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CELLULAR AND MOLECULAR

Evidence That Nuclear Factor-B Activation Is Critical in Oxidant-Induced Disruption of the Microtubule Cytoskeleton and Barrier Integrity and That Its Inactivation Is Essential in Epidermal Growth Factor-Mediated Protection of the Monolayers of Intestinal Epithelia

Departments of Internal Medicine (Section of Gastroenterology and Nutrition), Pharmacology, and Molecular Physiology, Rush University Medical Center, Chicago, Illinois

Received January 29, 2003; accepted March 18, 2003.

	Abstract

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Using monolayers of intestinal (Caco-2) cells, we showed that oxidants disrupt the microtubule cytoskeleton and barrier integrity; epidermal growth factor (EGF) was protective via stabilization of the microtubules. Because proinflammatory conditions activate nuclear factor-B (NF-B), we hypothesized that oxidants disrupt barrier integrity through activation of NF-B and that EGF protects by suppressing NF-B. Parental cells were pretreated with EGF or NF-B or inhibitory B (I-B) modulators. Other cells were stably transfected with varying levels of a dominant negative mutant for the NF-B inhibitor I-B. Both types of cells were grown as monolayers and then exposed to oxidant (H₂O₂). We then monitored monolayer barrier integrity (permeability), stability of the microtubule cytoskeleton (confocal microscopy, immunoblotting), intracellular levels of the I-B (immunoblotting), translocation, and activity of NF-B (immunoblotting, sensitive enzyme-linked immunosorbent assay). Monolayers were also fractionated and processed to assess alterations in 1) polymerized tubulin (S2; an index of cytoskeletal integrity) and 2) monomeric tubulin (S1; an index of disassembly) (polyacrylamide gel electrophoresis fractionation and immunoblotting). We found the following: 1) Oxidants caused I-B degradation, NF-B translocation, NF-B (p50 and p65 subunits) activation, tubulin disassembly ( S1, S2), microtubule architectural instability, and barrier disruption. I-B stabilizers and NF-B inhibitors [e.g., carbobenzyloxy-leuleu-leucinol (MG-132), lactacystin] suppressed oxidants injurious effects. 2) EGF (10 ng/ml) stabilized I-B and prevented both NF-B translocation and activation while protecting monolayers against oxidants. 3) In stably transfected cells, transfection-induced stabilization of I-B by itself led to EGF-like protective effects. In these mutant cells, protection was not potentiated by EGF (10 ng/ml). Conclusions are 1) oxidants induce disruption of the cytoskeleton and intestinal barrier integrity, in part, through I-B degradation and subsequent NF-B activation, 2) I-B stabilization is by itself protective, mimicking EGF, and 3) EGF protects cell monolayers through I-B stabilization and NF-B inactivation. To our knowledge, this is the first report that NF-B can affect the dynamics of cytoskeletal assembly and intestinal barrier integrity.

Although the pathophysiology of gut barrier dysfunction in IBD remains poorly understood, several studies have shown that gut inflammation is linked with the activation of the nuclear transcription factor nuclear factor-B (NF-B) as well as with high levels of oxidants such as H₂O₂ and that this oxidative damage seems to be a key contributor to tissue injury (Banan et al., 2000a,c, 2001d; Barnes and Karin 1997; Keshavarzian et al., 1992, 2003; McKenizie et al., 1996; Rogler et al., 1998; Schreiber et al., 1998). Oxidative injury and NF-B induction are of substantial clinical importance because increases in oxidative stress and NF-B activation are common in inflammation, and these changes may lead to mucosal barrier hyperpermeability and, in turn, to the initiation and/or perpetuation of mucosal inflammation and dysfunction. Indeed, inflammation that was induced by proinflammatory agents (e.g., endotoxin, silica) seems to be dependent upon the activation of the NF-B (Chen et al., 1995; Barnes and Karin 1997; Jobin et al., 1999; Neurath et al., 1999). Once activated, NF-B seems to regulate several important cellular processes involved in inflammatory responses such as the up-regulation of iNOS, IL-8, TNF-, and cyclooxygenase-2. Thus, activation of NF-B and its consequences are highly relevant to inflammation (Barnes and Karin, 1997; Schreiber et al., 1998). NF-B is typically composed of two subunits (p50 and p65), and its activation is tightly regulated by an endogenous cytoplasmic inhibitor, IB-, which complexes with NF-B and traps it in the cytoplasm in an inactive form (Jobin et al., 1999; Moon et al., 1999). Interestingly, reactive oxygen species (ROS) and in particular H₂O₂ can function as second messengers and regulate the activation of NF-B (Schreck et al., 1991; Kretz-Remy et al., 1996; Schmidt et al., 1996; Bonizzi et al., 2000). For example, generation of H₂O₂ second messenger induced by proinflammatory cytokines such as TNF- or IL-1 seems to be key for NF-B induction in cell cultures (Schmidt et al., 1996; Bonizzi et al., 2000).

