Abstract. Background: Peroxynitrite has been proposed toactivate Nuclear Factor κappa beta (NF-κB) in a non-canonicalor aberrant pathway and to contribute to pathology of manydiseases. Methods: The activation of NF-κB by peroxynitritewas assessed by Western blot, immunoprecipitation, RT-PCR,and image stream analysis. Results: Our work showed that inHT-29 cancer cells, peroxynitrite can cause nitration ofInhibitory protein kappa B alpha (IκBα) at the expense of itsphosphorylation. This led to a decrease in the degradation andre-synthesis of IκBα. Similar findings were observed for mRNAlevels assessed by RT-PCR. Exposure of HT-29 cells to p38inhibitor SB202190, prior to stimulation, resulted in a dramaticdecrease of IκBα kinase and IκBα phosphorylation and causedan increase of peroxynitrite-mediated nitration of IκBα,indicating that peroxynitrite may activate NF-κB via dualmechanism of IκBα phosphorylation which is p38 dependent,as well as IκBα nitration. Conclusion: Our findingsdemonstrate a possible interaction of the p38 pathway with theNF-κB pathway under peroxynitrite stimulation.

Reactive nitrogen species (RNS) are highly active compoundsthat can cause cellular damage through oxidation and nitrationof macromolecules. RNS include among others, NO and itsderivative peroxynitrite (ONOO¯) (1). Peroxynitrite is also apotent oxidizing agent and mediator of cellular damage thattriggers apoptosis in many cell types via different mechanisms.It is produced under inflammatory conditions and has beenimplicated in promoting the development of differentpathologies of a variety of diseases, including cancer (2).

The transcription factor nuclear factor-kappa B (NF-κB) isan important regulator of many physiological and

pathophysiological processes, including control of the adaptiveand innate immune responses, inflammation, proliferation,tumorigenesis, and apoptosis. Activation of NF-κB requiresthe activation of the IκBα kinase (IKK) which phosphorylatesIκBα on two conserved serines (Ser 32 and Ser 36 in IκBα).This phosphorylation marks IκBα for ubiquitination andproteasomal degradation, resulting in activation of NF-κB andnuclear translocation. This is considered the classic pathwayof NF-κB activation. Several alternative pathways have beendescribed including modulation of NF-κB activity by NO andperoxynitrite (3, 4). Another aberrant mechanism has beenreported by Bar Shai and Reznick (5), where peroxynitritecaused activation of NF-κB through its IκBα nitrationindependent of IκBα phosphorylation and degradation (5). Asimilar report revealed that ionizing radiation and peroxynitritestimulate NF-κB activity by a mechanism in which IκBtyrosine 181 was nitrated. This mechanism also did not requireIKK-dependent phosphorylation and proteolytic degradationof IκBα (6).

Peroxynitrite is formed by interaction of superoxide O2˙¯and NO·, a product of the nitric oxide synthase (NOS)enzyme family. This family includes endothelial NOS(eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS).Unlike eNOS and nNOS, iNOS is not constitutivelyexpressed in normal tissues and its concentration is regulatedmainly at the transcriptional and translational levels.Transcription of iNOS is regulated by various signalingpathways, including the NF-κB pathway. In addition, theiNOS isoform is mostly associated with malignant tissuetransformation and is rarely detected in normal tissues in theabsence of pathology (7).

Recent data show that iNOS is also expressed in a varietyof acute and chronic inflammatory human diseases, as wellas in various types of cancer. In addition, nitrated tyrosineresidues have been detected in inflamed and in cancer tissues(7). Consequently, a positive correlation between NOS andnitrotyrosine (a putative chemical marker for the formationof peroxynitrite) expression, and human tumor grade havealso been demonstrated (2, 8). Inflammatory bowel diseases

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Correspondence to: Abraham Reznick, Department of Anatomy andCell Biology, Faculty of Medicine, Technion, Haifa, Israel. Tel: +97248295388, Fax: +972 48295403, e-mail: Reznick@tx.technion.ac.il

Key Words: NFκB, IκBα, IKKβ, p38, peroxynitrite, nitration, coloncancer.

