Novel effect of NF-{kappa}B activation: carbonylation and nitration injury to cytoskeleton and disruption of monolayer barrier in intestinal epithelium

A. Banan, L. J. Zhang, M. Shaikh, J. Z. Fields, A. Farhadi, and A. Keshavarzian

Section of Gastroenterology and Nutrition, Department of Internal Medicine, Department of Pharmacology, and Department of Molecular Physiology, Rush University Medical Center, Chicago, Illinois 60612

Submitted 18 March 2004 ; accepted in final form 31 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Using monolayers of intestinal cells, we reported that upregulation of inducible nitric oxide synthase (iNOS) is required for oxidative injury and that activation of NF-{kappa}B is key to cytoskeletal instability. In the present study, we hypothesized that NF-{kappa}B activation is crucial to oxidant-induced iNOS upregulation and its injurious consequences: cytoskeletal oxidation and nitration and monolayer dysfunction. Wild-type (WT) cells were pretreated with inhibitors of NF-{kappa}B, with or without exposure to oxidant (H2O2). Other cells were transfected with an I{kappa}B{alpha} mutant (an inhibitor of NF-{kappa}B). Relative to WT cells exposed to vehicle, oxidant exposure caused increases in I{kappa}B{alpha} instability, NF-{kappa}B subunit activation, iNOS-related activity (NO, oxidative stress, tubulin nitration), microtubule disassembly and instability (increased monomeric and decreased polymeric tubulin), and monolayer disruption. Monolayers pretreated with NF-{kappa}B inhibitors (MG-132, lactacystin) were protected against oxidation, showing decreases in all measures of the NF-{kappa}B -> iNOS -> NO pathway. Dominant mutant stabilization of I{kappa}B{alpha} to inactivate NF-{kappa}B suppressed all measures of the iNOS/NO upregulation while protecting monolayers against oxidant insult. In these mutants, we found prevention of tubulin nitration and oxidation and enhancement of cytoskeletal and monolayer stability. We concluded that 1) NF-{kappa}B is required for oxidant-induced iNOS upregulation and for the consequent nitration and oxidation of cytoskeleton; 2) NF-{kappa}B activation causes cytoskeletal injury following upregulation of NO-driven processes; and 3) the molecular event underlying the destabilizing effects of NF-{kappa}B appears to be increases in carbonylation and nitrotyrosination of the subunit components of cytoskeleton. The ability to promote NO overproduction and cytoskeletal nitration/oxidation is a novel mechanism not previously attributed to NF-{kappa}B in cells.

tubulin cytoskeleton; microtubules; oxidation/nitration; inducible nitric oxide synthase/peroxynitrite; inflammatory bowel disease; Caco-2 cells; gut barrier; nuclear factor-{kappa}B/I{kappa}B{alpha}


A FUNDAMENTAL PROPERTY of the gastrointestinal (GI) epithelium is to function as a highly selective permeability barrier that prevents the passage of harmful proinflammatory molecules (e.g., bacterial antigens) but permits the absorption of nutrients and electrolytes into the mucosa and the systemic circulation. Disruption of the gut barrier function, in contrast, allows the penetration of the normally excluded luminal antigens into the mucosa and leads to the initiation or perpetuation of inflammatory events and tissue injury (2, 28, 29, 3537). It is therefore not surprising that disruption of mucosal barrier function, the so-called "leaky gut," has been implicated in the pathogenesis of a wide spectrum of GI disorders, including inflammatory bowel disease (IBD) and multiple organ system dysfunction as well as systemic disorders (e.g., alcohol-induced liver disease) (2729, 32, 36, 47, 50, 53, 54). The underlying difficulty in managing these disorders, especially IBD, is due in large part to a lack of effective preventive strategies, which is due, in turn, to our limited understanding of their pathophysiology.

Although the pathophysiology of mucosal barrier disruption in IBD is not fully characterized, it was recently discovered that a leaky and hyperpermeable gut barrier can cause intestinal inflammation and that promoting a normal mucosal barrier function is essential for intestinal health. In animal models, intestinal barrier hyperpermeability induced by the injection of proinflammatory substances (e.g., peptidoglycan-polysaccharide, PG-PS) into the mucosa elicits oxidative and inflammatory conditions similar to those of IBD (54). Moreover, transgenic mice with a leaky gut exhibit symptoms of intestinal inflammation (27). It is also known that chronic gut inflammation in IBD is associated not only with high levels of oxidants (e.g., H2O2) but also with increased cytoskeletal instability, which together appear to be key contributors to mucosal injury (35, 3739). Indeed, upregulation of oxidants and the consequent oxidative stress and cytoskeletal disruption have been implicated in mucosal inflammation and damage in IBD (2, 8, 11, 12, 17, 18, 3539). Accordingly, understanding how gut barrier and cytoskeletal function are destabilized under oxidative, proinflammatory conditions is of substantial clinical and biological value.

During the past decade, we have been investigating injurious mechanisms such as oxidant-induced mucosal damage and barrier disruption not only to better understand endogenous defensive mechanisms (e.g., against oxidative disruption by H2O2) but also to devise a rational basis for the development of potentially more effective treatment regimes for IBD. Using monolayers of intestinal Caco-2 cells as a well-established model of gut cytoskeleton and barrier function, we previously reported several original findings that cytoskeletal oxidation and disassembly are required in oxidant-induced loss of barrier function (2, 4, 5, 7, 9) and that oxidants (H2O2, HOCl, and others) induce oxidative stress damage, in large part through the upregulation of inducible nitric oxide synthase (iNOS)-driven reactions (e.g., reactive nitrogen metabolites such as NO) (8, 14).

It is noteworthy that activation of NF-{kappa}B (a key proinflammatory factor) is essential to the promotion of an oxidative and inflammatory response in gut disorders such as in IBD (ulcerative colitis and Crohn's disease) (11, 12, 43, 46, 48). Once activated, NF-{kappa}B appears to regulate several important processes involved in inflammatory response, including loss of barrier function (3, 15, 16). Indeed, we recently reported novel findings on the importance of NF-{kappa}B-dependent mechanisms in oxidant-induced barrier disruption (3, 11). We showed, using intestinal Caco-2 cells, that oxidants induce the nuclear translocation and activation of NF-{kappa}B and lead to disruption of monolayer barrier (permeability) function (3). Despite the importance of NF-{kappa}B to intestinal barrier integrity, the molecular mechanisms underlying NF-{kappa}B-mediated, oxidant-induced disruption of monolayer barrier and cytoskeletal function are poorly understood.

In light of the aforementioned considerations, we tested the hypothesis that not only is NF-{kappa}B activation critical to oxidant-induced iNOS and NO upregulation but also that it is key to the injurious consequences of this upregulation, namely, microtubule cytoskeletal oxidation, nitration, disassembly and disarray, and monolayer dysfunction.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Caco-2 cells were obtained from American Type Culture Collection (ATCC, Rockville, MD) at passage 15. This cell line was chosen because the cells form monolayers that morphologically resemble small intestinal cells, with defined apical brush borders and a highly organized microtubule network upon differentiation (13, 25, 41). Cells were maintained at 37°C in complete Dulbecco's modified Eagle's medium (DMEM) in an atmosphere of 5% CO2 and 100% relative humidity. Wild-type cells or transfected cells (see Plasmids and transfection) were split at a ratio of 1:6 upon reaching confluency and were set up in either 6- or 24-well plates for experiments or in T-75 flasks for propagation. The utility and characterization of this cell line have been previously reported (31, 34, 41). In select experiments, IEC-6 cells were used. The IEC-6 cell line was purchased from ATCC at passage 13. Stock cells were maintained in DMEM supplemented with 5% FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin. The IEC-6 cell line is derived from normal rat intestinal crypt cells and has been characterized by Quaroni et al. (44).

Plasmids and transfection. The dominant negative mutant (superrepressor) of I{kappa}B{alpha} was used as previously described (3, 42). This mutant contains double point mutations substituting key serines 32 and 36 with alanine residues, which stabilizes I{kappa}B{alpha} (and prevents the activation of NF-{kappa}B). The construct was cloned into a cytomegalovirus (CMV) expression vector to overexpress the dominant negative mutant. An expression vector containing CMV plasmid alone served as a control. Stable transfectants were determined by immunoblot analysis of cell fractions.

For transfection, cultures of cells grown to 50–60% confluency were stably transfected with varying amounts (1–4 µg) of expression plasmid encoding a dominant negative mutant of I{kappa}B{alpha} by using Lipofectin reagent (25 µl Lipofectamine/25-cm2 flask; GIBCO BRL) as we recently described (3, 42). Control conditions included vector alone. I{kappa}B{alpha} protein expression and lack of its degradation (i.e., stability) were verified by Western blot analysis of cellular cytosolic fractions. Multiple clones were subsequently plated and allowed to form confluent monolayers and were then used for experiments.

Experimental design. In the first series of experiments, postconfluence monolayers of wild-type cells were incubated with oxidant (H2O2, 0–0.5 mM) or vehicle (isotonic saline) for 30 min, and then outcomes were assessed over time (e.g., from 0 to 8 h). Outcomes measured are described below. As we previously showed, H2O2 at 0.5 mM disrupts microtubules and barrier integrity and upregulates iNOS in wild-type intestinal cells (2, 8, 14). These experiments were then repeated using monolayers composed of clones of transfected cells (i.e., I{kappa}B{alpha} mutants). In all experiments, we assessed 1) microtubule cytoskeletal stability (cytoarchitecture, assembly), 2) polymerized (S2) and monomeric (S1) tubulin pools, 3) iNOS activity and protein, 4) NO levels, 5) reactive nitrogen metabolite (RNM) levels (e.g., ONOO), 6) oxidative stress [dichlorofluorescein (DCF) fluorescence], 7) tubulin nitration (nitrotyrosine formation), 8) tubulin oxidation (carbonylation), 9) I{kappa}B{alpha} distribution (cytosolic expression and instability), and 10) NF-{kappa}B p65 subunit activity (nuclear translocation and activity) and NF-{kappa}B p50 subunit activity (nuclear translocation and activity).

