppressed in the NHBE cells used in this study as well as in many other cell lines that do not respond to H₂O₂ by activating NF-B.

In conclusion, the data presented here demonstrate that in airway epithelial cells H₂O₂ has an inhibitory effect on the NF-B activation cascade by preventing IB breakdown, despite activation of IKK and phosphorylation of IB by H₂O₂. It seems counterintuitive that H₂O₂ enhances IKK activity and phosphorylation of IB without increasing NF-B-dependent gene expression. Although H₂O₂ activated IKK and inhibited NF-B in a dose-dependent manner, it is possible that there are concentrations of H₂O₂ below the ones tested in this study that activate IKK in NHBE cells without inhibiting the IB breakdown, thus leading to activation of NF-B. On the other hand, studies using IKK knockout mice suggest that IKK has additional functions independent of IB phosphorylation (58). For example, fibroblasts derived from IKK / mice have impaired TNF--induced NF-B DNA binding without any defects in IB phosphorylation. In addition, these mice display severe defects in epidermal differentiation and skin morphogenesis, indicating that IKK plays an important role in skin development. Future studies are necessary to establish whether TNF- and H₂O₂ differentially activate IKK and IKK in airway epithelial cells. In addition, we have observed that IKK and  mRNA are upregulated during differentiation of NHBE cells into a pseudostratified ciliated epithelium (Dr. Jaspers, unpublished observation). Hence, activation of IKK in human airway epithelium may be a process whose importance goes beyond the activation of NF-B.