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NF-κB Activation by Peroxynitrite through IκBα-dependentPhosphorylation versus Nitration in Colon Cancer Cells

EINAT GOCHMAN1,2, JAMAL MAHAJNA2,3 and ABRAHAM Z. REZNICK1

1Department of Anatomy and Cell Biology, The Rappaport Faculty of Medicine, Technion, Haifa, Israel; 2Cancer Drug Discovery Program, Migal Galilee Technology Center, Kiryat Shmona, Israel;

3Department of Biotechnology, Tel Hai Academic College, Kiryat Shmona, Israel

0250-7005/2011 $2.00+.40

such as ulcerative colitis and Crohn’s disease are chronicinflammatory conditions which are associated with increasedrisk of developing cancer of the colon and rectum. A markedincrease in the activity of iNOS in inflamed colonic mucosafrom patients with ulcerative colitis has been reported. Inaddition, nitrotyrosine has been detected by immuno-histochemical staining in an animal model of chronic gutinflammation and found to co-localize with iNOS (9). Thus,excessive nitrative stress is one of the characteristics of apersistent inflammatory microenvironment. The net outcomeof this microenvironment enhances tumor promotion,accelerates tumor progression, invasion of the surroundingtissues, angiogenesis, and often metastasis [Danese #49]. Ithas been suggested that increased cancer incidence inchronic ulcerative colitis is due to mutations in, orinactivation of, key genes such as the tumor suppressor genep53 or β-catenin, a key mediator of the Wnt-signalingpathway. In fact, patients with ulcerative colitis had astandardized incidence ratio for colorectal cancer of 5.7 andan earlier age of onset than the general population.

Since the exact mechanism by which RNS activate the NF-κB pathway is still obscure, in the present study, we attemptedto investigate the mechanism of NF-κB/IκB activationpathway by RNS in a colon cancer cell line, HT-29.

Materials and MethodsMaterials. Peroxynitrite was purchased from Cayman ChemicalCompany (Ann Arbor, MI, USA). Stock solutions of 10 mM wereprepared in 0.3 M NaOH and stored at –70˚C. Tumor necrosis factorα (TNF-α) was purchased from Biological Industries (Kibbutz BeitHaemek, Israel). Pharmaceutical inhibitors IMD-0354, SP600125,SB202190 inhibitors of IKKβ, JNK, and p38, respectively, werepurchased from Calbiochem (Richmond, CA, USA).

Cell cultures. HT-29 human colon cancer cell line (ATCC,Manassas, VA, USA) was maintained in MacCoy’s 5A with L-glutamine medium (Biological Industries), supplemented with 10%fetal calf serum (FCS), 1% L-glutamine, and 1% PenStrep(penicillin + streptomycin) (Biological Industries).

Sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) and Western blot analysis. HT-29 cells (2.5×105 cells/ml)were seeded in 5 ml of MacCoy 5A medium in 25 ml plastic flasksand maintained in a humidified incubator at 37˚C with 5% of CO2in air. After 24 h, the growth medium was substituted with a serum-free medium (containing 0.5% FCS). Peroxynitrite and TNF-α wereadded to the growth medium at final concentrations of 10 μM–100μM and 10 ng/ml, respectively, and incubated with the cells forincreasing periods of time in the presence and absence of 10 μM ofpharmaceutical inhibitors IMD-0354, SP600125, SB202190, whichwere added 30 min prior to stimulation. After incubation, cells werecollected, washed, and total cell lysates were prepared (10). Proteinconcentrations of the samples were determined using the Bio-Radprotein assay (Dye Reagent Concentrate, Bio-Rad LaboratoriesGmbH, München, Germany). Equal amounts of proteins (40-50 μg)were separated on 10% SDS-PAGE, transferred to a nitrocellulose