In the second series of experiments, we investigated the potential importance of the NF-{kappa}B in oxidant-induced oxidative stress upregulation (e.g., NO overproduction) as well as cytoskeletal oxidation/nitration injury by using several pharmacological inhibitors (all 30-min preincubations). Monolayers of wild-type cells were preincubated with four different NF-{kappa}B/I{kappa}B{alpha} inhibitors and then incubated with or without oxidant or vehicle. These inhibitors included known inhibitors of the activation of NF-{kappa}B (1, 33), alone or in combination with H2O2: curcumin (20 µM), 1-pyrrolidinedithiocarbamate (PDTC; 20 µM), carbobenzyloxy-leu-leu-leucinol (MG-132; 10 µM), and lactacystin (10 µM). Controls were treated with vehicle. We confirmed that these doses of inhibitors were not toxic to cells. Outcomes measured were as described for the first series of experiments.

In a third series of experiments, we incubated monolayers of I{kappa}B{alpha} dominant negative transfected cells (I{kappa}B{alpha} mutants) with oxidant or vehicle. In all experiments, I{kappa}B{alpha} levels and NF-{kappa}B activity were determined. Other outcomes measured were as described for the first series of experiments. In corollary experiments, we investigated the effects of NF-{kappa}B activation or inactivation on the state of 1) tubulin oxidation and tubulin nitration, 2) iNOS upregulation and NO overproduction, 3) tubulin assembly and disassembly, and 4) stability of the cytoarchitecture of the microtubule cytoskeleton. Monomeric (S1) and polymerized (S2) fractions of tubulin (the structural protein subunit of microtubules) were isolated and then analyzed for outcomes (e.g., oxidation and nitration by immunoblot analysis) (2, 14). Microtubule integrity was assessed by 1) immunofluorescent labeling and fluorescence microscopy to assess cells with normal microtubules, 2) detailed morphological analysis of cytoskeleton by ultrahigh-resolution laser scanning confocal microscopy, 3) immunoblot analyses of monomeric and polymerized tubulin pools, and 4) immunoblot analyses of oxidation and nitration of tubulin.

Assay of NOS activity. Wild-type and transfected cells grown to confluence were removed by scraping, centrifuged, and homogenized on ice in a buffer containing 50 mM Tris·HCl, 0.1 mM EDTA, 0.1 mM EGTA, 12 mM 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride (pH 7.4). Conversion of L-[3H]arginine (Amersham, Arlington Heights, IL) to L-[3H]citrulline was measured in the cell homogenates by scintillation counting. Experiments in the presence of NADPH, without Ca2+ and with 5 mM EGTA, determined Ca2+-independent NOS (iNOS) activity (1, 6, 8).

Western blot analysis of iNOS level. After treatments, the cells were washed once with cold PBS, scraped into 1 ml of cold PBS, and harvested in a standard antiprotease cocktail. For immunoblot analysis, samples (20 µg protein/lane) were added to SDS buffer (250 mM Tris·HCl, pH 6.8, 2% glycerol, and 5% mercaptoethanol), boiled for 5 min, and then separated on 7.5% SDS-PAGE. Subsequently, proteins were transferred to nitrocellulose membranes and then blocked in 3% BSA for 1 h, followed by several washes (Tris-buffered saline, TBS). The immunoblotted proteins were incubated for 2 h in Tween 20-TBS (TBST) and 1% BSA with the primary antibody (mouse monoclonal anti-human iNOS, at 1:3,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). A horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR) was used as a secondary antibody, at 1:3,000 dilution. Membranes were visualized by enhanced chemiluminescence (ECL) and then autoradiographed and processed for densitometry (8, 14).

Chemiluminescence analysis of NO. NO production was assessed using a unique chemiluminescence procedure (8, 14). Briefly, cells were homogenized, and endogenous nitrate (NO3) and nitrite (NO2), the metabolic degradation products of NO, were then reduced to NO using vanadium (III) (Sigma, St. Louis, MO) and HCl at 90°C before NO concentration was measured using a Sievers NOA 280 analyzer (Sievers Instruments, Boulder, CO). NO was expressed as a micromolar concentration and was calculated by comparison to the chemiluminescence of a standard solution of NaNO2. The absolute NO values were reported as micromoles per 1 x 106 cells.

Determination of cell oxidative stress. Oxidative stress was assessed by measuring the conversion of a nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (DCFD; Molecular Probes) into a fluorescent dye, DCF (1, 6, 14). Briefly, monolayers grown in 96-well plates were preincubated with the membrane-permeable DCFD (10 µg/ml for 30 min) before the subsequent treatments were carried out. Fluorescent signals (i.e., DCF fluorescence) from samples were quantitated using a fluorescence multiplate reader set at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. As we previously showed (6, 8), the dependence of the DCF assay on oxidative stress of reactive oxygen metabolite (ROM) production (e.g., ·O2 generation) was demonstrated by adding the active superoxide radical scavenger superoxide dismutase (SOD) or, as a control condition, heat-inactivated SOD (iSOD). Similarly, we previously showed the usefulness of this assay for detecting other forms of oxidative stress such as RNM production (e.g., NO or ONOO generation) by adding either an RNM scavenger (e.g., cysteine or urate) or an inhibitor of RNM biosynthesis [e.g., L-N6-(1-iminoethyl)lysine]. These studies thus indicated that oxidative stress seen under conditions of H2O2 challenge is most likely due to the generation of new ROM species (e.g., ·O2) as well as RNM species (e.g., NO).

Analysis of NF-{kappa}B activation. NF-{kappa}B (p65 and p50 subunit) activation was assessed by a unique ELISA-based procedure as we described previously (3, 15). Monolayers of wild-type and transfected cells grown in 25-cm2 flasks were processed for the isolation of cytosolic and nuclear fractions. Cell fractions were added to a 96-well plate to which oligonucleotides containing a consensus-binding site for NF-{kappa}B had been immobilized (Trans-Am; Active Motif, Carlsbad, CA). Specifically, the NF-{kappa}B activity test is based on a validated ELISA principle whereby NF-{kappa}B is captured by a double-stranded oligonucleotide probe containing the consensus-binding sequence for either NF-{kappa}B p65 or p50 subunit (45). Consequently, only the activated NF-{kappa}B is captured by the probe bound in microwell plates. The binding of NF-{kappa}B to its consensus sequence was then detected using a primary anti-NF-{kappa}B (p65 or p50) antibody (Santa Cruz Biotechnology), followed by a secondary antibody conjugated to HRP. The results were quantitated by a chromogenic reaction (45), which was then read for absorbance at 450 nm by using a multiplate reader (FL 600; BIO-TEK Instruments).

Western blot analysis of changes in NF-{kappa}B subunit levels and nuclear translocation. Cellular nuclear and cytosolic extracts from wild-type and transfected cells were prepared as described above. NF-{kappa}B nuclear distribution (translocation) was determined by comparing the levels of NF-{kappa}B protein (e.g., p65 subunit) expression in the cytosolic and nuclear extracts with the use of anti-p65 (or anti-p50) antibody on a nondenaturing gel (6%) (3, 15). Samples (20 µg protein/lane) were placed in a standard sample buffer, boiled, and then subjected to PAGE. Proteins were visualized by ECL (Amersham) and autoradiography. For comparison of different blots, standard (positive) loading controls (20 µg of HeLa cell extracts/lane) for NF-{kappa}B were included concurrently with each run. In addition, after the blots were stripped, actin (~43 kDa) immunoblotting was performed as an internal control for equal loading.

Electrophoretic mobility shift assays. LightShift chemiluminescent EMSA kits (Pierce, Rockford, IL), which use a nonisotopic method to detect specific DNA-protein interactions, were utilized (51). Biotin-end-labeled DNA is incubated with nuclear extracts. This reaction is then subjected to gel electrophoresis on a nondenaturing (6%) polyacrylamide gel and transferred to a nylon membrane. The biotin-end-labeled DNA is detected using the streptavidin-HRP conjugate and LightShift chemiluminescent substrate. To this end, thawed nuclear extracts (6–10 µg) were prepared as described above and incubated with 1 ng of biotin-end-labeled NF-{kappa}B-specific probes (provided in the kit) in a total volume of 25 µl in the presence of 10 mM Tris·HCl (pH 7.5), 80 mM NaCl, 1 mM EGTA, 1 mM DTT, 10% glycerol, and 1 µl of poly(dI-dC). DNA-nuclear protein complexes were separated by PAGE (6%, nondenaturing gels) and visualized by autoradiography. The specificity of binding interactions was further assessed by competition with an excess of unlabeled double-stranded oligonucleotide of the same identity.

Western blot analysis of I{kappa}B{alpha} degradation and expression levels. The level of I{kappa}B{alpha} expression in the cytosolic extracts as well as its degradation and/or instability (i.e., disappearance from the cytosolic fractions) was confirmed by anti-I{kappa}B{alpha} antibody (Santa Cruz Biotechnology) according to a Western blot protocol (10% gel) (3, 15). Briefly, samples (20 µg protein/lane) were added to a standard SDS buffer, boiled, and then separated on SDS-PAGE. As for NF-{kappa}B, proteins were visualized by ECL and subsequently autoradiographed and processed for densitometry. Standard (positive) loading controls (20 µg of HeLa cell extracts/lane) for I{kappa}B{alpha} were included in each run.

Immunofluorescent staining and high-resolution laser scanning confocal microscopy of microtubules. Cells from monolayers were fixed in cytoskeletal stabilization buffer and then postfixed in 95% ethanol at –20°C as we previously described (1–17). Cells were then processed for incubation with a primary antibody, monoclonal mouse anti-{beta}-tubulin (human reactive; Sigma) at 1:200 dilution for 1 h at 37°C and then incubated with a secondary antibody (FITC-conjugated goat anti-mouse; Sigma) at 1:50 dilution for 1 h at room temperature. Slides were washed three times in Dulbecco's PBS and subsequently mounted in aquamount. After staining, cells were observed with an argon laser ({lambda} = 488 nm) using a x63 oil-immersion Plan Apochromatic objective, NA 1.4 (Carl Zeiss, Oberkochen, Germany). Cells from desired areas of monolayers were processed using the image processing software on a Zeiss ultrahigh-resolution laser scanning confocal microscope. The cytoskeletal elements were examined in a blinded fashion for their overall morphology, orientation, and disruption as we described (1–17). The identity of the treatment groups for all slides was decoded only after examination was complete.

Microtubule (tubulin) fractionation and quantitative immunoblot analysis of tubulin assembly. Polymerized (S2) and monomeric (S1) fractions of tubulin were isolated using a series of extraction and ultracentrifugation steps as we described previously (2, 13). Briefly, cells were gently scraped and pelleted with centrifugation at low speed (700 rpm, 7 min, 4°C) and resuspended in microtubule stabilization-extraction buffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% DMSO, 1 mM MgSO4, 10 µg/ml antiprotease cocktail, 1 mM EGTA, and 1% Triton X-100) at room temperature for 20 min. Tubulin fractions were separated after a series of centrifugation and extraction steps. Specifically, cell lysates were centrifuged at 105,000 g for 45 min at 4°C, and the supernatant containing the soluble monomeric pool of tubulin (S1 fraction) was gently removed. The remaining pellet was then resuspended in 0.3 ml of Ca2+-containing depolymerization buffer (0.1 M PIPES, pH 6.9, 1 mM MgSO4, 10 µg/ml antiprotease cocktail, and 10 mM CaCl2) and incubated on ice for 60 min. Subsequently, samples were centrifuged at 48,000 g for 15 min at 4°C, and the supernatant (S2 fraction or cold/Ca2+-soluble fraction) was removed. To ensure complete removal of the S2 fraction, we treated the remaining pellet with the Ca2+-containing depolymerization buffer twice more by resuspension and centrifugation. The "microtubules" were recovered by separate incubation (at 37°C for 30 min) of the S1 and S2 fractions with the stabilizing agents taxol (10 µM) and GTP (1 mM) in microtubule stabilization buffer (MSB: 0.1 M PIPES, pH 6.9, 30% glycerol, 5% DMSO, 10 µg/ml antiprotease cocktail, 1 mM EGTA, 1 mM MgCl2, and 1 mM GTP) to promote polymerization of tubulin. Tubulin was then recovered by centrifugation and resuspended in MSB. Fractionated S1 and S2 samples were then flash frozen in liquid N2 and stored at –70°C until immunoblotting. For immunoblotting, samples (5 µg protein/lane) were placed in a standard SDS sample buffer, boiled for 5 min, and then subjected to PAGE on 7.5% gels. Procedures for Western blotting were performed as previously described (2, 13). The immunolabeled tubulin was then processed for autoradiography. Standard (purified) tubulin loading controls (5 µg/lane) were included concurrently with each run.

Immunoblotting determination of protein tubulin oxidation and tubulin nitration. Oxidation and nitration of the microtubule (tubulin) cytoskeleton were assessed, respectively, by measuring protein carbonyl and nitrotyrosine formation using a method we developed (2, 14). To prevent unwanted oxidation of tubulin samples, all buffers contained 0.5 mM DTT and 20 mM 4,5-dihydroxy-1,3-benzene sulfonic acid (Sigma). To determine the carbonyl content, we blotted samples to a polyvinylidene difluoride membrane, followed by successive incubations in 2 N HCl and 2,4-dinitrophenylhydrazine (DNPH, 100 µg/ml in 2 N HCl; Sigma) for 5 min each. Membranes were then washed three times in 2 N HCl and subsequently washed seven times in 100% methanol (5 min each), followed by blocking for 1 h in 5% BSA in 10x PBS containing Tween 20 (PBS-T). Immunologic evaluation of carbonyl formation was performed for 1 h in 1% BSA/PBS-T buffer containing anti-DNPH (1:25,000 dilution; Molecular Probes). Membranes were then incubated with a HRP-conjugated secondary antibody (1:4,000 dilution, 1 h; Molecular Probes). To determine nitrotyrosine content, after the blocking step above (i.e., BSA/PBS-T buffer), membranes were probed for nitrotyrosine by incubation with 2 µg/ml monoclonal anti-nitrotyrosine antibody for 1 h (Upstate Biotechnology, Lake Placid, NY), followed by the HRP-conjugated secondary antibody (as above). Wash steps and film exposure were as described in a standard Western protocol (2, 14). The relative levels of oxidized or nitrated tubulin were then quantified by measuring, with a laser densitometer, the optical density (OD) of the bands corresponding to anti-DNPH or anti-nitrotyrosine immunoreactivity. Immunoreactivity was expressed as the percentage of carbonyl or nitrotyrosine formation (OD) in the treatment group compared with the maximally oxidized or nitrated tubulin standards, which also served as loading controls. Oxidized or nitrated tubulin standards (5 µg/lane) were run concurrently with corresponding treatment groups. To further verify equal loading of lanes (22), blots were routinely stained with 0.1% india ink in TBST buffer. Oxidized tubulin standard was prepared by utilizing purified tubulin (ICN, Costa Mesa, CA) that was subsequently carbonylated by exposure to 0.5 mM H2O2 and 1 mM FeSO4 (30 min at room temperature). Nitrated tubulin standard was prepared by reacting purified tubulin with 0.1 mM ONOO (30 min at 37°C). These oxidized standards were then precipitated with trichloroacetic acid (TCA) followed by the decanting of supernatant and were washed (3 times) with TCA to remove excess oxidizing agents.

Statistical analysis. Data are presented as means ± SE. All experiments were carried out with a sample size of at least six observations per treatment group. Statistical analysis comparing treatment groups was performed using analysis of variance followed by Dunnett's multiple range test (26). Correlational analyses were done using the Pearson test for parametric analysis or, when applicable, the Spearman test for nonparametric analysis. P values < 0.05 were deemed statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the current investigation, utilizing both pharmacological and molecular biological interventions, we have studied the mechanisms by which NF-{kappa}B injures the monolayer cytoskeleton and barrier function.

Pharmacological inhibition of NF-{kappa}B protects against oxidative damage to the cytoskeleton: Prevention of both tubulin nitration and oxidation. Because NF-{kappa}B is key in oxidant-induced barrier disruption (3), we surmised that this disruption may be due to the activation of oxidant-activated pathways such as the one triggered by reactive metabolites. Using intestinal cells, we initially measured the "footprints" of RNM formation, namely, nitrotyrosine moieties under conditions of oxidant challenge with or without several pharmacological inhibitors of NF-{kappa}B. We also measured oxidation footprints by assessing carbonylation levels. These were performed by sequentially fractionating and purifying the 50-kDa tubulin molecule from cell monolayers and subsequently immunoblotting these fractions to assess the oxidation state of microtubule (tubulin-based) cytoskeleton. Oxidant (H2O2, 0.5 mM) alone resulted in substantial levels of nitration and oxidation of the tubulin cytoskeleton (Fig. 1A). In contrast, pharmacological inhibitors of NF-{kappa}B afforded protection against oxidant-induced tubulin nitration and tubulin carbonylation as demonstrated by decreased oxidation levels, which were comparable to those in control (vehicle treated) cells. For example, only cells pretreated with inhibitors of NF-{kappa}B activation (e.g., curcumin or PDTC) were protected against oxidant-induced nitration and oxidation injuries (95% less oxidation). Similarly, preincubation with inhibitors of the NF-{kappa}B modulator I{kappa}B{alpha} (e.g., lactacystin or MG-132) also led to protection against oxidation (by ~90–93%). In the absence of oxidants, these inhibitors by themselves did not affect tubulin (not shown).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1. A: pharmacological inhibitors of NF-{kappa}B protect against oxidant-induced nitration (nitrotyrosination) and oxidation (carbonylation) injury to the microtubule (tubulin-based) cytoskeleton of intestinal cell monolayers. Caco-2 cells were incubated with 4 different inhibitors of either NF-{kappa}B [curcumin or 1-pyrrolidinedithiocarbamate (PDTC)] or its endogenous modulator, I{kappa}B{alpha} (lactacystin or MG-132) before subsequent exposure to oxidant (H2O2) or vehicle and were then processed for immunoblot analysis. Nitration or oxidation immunoreactivity was normalized to a maximally nitrated or oxidized purified tubulin loading standard and expressed as a percentage. Values are means ± SE; n = 6 observations per treatment group in all figures unless otherwise indicated. *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2 alone. Representative immunoblots of the tubulin nitration (B) and tubulin oxidation (carbonylation; C) following the treatments described in A are shown. Bands representing tubulin nitration immunoreactivity (anti-nitrotyrosine) or tubulin carbonylation immunoreactivity [anti-dinitrophenylhydrazone (DNP)] are from cells exposed to vehicle (lane a), cells exposed to 0.5 mM H2O2 (lane b), cells preexposed to NF-{kappa}B inhibitor (curcumin) + 0.5 mM H2O2 (lane c), cells preexposed to NF-{kappa}B inhibitor (PDTC) + 0.5 mM H2O2 (lane d), cells preexposed to NF-{kappa}B inhibitor (lactacystin) + 0.5 mM H2O2 (lane e), cells preexposed to NF-{kappa}B inhibitor (MG-132) + 0.5 mM H2O2 (lane f), and corresponding nitrated or oxidized tubulin (5 µg/lane) loading standard (approximate molecular weight ~50 kDa) (lane g). Pharmacological inhibitors of NF-{kappa}B protected microtubule cytoskeleton against both nitration and oxidation injury induced by oxidant challenge (lanes c–f in B and C). This low level of oxidation is comparable to that of control cells (vehicle treated, corresponding lane a). Representative blots are shown.