membrane (Schleicher & Schuell BioScience GmbH, München,Germany), and subjected to Western blot analysis using anti-pIκBα(Ser 32/36), anti-pIKKα/β (Ser 176/180), anti-pSAPK/JNK (CellSignaling Technology Inc., Danvers, MA, USA), anti-IκBα, anti-iNOS (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-phospho-p38 (R&D Systems Minneapolis, MN, USA) and anti-nitrotyrosine antibodies (Chemicon Billerica, MA USA) accordingto the manufacturers’ instructions. The equality of sample loadingin all lanes was confirmed by stripping and re-blotting with anti-α-tubulin (Santa Cruz Biotechnology, Inc.) and anti-actin antibodies(Millipore, Temecula, CA, USA). Secondary antibodies were goatanti-rabbit IgG, rabbit anti-goat IgG, and goat anti-mouse IgG(Jackson ImmunoResearch Laboratories, Inc., PA, USA).

Immunoprecipitation. Total cell lysates (approximately 500 μgprotein) were incubated with 5 μg of anti IκBα at 4˚C overnightwith agitation. Subsequently, 40 μl of washed protein A agaroseslurry (Santa Cruz Biotechnology, Inc.) was added and the mixtureswere incubated at 4˚C for 1.5 h with agitation. Finally, cytoplasmicfractions were centrifuged at 10,000 × g for 5 min, and theprecipitates were separated and washed and dissociation buffer wasadded. The precipitated IκBα was used to determine its nitration orphosphorylation status with SDS-PAGE and immunoblotting.

RT-polymerase chain reaction (PCR) of IκBα. HT-29 cells (2.5×105

cells/ml) were starved for 24 h before treatment with TNF-α (10 μg/ml)and peroxynitrite (25 μM) for 30-180 min. After incubation, total RNAwas extracted from the cells using Tri Reagent (Sigma, Rehovot, Israel).Single-stranded complementary DNA (cDNA) was synthesized fromthe total RNA. In brief, 0.5-1 μg RNA was pre-incubated with 1 μloligo(dT)17 primer and diethylpyrocarbonate (DEPC)-treated waterwas added to a total volume of 15 μl at 70˚C for 10 min, then rapidlychilled on ice. To the annealed primer/template, 2 μl AMV RT5×reaction buffer, 2 μl dNTP (25 mM), 28 units of rRNasinribonuclease inhibitor, 30 units of AMV RT and DEPC-treated waterwere added to a final volume of 10 μl. The reaction mixture wasincubated at 42˚C for 60 min. The resulting cDNA was amplified witha PCR kit (Bioline, USA) using the following primers: human IκBαforward: 5’-CTGTGATCA CCAACCAGCCAGA-3’; human IκBαreverse: 5’-GTAGCCATG GATAGAGGCTAAG-3’. A total of 30cycles of amplification were performed with initial incubation at 94˚Cfor 2 min and a final extension at 72˚C for 15 min, each cycle consistedof denaturation at 94˚C for 30 s, annealing at 57˚C for 30 s andextension at 72˚C for 2:30 min. The housekeeping gene GAPDH wasused as a loading control: human GAPDH forward: 5’-GAGTCAACGG ATTTGGTCGT-3’; human GAPDH reverse: 5’-GGTGCCATGGA ATTTGCCAT-3’. A total of 5 μl of the PCRproducts were analyzed by electrophoresis on a 1.5% agarose gel.

Activation and immunofluorescent detection of NF-κB nucleartranslocation. HT-29 cells (2.5×105 cells/ml) in exponential growthphase were treated with 10 ng/ml TNF-α and 25 μM peroxynitritefor 15 or 60 min respectively in 14 cm diameter dishes. Cells werewashed with (PBS) and were gently scraped in cold PBS from theflask, followed by fixation with 3% paraformaldehyde for 20 min,washed and incubated with PBS containing 0.1% Triton X-100/2%BSA for 20 min. After washing, the cells were incubated for 20 minwith 10 μg/ml mouse anti-NF-κB p65 monoclonal antibodies (SantaCruz Biotechnology, Santa Cruz, CA, USA) in PBS containing 2%bovine serum albumin (BSA) for 1hour. The cells were then washed

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and incubated for 1hour with 7.5 μg/ml fluorescein isothiocyanate(FITC)-conjugated F (ab’)2 donkey anti-mouse IgG (JacksonImmunoResearch, West Grove, PA, USA) in PBS/2% BSA for 1 h,then washed and resuspended in PBS. Propidium iodide at 0.1 mg/ml(Sigma-Aldrich) was added to the cells for 5 min and the sample ofcells was run directly on the Image Stream system. All fixation andstaining reactions were carried on ice in the dark at a cell density of8×105 cells/ml. All washes were performed with cold PBS.

Image Stream data acquisition and analysis. The fluorescenceimage-based method for quantifying nuclear translocation describedhere relies on the differential spectral isolation of NF-κB and PIimages of nuclear images by the Image Stream technology (11).Determination of NF-κB translocation from a similarity score (11):The assessment of nuclear translocation was determined in aqualitative manner by comparing a cell’s nuclear fluorescence (PIfluorescence) image to the pattern of fluorescence produced by theNF-κB label (FITC-conjugated F (ab’)2 donkey anti-mouse IgGfollowing mouse anti p65 monoclonal Ab). Nuclear translocation isjudged to have occurred if the NF-κB and nuclear fluorescencesignals overlap with similar shape. Conversely, if the NF-κB signalsurrounds the nucleus, it is judged not to exhibit significant

translocation. The similarity score is a method of quantitativelyperforming this assessment. The similarity score has a high positivevalue if the NF-κB and nuclear images are alike. In contrast, if theNF-κB and image is dim where the nuclear image is bright, thescore has a large negative value. The data pairs are the pixelintensities at the same location in each image of the two differentfluorescence intensities of each pixel. Plotting the pixel intensitiesof the nuclear image against the transcription factor image revealsan inverse correlation for un translocated protein and a positivecorrelation for translocated protein in a cell.

Each calculation of the similarity score is based on measuringthe value of 5,000 individual cells in each experiment, and theirdistribution is presented as the geometric mean (G-mean) of the5000 cells. Under high conditions of translocation, the G-meanvalues are above 1.0 and under those of low translocation, the G-mean values are below 1.0.

Statistical analysis. Comparisons of the means of histogram analysiswere made using an unpaired two-tailed Student’s t-test andANOVA analysis with significant values set at p<0.05. The resultsof the experiments are expressed as mean values±standard deviationof band density relative to the house keeping protein.

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Figure 1. Peroxynitrite (ONOO¯) induced IκBα phosphorylation in HT-29 colon cancer cells. A: HT-29 cells (3×105 cells/ml) were stimulated with(ONOO¯) for 20 min. Levels of pIκB and β-actin were monitored as described in the Materials and Methods. The results are representative of oneof three similar experiments. B: HT-29 cells (1.5×106 cells/ml) were stimulated with of TNF-α and ONOO for 20 min. Cells were lysed and celllysates were immune-precipitated with anti IκB as described in Materials and Methods: The supernatants were run on PAGE and electrophoresedto nitrocellulose membrane, blotted with pIκB antibody (left panel) and Nitro-Tyrosine antibody (right panel) and quantified densitometrically asdescribed in the Materials and Methods. The results are representative of one of three similar experiments.

Results

Peroxynitrite induces IκBα phosphorylation in HT-29 coloncancer cells. Since previous studies from our laboratory havedemonstrated that RNS such as peroxynitrite caused thenitration of IκBα in muscle cells on the expense of itsphosphorylation (5), we were interested in also verifying thisobservation also in cancer cell lines such as HT-29 humancolon cancer cells. The results shown in Figure 1A illustratethat presence of 10-100 μM of peroxynitrite causedsubstantial phophorylation of IκBα with maximal IκBαphophorylation being seen at 25 μM of peroxynitrite.