 
Figure 1, B and C, exhibits representative immunoblots of the alterations in tubulin nitration and carbonylation. As shown, the inhibitors of NF-{kappa}B substantially prevent both tubulin nitration (Fig. 1B) and oxidation (Fig. 1C) as demonstrated by reduced band (lane) densities to a level close to that of controls, indicating suppression of oxidative damage to the tubulin cytoskeleton (by pharmacological inhibition of NF-{kappa}B). As above, inhibitors of I{kappa}B{alpha} (a known endogenous regulator of NF-{kappa}B) also prevented the appearance of both tubulin oxidation and nitration bands in the immunoblots of cell fractions. In contrast, oxidant alone caused the oxidation and nitration of tubulin as shown by increased oxidation band densities.

NF-{kappa}B-induced oxidative injury involves upregulation of iNOS-driven processes: Increases in iNOS, NO, RNM (ONOO), and oxidative stress. We next probed potential mechanisms through which NF-{kappa}B promotes oxidative stress to the cytoskeleton. Because oxidants such as H2O2 upregulate iNOS (8, 14), we hypothesized that activation of iNOS-driven pathways might be a novel mechanism for NF-{kappa}B-induced oxidative injury to the tubulin-based cytoskeleton.

To this end, the same four pharmacological inhibitors of NF-{kappa}B also cause a substantial reduction in Ca2+-independent iNOS activity induced by oxidant (0.5 mM) challenge (~96–98% lower iNOS activity) (Fig. 2A). This is comparable to that of the control, displaying only low (basal) iNOS activity. In contrast, oxidant by itself causes large increases in iNOS activity. Similarly, NO levels (a product of the iNOS-catalyzed reaction) in monolayers exposed to H2O2 were also increased (not shown). Inhibitors of NF-{kappa}B activity markedly prevented oxidant-induced NO overproduction. Indeed, as for both tubulin oxidation/nitration and iNOS upregulation, NO overproduction was suppressed by these inhibitors.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2. A: protective effects of NF-{kappa}B inhibitors against upregulation of inducible nitric oxide synthase (iNOS) activity induced by oxidant (H2O2) in intestinal Caco-2 monolayers. Cells were preincubated with 4 different NF-{kappa}B inhibitors (curcumin, PDTC, lactacystin, or MG-132) and then exposed to H2O2 and subsequently processed for assessment of L-[3H]citrulline formation. *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2 alone. B: representative Western immunoblot showing protective effects of NF-{kappa}B inhibitors against upregulation of iNOS protein in Caco-2 cell monolayers. Bands representing iNOS upregulation are from cells exposed to vehicle (lane a), cells exposed to 0.5 mM H2O2 (lane b), cells preexposed to NF-{kappa}B inhibitor (curcumin) + 0.5 mM H2O2 (lane c), cells preexposed to NF-{kappa}B inhibitor (PDTC) + 0.5 mM H2O2 (lane d), cells preexposed to NF-{kappa}B inhibitor (lactacystin) + 0.5 mM H2O2 (lane e), and cells preexposed to NF-{kappa}B inhibitor (MG-132) + 0.5 mM H2O2 (lane f). In cells preincubated with pharmacological inhibitors of NF-{kappa}B (or of its modulator, I{kappa}B{alpha}) iNOS upregulation is completely suppressed. The region of gel shown was between the Mr 126,000 and 218,000 prestained molecular weights, which were run in adjacent lanes. Top arrow denotes approximate apparent molecular weight for iNOS. After blots were probed for iNOS protein and stripped, actin (~43 kDa) immunoblotting was performed as an internal control for equal loading. A representative blot is shown.

 
Figure 2B depicts a representative Western blot showing that H2O2 significantly increased iNOS protein levels in intestinal cells, whereas cells pretreated with any of the pharmacological inhibitors of NF-{kappa}B activity (e.g., curcumin or PDTC, lactacystin or MG-132) exhibited only low, basal levels of the iNOS protein. For example, the corresponding OD values were 346 ± 23 for the control, 4,235 ± 72 for 0.5 mM H2O2, and 392 ± 56 for inhibitor (e.g., MG-132)-pretreated cells incubated in H2O2. Inhibitors by themselves, similar to their lack of effects on basal iNOS activity and tubulin oxidation, did not affect basal iNOS protein (not shown).

In parallel with the prevention of oxidant-induced effects, NF-{kappa}B inhibitors suppressed oxidative stress as determined by reduction in the fluorescence of DCF (Fig. 3). In cells where H2O2 substantially increased DCF fluorescence, oxidative stress was prevented only by inhibitors of NF-{kappa}B activity. In the absence of oxidant (i.e., vehicle), we observed significantly lower and basal levels of cellular oxidative stress [presumably due to the normal generation of DCF-reactive oxygen radicals (e.g., ·O2) by well-known cellular metabolic processes such as the mitochondrial respiratory chain reactions (1, 6, 8, 14)]. Inhibitors by themselves did not affect basal oxidative stress (not shown).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 3. Oxidative stress in intestinal Caco-2 cells that is induced by oxidant insult is attenuated by NF-{kappa}B inhibitors, assessed by alterations in dichlorofluorescein (DCF) fluorescence intensity. NF-{kappa}B inhibitors (curcumin, PDTC, lactacystin, or MG-132) suppress oxidative stress as demonstrated by a reduction in the DCF fluorescence level, which was comparable to the low (basal) oxidative stress shown in the control. Only cells exposed to oxidant alone showed increased oxidative stress. *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2 alone.

 
Pharmacological inhibition of NF-{kappa}B activity protects assembly of tubulin pools and cytoarchitecture of microtubule cytoskeleton. Because it is known that oxidants in this intestinal model disrupt the cytoskeleton, we confirmed the state of tubulin polymerization and its intracellular architecture (3). Pharmacological inhibitors of NF-{kappa}B conferred protection to the assembly of tubulin pools (Fig. 4, A and B, both polymerized/S2 and monomeric/S1) as well as to the cytoarchitecture of microtubule cytoskeleton (Fig. 4C). For instance, to confirm effects of NF-{kappa}B inhibitors on the dynamic alterations in the polymerization states of the tubulin, we performed immunoblot analysis of tubulin cytoskeletal pools. To this end, the intracellular tubulin fractions (both polymerized S2 and monomeric S1, respectively, indexes of microtubule stability and instability) were isolated from monolayers. Figure 4 shows that pharmacological inhibition of NF-{kappa}B in monolayers that were exposed to oxidant led to a stable (protected) tubulin assembly as indicated by an enhancement in the polymerized (S2) tubulin (i.e., increased band density) (Fig. 4A) and a corresponding decrease in the monomeric (S1) tubulin (Fig. 4B). This preserved state of tubulin assembly is comparable to that of controls. In contrast, oxidant decreased stable polymerized tubulin (Fig. 4A), whereas it increased unstable monomeric tubulin (Fig. 4B), indicating disassembly of the microtubule cytoskeleton.



View larger version (64K):
[in this window]
[in a new window]
 
Fig. 4. A and B: stabilizing (protective) effects of NF-{kappa}B inhibitors on the assembly of tubulin cytoskeletal pools in intestinal Caco-2 monolayers. Microtubule cytoskeletal extracts, stable polymerized tubulin (S2; A) and unstable monomeric tubulin (S1; B) from cells were subjected to SDS-PAGE fractionation and then immunoblotted using a monoclonal anti-{beta}-tubulin antibody followed by a HRP-conjugated-secondary antibody and were subsequently processed for autoradiography. Bands representing the polymerized S2 as well as monomeric S1 tubulin are from cells exposed to vehicle (lane a), cells exposed to 0.5 mM H2O2 (lane b), cells preexposed to NF-{kappa}B inhibitor (curcumin) + 0.5 mM H2O2 (lane c), cells preexposed to NF-{kappa}B inhibitor (PDTC) + 0.5 mM H2O2 (lane d), cells preexposed to NF-{kappa}B inhibitor (lactacystin) + 0.5 mM H2O2 (lane e), and cells preexposed to NF-{kappa}B inhibitor (MG-132) + 0.5 mM H2O2 (lane f). Lane g represents purified tubulin (5 µg/lane) loading standard (~50 kDa). In cells preincubated with NF-{kappa}B inhibitors, the destabilizing effects induced by H2O2 on the assembly of polymerized and monomeric tubulin were not seen (lanes c–f in A and B). This protection is demonstrated by a preserved assembly of tubulin pool (S2, S1), which is comparable to the corresponding control (lane a). C: intracellular distribution of the microtubule cytoskeleton as confirmed by ultrahigh-resolution laser scanning confocal microscopy (LSCM) in intestinal Caco-2 cell monolayers. Cell monolayers were exposed to vehicle (a), exposed to 0.5 mM H2O2 (b), or incubated with a representative NF-{kappa}B inhibitor (lactacystin) before exposure to 0.5 mM H2O2 (c). In cells exposed to H2O2 (b), the microtubule cytoarchitecture is clearly disorganized, fragmented, and collapsed. In the presence of lactacystin (c), on the other hand, microtubule integrity appears to be highly preserved (protected). This appearance is indistinguishable from that of the control (a), in which intact microtubules were dispersed in a stellar fashion throughout the cytosol. Bar = 25 µm. Representative photomicrographs are shown.