Nitration and phosphorylation of IκBα by peroxynitrite andTNF-α using immunoprecipitation. IκBα phophorylation on

serine 32 and 36 is a crucial step in the classical activation ofNF-κB. Therefore, IκBα phophorylation as well as IκBαnitration following peroxynitrite treatment were measured inthe same experiment depicted in Figure 1B. Treatment withTNF-α led to increased IκBα serine phosphorylation, asexpected from agents that induce the classic activationpathway. Serine phosphorylation was also observed afterperoxynitrite stimulation; nevertheless, the phosphorylationwas lower by about 40-45% in peroxynitrite treated cellscompared to TNF-α stimulation, and occurred in a dose-dependent manner (Figure 1B). On the other hand, reciprocalfindings were observed while assessing tyrosine nitration.For example, stimulation of TNF-α or peroxynitrite led totyrosine nitration of IκBα, yet it was increased byapproximately 50-55% after peroxynitrite treatment

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Figure 2. Effect of TNF-α, peroxynitrite (ONOO¯) on IκBα degradation and re-synthesis. A: HT-29 cells (3×105 cells/ml) were stimulated with 10ng/ml of TNF-α, 25 μM ONOO¯ for 10-60 min. Cells were lysed as described in the Materials and Methods and levels of IκBα and β-actin weremonitored by Western blotting. B: Quantification was carried out by densitometry as described in the Materials and Methods and data representaverage+SD of three different experiments. C: Cells were stimulated with TNF-α (10 μg/ml) and ONOO¯ (25 μM) for 30-180 min. Levels of IκBαtranscript were determined using RT-PCR, and compared to levels of the house keeping gene, GAPDH, as described in the Materials and Methods.

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Figure 3. Effect of MAPK inhibitors on pyroxiynitrite (ONOO¯)-induced IKK and IκBα phosphorylation in HT-29 cells. A: Western blot analysis ofHT-29 cells (3×105 cells/ml) stimulated with ONOO¯ (25 μM) for 15 and 60 min in the presence and absence of IMD-0354 (IKK- specific inhibitor),SP600125 (JNK inhibitor), and SB202190 (p38- specific inhibitor) were added to appropriate samples for 30 min prior to peroxynitrite treatment.Levels of pIKK and α-tubulin were assessed as described in the Materials and Methods. B: Densitometric quantitation of three similar Western blotexperiments shown in A. Data represent average±SD of the experiments. C: Western blot analysis of HT-29 cells (3×105 cells/ml) stimulated withONOO¯ (25 μM) for 15 and 60 min in the presence and absence of IKK and p38 inhibitors. Inhibitors were added to appropriate samples for 30 minprior to peroxynitrite treatment. Levels of IκBα and α-tubulin were assessed as described in the Materials and Methods. D: Densitometricquantitation of three different Western blot experiments seen in B. Data represent the average±SD of the experiments.

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Figure 4. continued

compared to TNF-α stimulation (Figure 1B). Interestingly,some low basal tyrosine nitration of IκBα was observed inuntreated HT29 colon cancer cells and a noticeable nitrationwas also observed with TNF-α stimulation (Figure 1B).

Effect of authentic peroxynitrite on IκBα degradation in HT-29cells. Regulation of NF-κB activity is mediated by itscytoplasmic localization, which is maintained by IκBα. IκBαphosphorylation triggered by variety of stimuli results in IκBαdegradation and subsequently NF-κB nuclear translocation. Toinvestigate the ability of peroxynitrite to affect IκBαdegradation, cells were exposed peroxynitrite and to TNF-α.Exposure of HT29 cells to TNF-α for 10 or 20 min resulted ina decrease of IκBα levels, as expected from the classicalactivation pathway of IκBα degradation, and its expressionreturning to normal at 30 min. However, treating the cells withperoxynitrite caused insignificant change in IκBα level

throughout the 60 min experiment (Figure 2A). This may stemfrom nitration at the expense of phosphorylation.

Quantitation of the blotting of the results of Figure 2A isshown in Figure 2B. These results represent the means of 3experiments±SD.

Effect of peroxynitrite on IκBα transcription in HT-29 cells.Results shown in Figure 2C illustrate that stimulation withTNF-α caused a rapid increase in IκBα transcript in HT29cells. This observation is consistent with the fact that TNF-αactivated NF-κB through the classic pathway in ourexperimental model (Figure 2A).

However, treating HT29 cells with peroxynitrite caused alower increase in IκBα transcript compared to that with TNF-α stimulation, suggesting NF-κB activation by peroxynitritestimulation, which was mild, and is delayed in time incomparison to TNF-α activation (Figure 2C).

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Figure 4. Image Stream fluorescence imaging of TNFα and pyroxiynitrite ONOO¯- induced NF-κB nuclear translocation in HT-29 cells. Quantificationof nuclear translocation using the similarity algorithm. A: Calculation of the NF-κB/PI similarity score from images of untreated cell with acytoplasmic NF-κB distribution (left) and TNFα-treated (10 μg/ml for 15 min) cell with a nuclear NF-κB distribution (right). Pixel intensities fromthe NF-κB images are plotted against the corresponding pixel intensities from the PI or dark-field images. B: The region High Sim drawn on the darkfield/PI similarity plot represents the region for positive image correlation for the similarity algorithm. This region is applied to the NF-κB/PI similarityplot. The percentages of cells that fall within the High Sim region are displayed on the right of each histogram. C: Control unstimulated HT-29 cells(left panel), HT-29 cells treated with 10 ng/ml TNF-α for 15 min (middle panel), and HT-29 cells treated with 25 μM ONOO¯ for 60 min (right panel)were probed for NF-κB expression and PI as described in the Materials and Methods and run on the Image Stream. The multispectral imaging systemacquires up to 6 images per cell in three different imaging modes: dark field (side scatter) (1), bright field (morphology) (2), and fluorescence; NF-κB (green-FITC) (3), PI (red) (4), and NF-κB/PI composite images (5) for five representative (of 5,000 images) cells and are shown for each treatmentgroup. D: NF-κB nuclear translocation in HT-29 cells. The G-means represent the geometric means of the similarity of nuclear/cytoplasmic NF-κBintensity ratio to PI intensity. HT-29 cells were either left unstimulated or were stimulated with TNF-α 10 ng/ml, ONOO– 25 μM with/ without priorto stimulation with 10 μM SB202190. The results show the mean±SD of 3-6 independent experiments in which the similarity ratio was calculatedfrom 5,000 cells in each independent experiment. *p<0.02 Compared to control, **p<0.001 Compared to control.

Peroxynitrite induced MAPK p38-dependent IKKβ and IκBαphosphorylation. Recent studies demonstrated a cross talkbetween the MAPK p38 and the NF-κB pathways (12). Toinvestigate the involvement of MAPK p38 in IKKactivation, as well as its downstream proteinphosphorylation target IκBα, cells were incubated withIMD-0354, IKKβ-specific inhibitor, SP600125, JNKinhibitor, and SB202190, p38 inhibitor for 30 min. Resultsshown in Figure 3A demonstrate that pre-exposure to bothIMD-0354 and SB202190 dramatically reduced IKKphosphorylation compared to stimulation by peroxynitritealone. On the other hand, the presence of SP600125 did notchange the level of IKK phosphorylation relative toperoxynitrite alone, indicating that activation of the NF-κBpathway by peroxynitrite might occur via the p38 pathway.Quantitations of 3 Western blot experiments of target proteinexpression (relative to housekeeping protein – α-tubulin) areshown in Figure 3B.

When IκBα phosphorylation was assessed, comparableresults were obtained. Immunobloting analysis using anti-pIκBα revealed that exposure to peroxynitrite caused markedphosphorylation after 15 min which increased at 60 min.This phosphorylation was dramatically reduced when cellswere pre-incubated with 10 μM of p38 inhibitor 10 μM ofIKK inhibitor (Figure 3C). Quantitation of 3 separateexperiments are represented in Figure 3D.