 
High-resolution laser scanning confocal microscopy confirms (Fig. 4C) that intestinal cells pretreated with NF-{kappa}B inhibitors show a normal architecture of the microtubule cytoskeleton, even after exposure to oxidant (Fig. 4Cc, lactacystin). This preserved appearance is indistinguishable from that of control (vehicle treated) cells (Fig. 4Ca), which also show an intact organization of the microtubule cytoskeleton. On the other hand, cell monolayers that are challenged with H2O2 alone exhibit instability, fragmentation, and collapse of the microtubule cytoskeleton (Fig. 4Cb). The noted findings on protection of both the dynamic assembly and the cytoarchitecture of microtubule cytoskeleton by NF-{kappa}B inhibitors parallel the protective effects of the same inhibitors against oxidant-induced iNOS and NO upregulation as well as tubulin oxidation and nitration.

Intracellular distribution and activation of NF-{kappa}B subunit proteins (p50, p65) in intestinal cells parallel several different indexes of iNOS/NO and oxidative stress in monolayers. Assessment of alterations in NF-{kappa}B subunit distribution in the nuclear fractions (Figs. 5) shows that NF-{kappa}B inhibitors suppressed oxidant-induced nuclear distribution of the NF-{kappa}B subunits p50 (Fig. 5A) and p65 (Fig. 5B) as shown by the corresponding reductions in band densities, which are comparable to those of controls. On the other hand, exposure to oxidant led to increased nuclear distribution of NF-{kappa}B subunits, paralleling findings on oxidant-induced activation of oxidative stress pathways.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 5. A and B: representative immunoblots of the distribution of NF-{kappa}B protein subunits p50 (A) and p65 (B) in nuclear fractions of intestinal Caco-2 cells. Bands representing p50 or p65 protein bands are from cells exposed to vehicle (lane a), cells exposed to 0.5 mM H2O2 (lane b), cells preexposed to NF-{kappa}B inhibitor (curcumin) + 0.5 mM H2O2 (lane c), cells preexposed to NF-{kappa}B inhibitor (PDTC) + 0.5 mM H2O2 (lane d), cells preexposed to NF-{kappa}B inhibitor (lactacystin) + 0.5 mM H2O2 (lane e), and cells preexposed to NF-{kappa}B inhibitor (MG-132) + 0.5 mM H2O2 (lane f). Inhibitors of NF-{kappa}B (curcumin or PDTC) or of its endogenous modulator, I{kappa}B{alpha} (lactacystin or MG-132), prevented NF-{kappa}B subunit nuclear distribution under conditions of oxidant (H2O2) exposure. This level of distribution is comparable to that in control (vehicle treated) cells, which exhibited basal levels of NF-{kappa}B (p50 and p65 subunits) in the nuclear extracts. The region of gel shown was between the Mr 43,000 and 75,000 prestained molecular weights, which were run in adjacent lanes. After blots were probed for NF-{kappa}B protein subunits and stripped, actin (~43 kDa) immunoblotting was performed as an internal control for equal loading. Representative blots are shown.

 
Using a highly sensitive ELISA, we previously showed (3) that there is a substantial increase in the activity levels of NF-{kappa}B (in the nuclear fractions) after exposure to H2O2. The p65 and p50 subunit proteins of NF-{kappa}B were activated to an almost identical degree. Pharmacological inhibitors of NF-{kappa}B (curcumin, PDTC, lactacystin, or MG-132) prevented the ability of oxidant to cause NF-{kappa}B activation as demonstrated by decreases in OD at 450 nm.

Using data across all experimental conditions, we found positive correlations (e.g., r = 0.92, P < 0.05) between NF-{kappa}B activity (activity assay or OD from the nuclear fractions) and iNOS upregulation, further indicating that activation of NF-{kappa}B is key in oxidant-induced iNOS upregulation and its deleterious consequences (e.g., nitration). Other correlations were seen when either NO overproduction or oxidative stress (DCF fluorescence) was correlated with the NF-{kappa}B activity (r = 0.91 or 0.88, respectively, P < 0.05 for each). When two other markers of oxidative stress, tubulin carbonylation and tubulin nitration, were correlated with NF-{kappa}B, additional correlations were observed (r = 0.96 and 0.97, respectively, P < 0.05 for each), further indicating that activation of NF-{kappa}B is key in NO overproduction and cytoskeletal nitration through upregulation of iNOS enzyme. Similarly, when markers of stability such as either microtubule integrity or tubulin assembly (e.g., S2 pool) were correlated with the NF-{kappa}B, still other correlations were seen (r = –0.87 or –0.90, respectively, P < 0.05 for each).

Inhibition of NF-{kappa}B activity by a dominant negative mutant (superrepressor) for I{kappa}B{alpha} after transfection of intestinal cells: Prevention of oxidative stress of iNOS and NO upregulation and cytoskeletal nitration/oxidation in I{kappa}B{alpha} mutant clones. The aforementioned findings collectively indicate that NF-{kappa}B activation could play a novel role in promotion of the oxidative stress of NO upregulation and cytoskeletal nitration/oxidation (and disarray). To independently demonstrate that NF-{kappa}B contributes to oxidative injury to cytoskeleton and to further probe the underlying events, we used stable I{kappa}B{alpha} mutant (dominant negative) transfected clones of intestinal cells that we recently developed (in which I{kappa}B{alpha} is stabilized against degradation) (3). Because oxidants not only reduce the stability of I{kappa}B{alpha} (a 37-kDa endogenous modulator of NF-{kappa}B) but also increase monolayer dysfunction in intestinal Caco-2 cells, we surmised that reduced stability of I{kappa}B{alpha} could be a unique mechanism underlying NF-{kappa}B-induced upregulation of oxidative stress and cytoskeletal oxidation.

Initially, intestinal Caco-2 cells were stably transfected with plasmid DNA encoding both neomycin resistance (for selection) and varying amounts (1, 2, 3, or 4 µg) of the I{kappa}B{alpha} mutant (3). Using this dominant negative approach, we were able to suppress NF-{kappa}B activation in Caco-2 clones as determined by an EMSA (a nonradioactive chemiluminescent gel shift), using biotin-end-labeled NF-{kappa}B-specific probes (Fig. 6, 3-µg mutant). Transfection of control vector alone (CMV plasmid), as might be expected, did not inhibit NF-{kappa}B activity. Indeed, NF-{kappa}B activity in the control vector clone, which was challenged with oxidant, was similar to that of wild-type cells under similar conditions. Utilizing a second technique involving a sensitive ELISA, we confirmed that in the I{kappa}B{alpha} mutant cells, oxidant could no longer increase the NF-{kappa}B activity in nuclear fractions of Caco-2 cells (Table 1). We observed similar effects by I{kappa}B{alpha} mutant transfection in a normal intestinal cell line, IEC-6 (Table 2).



View larger version (59K):
[in this window]
[in a new window]
 
Fig. 6. Inactivation of NF-{kappa}B in intestinal Caco-2 cells by a dominant negative mutant (superrepressor) for I{kappa}B{alpha}, assessed using a modified electrophoretic mobility shift assay (EMSA) with biotin-end-labeled NF-{kappa}B-specific probes. We used I{kappa}B{alpha} mutant Caco-2 clones recently developed in our laboratory that show almost complete suppression of NF-{kappa}B activity. Monolayers were exposed to H2O2 (0.5 mM), processed for extraction, and then assessed for sequence-specific NF-{kappa}B binding activity. Thus nuclear extracts were prepared, and a modified nonisotopic EMSA assay (LightShift chemiluminescent EMSA) was performed using 10 µg of nuclear proteins and 1 ng of biotin-end-labeled NF-{kappa}B probe. The position of the specifically bound DNA-protein complex is indicated. Bands representing NF-{kappa}B are from wild-type cells exposed to vehicle (lane a), vector cells exposed to vehicle (lane b), mutant cells exposed to vehicle (lane c), wild-type cells exposed to H2O2 (lane d), mutant cells exposed to H2O2 (lane e), and vector cells exposed to H2O2 (lane f). Wild type, cells not transfected with dominant plasmid; mutant, dominant I{kappa}B{alpha} mutant inhibiting NF-{kappa}B activity; vector, control vector alone; n = 5 observations per group.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Effects of varying amounts of a mutant I{kappa}B{alpha} on iNOS activity, NO levels, and NF-{kappa}B activity in intestinal Caco-2 monolayers

 

View this table:
[in this window]
[in a new window]
 
Table 2. Effects of varying amounts of a mutant I{kappa}B{alpha} on iNOS activity, NO Levels, NF-{kappa}B activity, and tubulin nitration in a normal intestinal cell line, IEC-6

 
Tables 1 and 2 further demonstrate the dose-dependent protective effects of varying amounts (1, 2, 3, or 4 µg) of I{kappa}B{alpha} mutant plasmid on suppression of oxidant-induced iNOS upregulation and NO overproduction in intestinal cell clones. For example, I{kappa}B{alpha} mutant suppression of NF-{kappa}B activity substantially prevented iNOS upregulation by oxidant (0.5 mM H2O2). In wild-type cells, on the other hand, this same concentration of oxidant upregulated iNOS. A very large percentage (~96%) of iNOS upregulation induced by oxidant was NF-{kappa}B/I{kappa}B{alpha} dependent. In both Caco-2 cells (Table 1) and IEC-6 cells (Table 2), as might be expected, the clones that were transfected with 3 µg of I{kappa}B{alpha} dominant negative plasmid (i.e., 3-µg clone) resulted in a maximum inability of oxidant to activate NF-{kappa}B, upregulate iNOS, or overproduce NO.