NF-κB nuclear translocation stimulated by peroxynitrite isdelayed in comparison to that by TNF-α. Following thefinding that IKK activation by peroxynitrite is dependent onMAPK p38 activity, we were interested in observing NF-κBnuclear translocation in response to peroxynitrite stimulation.NF-κB nuclear translocation was monitored and quantitatedusing the similarity score by Image Stream technologydescribed in the Material and Methods (Figure 4A and 4B).Figure 4C illustrates Image Stream images of four control

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Figure 5. Exposure to p38 inhibitor prior to peroxynitrite stimulation raises IκBα nitration in HT-29 colon cancer cell line. A: HT-29 cells (1×106

cells/ml) were stimulated with 10 ng/ml TNFα and 25 μM ONOO– for 15 and 60 min and 10 μM SB202190 prior to the latter stimulation for 15 and60min. Cells were lysed as described in Materials and Methods and were immune-precipitated with anti-IκBα. The supernatants were run on PAGEand electrophoresed to nitrocellulose membrane and blotted with Nitro-Tyrosine antibody. A: Results are representative of one of three similarexperiments. B: Quantitation of three similar experiments are shown with means±SD. *p<0.035 Compared to control.

cells (left panel), and five TNF-α treated cells at 15 min(middle panel), and peroxynitrite treated cells at 60 min(right panel).

In the classical pathway, IκBα being phosphorylated inresponse to extracellular stimuli (13) by IKK. Thisphosphorylation results in fast nuclear translocation andactivation of NF-κB. Stimulation of HT-29 cells with 10ng/ml of TNF-α clearly results in nuclear translocation ofNF-κB within 15 min which was reduced as expectedafter 60 min as measured by the G-mean (Figure 4D).Unlike stimulation with TNF-α, peroxynitrite stimulationresulted in a relatively low level of NF-κB translocationat 15 min. However significant translocation was evidentafter 60 min stimulation with peroxynitrite (Figure 4D)compared to the control (p<0.02). Prolonged exposure ofthe cells to peroxynitrite for 120 min and 180 minrevealed a decrease in NF-κB nuclear translocation;

compared to 60mins. The most interesting finding wasthat p38 inhibitor partially reduced peroxynitrite-inducedNF-κB translocation in the short term (Figure 4D).However, assessment of NF-κB nuclear translocation, withpre-exposure of cells to p38 inhibitor (SB202190) beforeperoxynitrite treatment revealed lower tranlocation.Nevertheless, it also peaked at 60 min and wassignificantly greater (p<0.001) compared to the control.The latter activation occurs to a lesser extent and isdelayed compared to the activation via p38 (Figure 4D).

Exposure to p38 inhibitor prior to peroxynitritestimulation raises IκBα nitration in the HT-29 colon cancercell line. Since stimulation by peroxynitrite leads to nitration,and since former work from our lab demonstrated thatphosphorylation and nitration occurs one at expense of eachother, we suspected that inhibition of phosphorylation wouldlead to increased nitration.

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Figure 6. Suggested model of the dual mechanism by which peroxynitrite activates the NF-κB pathway. The left hand cascade represents the classicaltransient activation of peroxynitrite through the p38 pathway, resulting in IκBα phosphorylation and rapid p65/p50 nuclear translocation. The righthand cascade represents the aberrant somewhat delayed activation of peroxynitrite through nitration of IκBα. IκBα- Inhibitory protein kappa Balpha. IKK- IκB kinase; TNF-α- Tumor necrosis factor α; Tyr-NO2· - Nitro-Tyrosime; P50/p65- NF-κB heterodimer.

To examine this hypothesis, HT-29 cells were treated withTNF-α or peroxynitrite for in the presence and absence ofSB202190, MAPK p38-selective inhibitor. Inhibition of p38prior to peroxynitrite stimulation significantly increased(p<0.04) the level of IκBα nitration compared to the control(Figure 5A and B).

Discussion

RNS have been implicated in a wide variety of pathologicalconditions (14). In particular, RNS have been shown tomodulate inflammatory and cancer processes (15).

Our studies demonstrated that the NF-κB pathway isactivated by peroxynitrite. This activation occurred in a dose-dependent manner: the higher peroxynitrite concentrations were,the lower IκBα phosphorylation was but nitration was higher(Figure 1B). Moreover, stimulation with TNF-α, which is a wellknown stimulant of the classical NF-κB pathway throughphosphorylation of IκBα, caused it to be nitrated as well (Figure1B). This nitration was however, lower compared to its nitrationafter peroxynitrite stimulation. Unlike TNF-α stimulation,which involves IκBα phosphorylation and degradation (Figure2A and B), peroxynitrite led to delayed NF-κB activation whichwas assessed with IκBα degradation (Figures 2A, B, and C).Peroxynitrite stimulation, in spite of its high phosphorylationcaused only a slight degradation of IκBα. This may stem fromits high nitration which may interfere with the phosphorylationsites of recognition by the ubiquitin system. Since TNF-α hasbeen shown to increase the oxidative status in cells (16), it maybe possible that it also raises the nitration level, leading to a lowlevel of IκBα nitration.