Furthermore, immunoblot analysis of the oxidative state of tubulin (Fig. 7A and Table 2) from the same I{kappa}B{alpha} mutant clone shows that suppression of NF-{kappa}B activity prevented (protected against) oxidant-induced tubulin oxidation. In parallel, immunoblot analysis of the state of tubulin pool assembly from these I{kappa}B{alpha} mutant cells demonstrates (Fig. 7B, Caco-2 cells) that inhibition of NF-{kappa}B activity attenuated tubulin depolymerization induced by oxidant challenge. In these mutant clones, oxidant cannot cause tubulin disassembly as shown by enhanced polymerized (stable) tubulin to near control levels.



View larger version (60K):
[in this window]
[in a new window]
 
Fig. 7. A: immunoblot analysis showing the suppressive (protective) effects of stable inactivation of NF-{kappa}B by an I{kappa}B{alpha} mutant on oxidant-induced tubulin nitration and oxidation in intestinal Caco-2 cells. This dominant mutant inhibition was protective against oxidant-induced tubulin nitration and carbonylation. Nitration or carbonylation immunoreactivity was normalized to a maximally nitrated or oxidized tubulin standard and expressed as a percentage. *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2 in wild-type cells. &P < 0.05 vs. H2O2 in mutant cells. WT, wild type. B: representative immunoblot confirming the state of tubulin polymerization in intestinal Caco-2 monolayers of either mutant or wild-type origin. Inactivation of NF-{kappa}B (by an I{kappa}B{alpha} mutant) protected against oxidant-induced reduction of tubulin assembly (lane e), which is comparable to that of controls (lanes a–c). For comparison, oxidant-induced reduction of polymerized tubulin in both wild-type (lane d) and vector clones (lane f) is also shown. Tubulin fractions were extracted from intestinal monolayers, subjected to SDS-PAGE using a monoclonal anti-{beta}-tubulin antibody followed by a HRP-conjugated-secondary antibody, and subsequently autoradiographed. Bands represent polymerized tubulin from wild-type cells exposed to vehicle (lane a), vector cells exposed to vehicle (lane b), mutant cells exposed to vehicle (lane c), wild-type cells exposed to H2O2 (lane d), mutant cells exposed to H2O2 (lane e), and vector cells exposed to H2O2 (lane f). Lane g represents purified tubulin (5 µg/lane) loading standard (~50 kDa). A representative blot is shown. C: inactivation of NF-{kappa}B by an I{kappa}B{alpha} mutant protects against oxidant-induced microtubule cytoskeletal disruption in intestinal Caco-2 cells as confirmed by LSCM. Control (vector alone) clone was exposed to vehicle (a), wild-type cells were exposed to 0.5 mM H2O2 (b), and I{kappa}B{alpha} mutant clone was exposed to 0.5 mM H2O2 (c). Wild-type cells exposed to H2O2 (b) show extensive disruption and fragmentation of microtubule cytoskeleton, whereas mutant I{kappa}B{alpha} cells (c), which were exposed to the same damaging dose of H2O2, show a highly preserved/protected cytoarchitecture of the microtubules. This intact appearance resembles that of the control (a). Bar = 25 µm. Representative photomicrographs are shown.

 
Assessment of the microtubule cytoarchitecture by high-resolution laser confocal microscopy confirms (Fig. 7C, Caco-2 cells) that suppressing NF-{kappa}B activity in mutant clones preserved (protected) the structural organization of the microtubule cytoskeleton (Fig. 7Cc), which is indistinguishable from the controls (Fig. 7Ca). In wild-type cells, on the other hand, oxidant caused instability and collapse of the microtubule architecture (Fig. 7Cb).

Finally, analysis of oxidative stress (Fig. 8,DCF fluorescence in Caco-2 cells) from these I{kappa}B{alpha} mutant clones additionally demonstrates that inhibition of NF-{kappa}B activity substantially attenuated oxidants' upregulation of oxidative stress. As for iNOS and NO upregulation, a large percentage (~95–98%) of oxidant-induced effects on DCF fluorescence upregulation appears to be NF-{kappa}B dependent.



View larger version (60K):
[in this window]
[in a new window]
 
Fig. 8. Prevention of the upregulation of oxidative stress (DCF fluorescence intensity) induced by oxidant in I{kappa}B{alpha} mutant clones of intestinal Caco-2 cells. Conditions were as described in Figs. 6 and 7. *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2 in wild-type cells. &P < 0.05 vs. H2O2 in mutant cells.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, using monolayers of intestinal cells as a reductionist model of gut, we demonstrated that the NF-{kappa}B appears to play a key role not only in oxidant-induced iNOS upregulation and NO overproduction but also in oxidative injury to the integrity of the microtubule cytoskeleton and intestinal epithelium. A second major finding is that activation of NF-{kappa}B by itself appears to upregulate NO and then disrupt cytoskeletal filaments and the stability of intestinal monolayers. The mechanism for the unique injurious effects of NF-{kappa}B appears to be the oxidation and nitration of the tubulin (50 kDa) subunit components of the cytoskeleton and the consequent disruption of microtubule assembly and architecture.

These conclusions are based on several independent lines of evidence. First, activation of NF-{kappa}B induces an oxidant-like injury, including oxidant-induced iNOS upregulation. NF-{kappa}B activation evokes an oxidative cascade of alterations, including hyperactivation of iNOS enzyme, overgeneration of NO, increases in RNM, and promotion of oxidative stress (DCF fluorescence). This oxidative injury appears to require activation of the NF-{kappa}B. Second, NF-{kappa}B induces footprints of oxidative damage to tubulin (50 kDa) protein subunits of the microtubule network. Activation of NF-{kappa}B (e.g., p65 subunit) promotes the nitration (nitrotyrosination) of tubulin and increases the oxidation (carbonylation) of tubulin (and as we previously showed, destabilizes the appearance of the microtubule cytoskeleton; Ref. 3). Third, dominant negative mutation of I{kappa}B{alpha}, which causes stabilization of I{kappa}B{alpha} and inactivation of NF-{kappa}B, substantially interfered with oxidant-induced increases in iNOS upregulation (by ~96%) and with nitration and carbonylation of tubulin (as well as instability of microtubules). Oxidant also did not overproduce NO or increase DCF fluorescence in these dominant negative clones. Indeed, the I{kappa}B{alpha} mutants (e.g., 3-µg clones) induce almost complete protection against oxidation and/or nitration. Fourth, pretreatment of intestinal monolayers with pharmacological inhibitors of NF-{kappa}B suppresses NF-{kappa}B activity and evokes protective cascade of alterations that are further consistent with the proposed mechanism. Not surprisingly, the effects of I{kappa}B{alpha} mutant clones against oxidation are selectively mimicked by the I{kappa}B{alpha} inhibitors (e.g., lactacystin) as well as NF-{kappa}B suppressors (e.g., curcumin). These various inhibitors of NF-{kappa}B thus substantially prevented cascade of oxidant stress injury, including cytoskeletal nitration. The concordance of our findings utilizing both pharmacological inhibition and molecular targeting further supports a key role for NF-{kappa}B in these injurious oxidative stress processes for the epithelial cytoskeletal dysfunction.

Finally, NF-{kappa}B activation quantitatively correlates with increases in all parameters indicating oxidative stress, which further supports our conclusions. Using both transfected clones and wild type cells, we found correlations (P < 0.05) between NF-{kappa}B activation and microtubule oxidation (r = 0.96) and several other outcomes of oxidative stress and cytoskeletal oxidation; for example, tubulin nitration and NF-{kappa}B activation (r = 0.97), tubulin oxidation and NF-{kappa}B activation (r = 0.96), and tubulin disassembly (increase in S1 monomeric pool) and NF-{kappa}B activation (r = 0.90). Importantly, other consistent correlations are also reached when oxidant-induced iNOS upregulation and NF-{kappa}B activation (r = 0.92), NO overgeneration and NF-{kappa}B activation (r = 0.91), or DCF fluorescence levels and NF-{kappa}B activation (r = 0.88) are utilized. The high strength of these various correlations, which explains 85–95% of the variance, indicates that NF-{kappa}B activation is required for the injury induced by iNOS upregulation and consequent oxidative stress (e.g., nitration, carbonylation) to the tubulin cytoskeleton and intestinal epithelial monolayer. In this view, activation of NF-{kappa}B leads to the overproduction of NO (a RNM) and instability of the cytoskeleton following oxidative stress of iNOS activation. Our previous and current studies on NF-{kappa}B are consistent with a unique model for cytoskeletal and monolayer dysfunction under proinflammatory conditions of oxidant stress in which

Our findings utilizing both pharmacology and targeted molecular approaches are further consistent with known properties of NF-{kappa}B. NF-{kappa}B is typically composed of two subunits (p50 and p65), and its activation is tightly regulated by an endogenous cytoplasmic inhibitor, I{kappa}B{alpha}, which complexes with NF-{kappa}B and traps it in the cytoplasm in an inactive form (3, 15, 33, 42). NF-{kappa}B is a crucial proinflammatory factor in the immune response induced by a wide variety of other agents including cAMP, phorbol esters, free radicals, and cytokines (e.g., TNF-{alpha}, IL-1, IL-6) as well as viral transactivators (19, 21, 24, 40). For example, cytokine-triggered nuclear distribution and activation of NF-{kappa}B has been shown to be key in the promotion of inflammatory processes in both non-GI (e.g., peritoneal macrophages) (40, 46, 52) and GI models (e.g., intestinal cells) (3, 33, 42). Also, bacterial lipopolysaccharide (LPS) induces instability of I{kappa}B{alpha}, resulting in distribution of "free" NF-{kappa}B into the nucleus, which, in turn, activates an inflammatory response (19). Consistent with our current and previous findings, H2O2 has been shown to activate NF-{kappa}B (through destabilization of I{kappa}B{alpha}) in several cellular models. For instance, activation of NF-{kappa}B induced by H2O2 or TNF-{alpha} has been shown in endothelium (20). Similarly, NF-{kappa}B activation induced by oxidant has been reported in colonic smooth muscles (49) as well as in Jurkat T-cells (23). Moreover, recent studies from our laboratory, which were based on both pharmacological and molecular biological approaches, showed that the 78-kDa "classical" {beta}1 isoform of protein kinase C (PKC-{beta}1) and the 72-kDa "atypical" {zeta} isoform of PKC (PKC-{zeta}) are both required for suppression of NF-{kappa}B activation in colonocytes (15, 16). We showed that both PKC-{beta}1 and PKC-{zeta} isoforms are key for growth factor (EGF, TGF-{alpha})-induced protection of the intestinal epithelium (4, 5, 15, 16). Nevertheless, altogether, our studies on damage support a new model showing that a fundamental mechanism in the cascade of events that underlies disruption of the GI epithelial monolayer and cytoskeletal integrity under oxidant stress involves an injurious cascade of events that is likely initiated by free radicals. In this injurious cascade, oxidants induce I{kappa}B{alpha} instability (degradation) and then activate NF-{kappa}B, a crucial inflammatory mediator. We have now expanded on previous studies and shown that oxidants cause oxidative stress injury, especially nitrotyrosination and carbonylation, to the cytoskeletal subunit assembly through the activation of the NF-{kappa}B, which then leads to NO overproduction and its injurious oxidation consequences. Thus it appears that activating NF-{kappa}B will have distinct injurious, oxidative stress effects on the intestinal epithelial cytoskeleton, including nitration. Overall, our findings, we believe, indicate a new function for NF-{kappa}B under pathophysiological conditions of oxidant challenge, namely, promotion of the stress of NO overproduction and consequent cytoskeletal nitration and carbonylation in intestinal epithelial monolayers. We have thus discovered a novel biological mechanism for cytoskeletal and cellular oxidation.

Our findings are potentially relevant for developing new treatment modalities for inflammation, in general, and IBD, in particular. The manifestations of IBD, including ulcerative colitis and Crohn's disease, wax and wane between active (symptomatic) phases of disease, when oxidant stress is high, and inactive (asymptomatic) phases, when oxidative stress is minimal. Our series of findings suggests a unique oxidative stress mechanism that could, if it occurred in vivo, promote oxidative injury and initiate or perpetuate manifestation of the IBD attacks. This concept is consistent with recent characterizations of the pathophysiology of IBD and of the proinflammatory nature of NF-{kappa}B (12, 21, 48). NF-{kappa}B activation is a critical event in the inflammatory response induced by an array of conditions, especially free radicals (3, 11, 15, 19, 20, 24, 33). NF-{kappa}B activation (as indicated by increased p65 nuclear levels) occurs in the inflamed mucosa of patients with IBD (ulcerative colitis or Crohn's disease) (11, 12, 43, 46, 48), in which excessive concentrations of oxidants (H2O2) as well as hyperpermeability of mucosal barrier function have been found (17, 28, 32, 35, 37, 39). We have found that the amount of oxidant stress and NF-{kappa}B activation closely parallel the degree of mucosal inflammation in patients with IBD (11, 12, 17, 35). Equally important, we recently found that the degree of gut mucosal cytoskeletal oxidation (and instability) correlates with the degree of inflammation and disease severity score in IBD patients (35). Thus induction of NF-{kappa}B appears to be key to the perpetuation of the active, symptomatic phase of IBD, in which intestinal oxidant stress creates a vicious cycle of inflammation dependent on sustained NF-{kappa}B activation, oxidant production, cytoskeletal instability, and, ultimately, tissue injury. The oxidative stress injury to key cytoskeletal structures mediated by NF-{kappa}B, as we have shown here in intestinal cells, could be pivotal in developing more effective strategies to suppress the continuation of inflammatory processes and structural damage in IBD mucosa.

In summary, our findings utilizing several independent approaches, including targeted molecular interventions, support the novel idea that NF-{kappa}B is responsible for a substantial portion of nitration/oxidation disruption of the intestinal mucosal epithelial cytoskeleton following NO upregulation. NF-{kappa}B, perhaps, is also crucial to promoting amplification and establishment of an uncontrolled, oxidant-initiated inflammatory stress cascade that causes structural (cytoskeletal) tissue damage in IBD, one that can be ignited by free radicals and other oxidants present in the GI tract under pathophysiological conditions.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported in part by a grant from Rush University Medical Center, Department of Internal Medicine, and by two National Institutes of Health R01 grants, DK-60511 (to A. Banan) and AA-13745 (to A. Keshavarzian).


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Banan, Rush University Medical Center, Department of Internal Medicine, Section of Gastroenterology and Nutrition, 1725 W. Harrison, Suite 206, Chicago, IL 60612 (E-mail: ali_banan{at}rush.edu)

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


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Banan A, Choudhary S, Zhang Y, Fields JZ, and Keshavarzian A. Ethanol-induced barrier dysfunction and its prevention by growth factors in human intestinal monolayers: evidence for oxidative and cytoskeletal mechanisms. J Pharmacol Exp Ther 291: 1075–1085, 1999.[Abstract/Free Full Text]

2. Banan A, Choudhary S, Zhang Y, Fields JZ, and Keshavarzian A. Oxidant-induced intestinal barrier disruption and its prevention by growth factors in a human colonic cell line: role of the microtubule cytoskeleton. Free Radic Biol Med 28: 727–738, 2000.[CrossRef][ISI][Medline]

3. Banan A, Farhadi A, Fields JZ, Mutlu E, Zhang L, and Keshavarzian A. Evidence that nuclear factor-{kappa}B activation is critical in oxidant disruption of the microtubule cytoskeleton and barrier integrity and that its inactivation is essential in growth factor-mediated protection of the monolayers of intestinal epithelia. J Pharmacol Exp Ther 306: 13–28, 2003.[Abstract/Free Full Text]

4. Banan A, Fields JZ, Talmage DA, Zhang L, and Keshavarzian A. PKC-{zeta} is required in EGF protection of microtubules and intestinal barrier integrity against oxidant injury. Am J Physiol Gastrointest Liver Physiol 282: G794–G808, 2002.[Abstract/Free Full Text]

5. Banan A, Fields JZ, Talmage DA, Zhang Y, and Keshavarzian A. PKC-{beta}1 mediates EGF protection of microtubules and intestinal epithelial barrier against oxidants. Am J Physiol Gastrointest Liver Physiol 281: G833–G847, 2001.[Abstract/Free Full Text]

6. Banan A, Fields JZ, Zhang Y, and Keshavarzian A. Nitric oxide and its metabolites mediate ethanol-induced microtubule disruption and intestinal barrier dysfunction. J Pharmacol Exp Ther 294: 997–1008, 2000.[Abstract/Free Full Text]

7. Banan A, Fields JZ, Zhang Y, and Keshavarzian A. Phospholipase C-{gamma} inhibition prevents EGF-mediated protection of microtubules and intestinal epithelial barrier against oxidants. Am J Physiol Gastrointest Liver Physiol 281: G412–G423, 2001.[Abstract/Free Full Text]

8. Banan A, Fields JZ, Zhang Y, and Keshavarzian A. iNOS upregulation mediates oxidant-induced disruption of F-actin and barrier of intestinal monolayers. Am J Physiol Gastrointest Liver Physiol 280: G1234–G1246, 2001.[Abstract/Free Full Text]

9. Banan A, Fields JZ, Zhang Y, and Keshavarzian A. Key role of PKC and Ca2+ in EGF protection of microtubules and intestinal barrier against oxidants. Am J Physiol Gastrointest Liver Physiol 280: G828–G843, 2001.[Abstract/Free Full Text]

10. Banan A, McCormack SA, and Johnson LR. Polyamines are required for microtubule formation during mucosal healing. Am J Physiol Gastrointest Liver Physiol 274: G879–G885, 1998.[Abstract/Free Full Text]

11. Banan A, Shaikh M, Fields JZ, Farhadi A, Mutlu E, and Keshavarzian A. Activation of NF-{kappa}B and I{kappa}B{alpha} degradation and cytoskeletal dysfunction in colonic mucosa of patients with inflammatory bowel disease (IBD) (Abstract). Gastroenterology 124, Suppl 1: S1338, 2003.

12. Banan A, Shaikh M, Zhang L, Fields JZ, Farhadi A, Mutlu E, and Keshavarzian A. NF-{kappa}B activation drives the vicious circle of oxidative tissue injury that initiates flare-up in the mucosa of patients with inflammatory bowel disease (IBD). Gut. In press.

13. Banan A, Smith GS, Rickenberg C, Kokoska ER, and Miller TA. Protection against ethanol injury by prostaglandin in a human intestinal cell line: role of microtubules. Am J Physiol Gastrointest Liver Physiol 274: G111–G121, 1998.[Abstract/Free Full Text]

14. Banan A, Zhang L, Fields JZ, Talmage DA, and Keshavarzian A. PKC-{zeta} prevents oxidant-induced iNOS upregulation and protects the microtubules and gut barrier integrity. Am J Physiol Gastrointest Liver Physiol 283: G909–G922, 2002.[Abstract/Free Full Text]

15. Banan A, Zhang LJ, Fields JZ, Shaikh M, Farhadi A, and Keshavarzian A. {zeta} Isoform of protein kinase C prevents oxidant-induced nuclear factor-{kappa}B activation and I-{kappa}B{alpha} degradation: a fundamental mechanism for epidermal growth factor protection of the microtubule cytoskeleton and intestinal barrier integrity. J Pharmacol Exp Ther 307: 53–66, 003.