Overall, these data suggest that peroxynitrite in this coloncancer cell line constitutes an independent signal for longerterm NF-κB activation. Activation of NF-κB requiresphosphorylation of IκBs by IKK. Therefore, the next step inour study was to investigate the pathway by whichperoxynitrite induces IKK activation.

The role of NF-κB in mediating inflammation has beenestablished using genetic approaches or selectivepharmaceutical inhibitors. In most cases, either epithelial cellsof the infected tissue or tissue-resident haematopoietic cells,such as mast cells or dendritic cells (DCs), initiate theinflammatory response by triggering pro-inflammatorypathways through NF-κB in response to inflammatory stimuli(17). These stimuli include inflammatory cytokines such asTNF-α, ROS such as H2O2, infection with bacteria and viruses,various carcinogens and tumor promoters such as benzo pyrene(17), the tobacco-specific nitrosamine N-nitrosonornicotine andphorbol ester, therapeutic agents such as taxol, apoptoticmediators such as anti-Fas, UV radiation and many others (15).

Since several studies have established a cross talk betweenthe MAPK p38 and the NF-κB pathways, we used specificpharmaceutical inhibitors of IKK, JNK, and p38 to shed light

on the connection between MAPK p38 and the NF-κBpathways in our system. Our results clearly indicate theinvolvement of the MAPK p38 pathway in IKK activation.Specific IKK inhibitor reduced IκBα phosphorylation afterperoxynitrite exposure by more than 40% compared toperoxynitrite stimulation alone. However, using p38 inhibitorprior to peroxynitrite treatment reduced its impact to 75%compared to peroxynitrite stimulation alone. Furthermore,investigating NF-κB nuclear translocation supported ourfinding for MAPK p38 involvement in NF-κB activation inresponse to peroxynitrite stimulation, indicating that activationof the NF-κB pathway by peroxynitrite might well occur viathe p38 pathway as was suggested by Mourkioti andRosenthal (12). In peripheral T-cells, T-cell stimulation byINF-γR1 can recruit Myeloid differentiation primary responsegene (88), and through Mixed-lineage protein kinase 3 activatep38, which activates IKK (12). In addition, an NF-κB targetgene was found to be expressed via p38 MAPK (18).

The exact mechanism by which peroxynitrite activatesp38, which then further activates NF-κB is still unknown andshould be investigated. Using the inhibitor of p38,SB202190, which eliminated phosphorylation, we stillobserved significant NF-κB translocation, pointing to the factthe mere nitration can also lead to NF-κB activation, thus,lending further support to the dual mechanism ofperoxynitrite activation of NF-κB (Figure 6).

The possible dual mechanism by which peroxynitriteactivates the NF-κB pathway clearly indicates continuousnitrative stress which occurs under chronic inflammation.This may cause longer term of activation of the NF-κBpathway. Consequently, this activation may prolong theinflammatory response. Such phenomena may create theenvironment for the first steps in carcinogenesis. Thus, thispathway should be tagged for further research to identifyinhibitors of the NF-κB pathway as well as chelators for theoxidative/ nitrative stress

AcknowledgementsThis work was supported by the Krol Foundation of Barnegat N.J.USA, and by a grant from the Rappaport Research Institute to AZR.We wish to thank Merkel Technologies Ltd, David Merkel Golanand Ariel Roytman, for their kind and professional assistance inoperating the ImageStream Multispectral Imaging Flow Cytometer,as well as in analysis of the data and reporting of results.

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Received January 25, 2011Revised April 20, 2011

Accepted April 21, 2011

Gochman et al: Peroxynitrite Activates NF-κB via Phosphorylation and Nitration

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