16. Banan A, Zhang LJ, Shaikh M, Fields JZ, Farhadi A, and Keshavarzian A. {theta}-Isoform of PKC-{beta}1 is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-{theta}. Am J Physiol Cell Physiol 287: C218–C234, 2004.[Abstract/Free Full Text]

17. Banan A, Zhang Y, Hutte R, and Keshavarzian A. Increased oxidation injury in intestinal mucosa of patients with inflammatory bowel disease (Abstract). Gastroenterology 118: 4266, 2000.

18. Banan A, Zhang Y, Losurdo J, and Keshavarzian A. Carbonylation and disassembly of the F-actin in oxidant-induced barrier dysfunction and its prevention by epidermal growth factor and transforming growth factor-{alpha} in a human intestinal cell line. Gut 46: 830–837, 2000.[Abstract/Free Full Text]

19. Baeuerle PA and Henkel T. Function and activation of NF-{kappa}B in the immune system. Annu Rev Immunol 12: 141–179, 1994.[CrossRef][ISI][Medline]

20. Barchowsky A, Munro SR, Morana SJ, Vincenti MP, and Treadwell M. Oxidant-sensitive and phosphorylation-dependent activation of NF-{kappa}B and AP-1 in endothelial cells. Am J Physiol Lung Cell Mol Physiol 269: L829–L836, 1995.[Abstract/Free Full Text]

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

22. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]

23. Brennan P and O'Neill LA. Effects of oxidants and antioxidants on nuclear factor {kappa}B activation in three different cell lines: evidence against a universal hypothesis involving oxygen radicals. Biochim Biophys Acta 1260: 167–175, 1995.[ISI][Medline]

24. Chen F, Sun SC, Kuh DC, Gaydos LJ, and Demers LM. Essential role of NF-{kappa}B activation in silica-induced inflammatory mediator production in macrophages. Biochem Biophys Res Commun 214: 985–992, 1995.[CrossRef][ISI][Medline]

25. Gilbert T, Le Bivic A, Quaroni A, and Rodreguez-Boulan E. Microtubule organization and its involvement in the biogenic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells. J Cell Biol 133: 275–288, 1991.

26. Harter JL. Critical values for Dunnett's new multiple range test. Biometrics 16: 671–685, 1960.[ISI]

27. Hermiston ML and Gordon JI. Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin. Science 270: 1203–1207, 1995.[Abstract]

28. Hollander D. The intestinal permeability barrier: a hypothesis as to its regulation and involvement in Crohn's disease. Scand J Gastroenterol 27: 721–726, 1992.[ISI][Medline]

29. Hollander D. Crohn's disease—a permeability disorder of the tight junction? Gut 26: 1621–1624, 1998.

30. Housey GM, Johnson MD, Hsiao WLW, O'Brian CA, Murphy JP, Kirschmeier P, and Weinstein IB. Overproduction of protein kinase C causes disordered growth control in rat fibroblasts. Cell 52: 343–354, 1988.[ISI][Medline]

31. Hurani MA, Noach AB, Blom-Roosemalen CM, DeBoer AG, Nagelkerke JF, and Breimer DD. Permeability enhancement in Caco-2 cell monolayers by sodium salicylate and sodium taurodihydrosulfate: assessment of effect-reversibility and imaging of transepithelial transport routes by laser confocal microscopy. J Pharmacol Exp Ther 267: 942–950, 1993.[Abstract]

32. Irvine EJ and Marshall JK. Increased intestinal permeability precedes the onset of Crohn's disease in a subject with familial risk. Gastroenterology 119: 1740–1744, 2000.[ISI][Medline]

33. Jobin C, Bradham CA, Russo MP, Juma B, Narula AS, Brenner DA, and Sartor RB. Curcumin blocks cytokine-mediated NF-{kappa}B activation and proinflammatory gene expression by inhibiting inhibitory factor I-{kappa}B kinase activity. J Immunol 163: 3474–3483, 1999.[Abstract/Free Full Text]

34. Kennedy M, Denenberg AG, Szabo C, and Salzman AL. Poly(ADP-ribose) synthetase activation mediates increased permeability induced by peroxynitrite in Caco-2BBe cells. Gastroenterology 114: 510–518, 1998.[ISI][Medline]

35. Keshavarzian A, Banan A, Kommandori S, Zhang Y, and Fields JZ. Increases in free radicals and cytoskeletal protein oxidation and nitration in the colon of patients with inflammatory bowel disease. Gut 52: 720–728, 2003.[Abstract/Free Full Text]

36. Keshavarzian A, Holmes EW, Patel M, Iber F, and Pethkar S. Leaky gut in alcoholic cirrhosis: a possible mechanism for alcohol-induced liver damage. Am J Gastroenterol 94: 200–207, 1999.[CrossRef][ISI][Medline]

37. Keshavarzian A, Sedghi S, Kanofsky J, List T, Robinson C, Ibrahim C, and Winship D. Excessive production of reactive oxygen metabolites by inflamed colon: analysis by chemiluminescence probe. Gastroenterology 103: 177–185, 1992.[ISI][Medline]

38. Kimura H, Hokari R, Miura S, Shigematsu T, Hirokawa M, Akiba Y, Kurose I, Higuchi H, Fujimori H, Tsuzuki Y, Serizawa H, and Ishii H. Increased expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in colonic mucosa of patients with active ulcerative colitis. Gut 42: 180–187, 1998.[Abstract/Free Full Text]

39. McKenizie SJ, Baker MS, Buffington GD, and Doe WF. Evidence for oxidant-induced injury to epithelial cells during inflammatory bowel disease. J Clin Invest 98: 136–141, 1996.[Abstract/Free Full Text]

40. Menon SD, Qin S, Guy RG, and Tan YH. Differential induction of nuclear NF-{kappa}B protein phosphatase inhibitors in primary and transformed human cells. Requirement for both oxidation and phosphorylation in nuclear translocation. J Biol Chem 268: 26805–26812, 1993.[Abstract/Free Full Text]

41. Meunier VM, Bourrie Y, Berger Y, and Fabre G. The human intestinal epithelial cell line Caco-2: pharmacological and pharmacokinetics applications. Cell Biol Toxicol 11: 187–194, 1995.[ISI][Medline]

42. Moon RM, Parikh AA, Pritts TA, Fischer JE, Cottongim S, Szabo C, Salzman AL, and Hasselgren PO. Complement component C3 production in IL-1{beta}-stimulated human intestinal epithelial cells is blocked by NF-{kappa}B inhibitors and by transfection with Ser 32/36 mutant I{kappa}B{alpha}. J Surg Res 82: 48–55, 1999.[CrossRef][ISI][Medline]

43. Neurath MF, Pettersson S, Buschenfelde KH, and Strober W. Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-{kappa}B abrogates established experimental colitis in mice. Nat Med 9: 998–1004, 1999.

44. Quaroni A, Wang JY, Trelstad RL, and Isselbacher HI. Epithelial cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell Biol 80: 248–265, 1979.[Abstract]

45. Renard P, Ernest I, Houbion A, Art M, Le Calvez H, Raes M, and Remacle J. Development of a sensitive multi-well colorimetric assay for active NF-{kappa}B. Nucleic Acids Res 29: 1–5, 2001.[Abstract/Free Full Text]

46. Rogler G, Brand K, Vogl D, Page S, Hofmeister R, Andus T, Knuechel R, Baeuerle PA, Scholrerich J, and Gross V. Nuclear factor {kappa}B is activated in macrophages and epithelial cells of inflamed intestinal mucosa. Gastroenterology 115: 357–369, 1998.[ISI][Medline]

47. Sanders SE, Madara JL, McGuirck DK, Gelman DS, and Colgan SP. Assessment of inflammatory events in epithelial permeability: a rapid screening method using fluorescein dextrans. Epithelial Cell Biol 4: 25–34, 1995.[ISI][Medline]

48. Schreiber S, Nikolaus S, and Hampe J. Activation of nuclear factor {kappa}B in inflammatory bowel disease. Gut 42: 477–484, 1998.[Abstract/Free Full Text]

49. Shi XZ, Lindholm PF, and Sarna SK. NF-{kappa}B activation by oxidative stress and inflammation suppresses contractility in colonic circular smooth muscle cells. Gastroenterology 124: 1369–1380, 2003.[CrossRef][ISI][Medline]

50. Soderholm JD, Peterson KH, and Olaison G. Epithelial permeability to proteins in the non-inflamed ileum of Crohn's disease? Gastroenterology 117: 65–72, 1999.[ISI][Medline]

51. Tarumi T, Kravtsov DV, Zhao M, Williams SM, and Gailani D. Cloning and characterization of the human factor XI gene promoter: transcription factor hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}) is required for hepatocyte-specific expression of factor XI. J Biol Chem 277: 18510–18516, 2002.[Abstract/Free Full Text]

52. Tang G and Leppla SH. Proteasome activity is required for anthrax lethal toxin to kill macrophages. Infect Immun 67: 3055–3060, 1999.[Abstract/Free Full Text]

53. Unno N, Menconi MJ, Smith M, Aguirre DE, and Fink MP. Hyperpermeability of intestinal epithelial monolayers is induced by NO: effect of low extracellular pH. Am J Physiol Gastrointest Liver Physiol 272: G923–G934, 1997.[Abstract/Free Full Text]

54. Yamada T, Sartor RB, Marshall S, Special RD, and Grisham MB. Mucosal injury and inflammation in a model of chronic granulomatous colitis in rats. Gastroenterology 104: 759–771, 1993.[ISI][Medline]