A major advance in recent years in GI inflammation (IBD) research was recognition that a leaky gut barrier can cause and/or perpetuate intestinal inflammation and that increased oxidative stress can cause barrier leakiness (hyperpermeability) in the intestinal tract. In animal models, intestinal barrier hyperpermeability induced by the injection of peptidoglycan-polysaccharide into the mucosa can elicit an oxidative and inflammatory condition similar to IBD (Yamada et al., 1993). Moreover, transgenic mice with a leaky gut exhibit symptoms of intestinal inflammation (Hermiston and Gordon 1995). Thus, characterizing how gut barrier integrity is lost under oxidative, proinflammatory conditions is of fundamental clinical and biological importance.

In our efforts to enhance understanding of endogenous defensive mechanisms that protect the cytoskeleton and barrier integrity, as well as in our efforts to develop more effective treatment regimens for inflammatory disorders of the GI tract, we have been investigating protective mechanisms such as EGF-mediated signaling against oxidant-induced barrier dysfunction pathways. Using monolayers of human intestinal (Caco-2) cells, we previously showed that oxidants induce disruption of the cytoskeleton and barrier function (Banan et al., 2000b,d, 2001d) and that growth factors (EGF or TGF-) protect barrier integrity by stabilizing the microtubule cytoskeleton (Banan et al., 1999, 2000a, 2001b,d, 2002c). We also showed that the instability of microtubule cytoarchitecture is required for mucosal damage under in vivo (Banan et al., 1996, 1998a) and in vitro conditions (Banan et al., 1998b, 1999, 2000a,b,c,d, 2001a,b,c,d, 2002a,b,c,d). Damage is based on the inability of cellular polymeric tubulin pools to resist disassembly, and the ability of the monomeric tubulin pools to increase, leading to microtubule instability, whereas protection is dependent on the ability of polymeric tubulin pools to assemble into orderly cytosolic arrays (Banan et al., 1999, 2000a, 2001b). Despite the critical importance of the microtubule cytoskeleton in the maintenance of intestinal barrier integrity, the role of the proinflammatory NF-B in oxidant-induced instability of the cytoskeleton and barrier function and in EGF-mediated protection against this injury remains elusive.

To this end, in the current study, we tested the hypothesis that oxidant induces disruption of epithelial cytoskeleton and barrier integrity through degradation of I-B and subsequent activation of NF-B. EGF mediates protection by suppressing NF-B activation via stabilization of I-B. To explore this intriguing possibility, we used both pharmacological and targeted molecular interventions in intestinal epithelial cells, including using stable mutant clones that are unable to degrade I-B. We were therefore able to selectively modulate the I-B/NF-B in opposite directions and then assess cytoskeletal and barrier alterations in our intestinal cells. Herein, we report new functions for NF-B: changes in cytoskeletal assembly and cytoarchitecture and barrier permeability under pathophysiological conditions.

	Materials and Methods

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Cell Culture. Caco-2 cells, which were obtained from American Type Culture Collection (Manassas, VA) at passage 15, were chosen because they form monolayers that morphologically resemble small intestinal cells, with defined apical brush borders and a highly organized microtubule network (Gilbert et al., 1991; Meunier et al., 1995; Banan et al., 1998b). 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. Parental cells or stably transfected cells (see below) were split at a ratio of 1:6 upon reaching confluence, and set up in either six- or 24-well plates for experiments, or T-75 flasks for propagation. Cells grown for barrier function experiments were split at a ratio of 1:2 and seeded at a density of 200,000 cells/cm² into 0.4 µM Biocoat Collagen I cell culture inserts (0.3-cm² growth surface; BD Biosciences-Discovery Labware, Bedford, MA), and experiments were performed at least 7 days postconfluence. The media were changed every 2 days. The utility and characterization of this cell line have been reported previously (Gilbert et al., 1991; Meunier et al., 1995; Banan et al., 1998b).

Plasmids and Stable Transfection by Selection. The dominant negative mutant (super-repressor) of I-B was constructed and used as described previously (Moon et al., 1999; Banan et al., 2001c). This mutant contains double point mutations substituting key serines 32 and 36 by alanine residues, which prevents the degradation of I-B. The construct was cloned into a 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 immunoblotting assessment of cellular cytosolic fractions.

Cultures of Caco-2 cells grown to 50 to 60% confluence were stably transfected with varying amounts (1–4 µg) of expression plasmid encoding a dominant negative mutant of I-B by Lipofectin (Lipofectin reagent; Invitrogen, Carlsbad, CA) (Moon et al., 1999; Banan et al., 2001c). Briefly, cells were incubated for 24 h at 37°C with the plasmid DNA in serum-free media in the presence of LipofectAMINE (25 µl/25-cm² flask). Subsequently, the DNA-containing solution was removed and replaced by fresh medium containing 10% fetal bovine serum to relieve cells from the shock of exposure to serum-free media. After transfection, cells were subjected to neomycin selection (1 mg/ml) for "colony picking". Resistant clones from this selection (e.g., 3-µg clone) were maintained in DMEM/fetal bovine serum and 0.2 mg/ml neomycin (selection medium). Control conditions included vector alone. I-B protein expression and lack of its degradation were verified by Western blot analysis of cellular cytosolic fractions (see below). Different stable clones were subsequently plated on cell culture inserts and allowed to form confluent monolayers and then used for experiments.

Experimental Design. In the first series of experiments, isotonic saline (vehicle) or oxidant (H₂O₂; 0–5 mM) was incubated with postconfluent monolayers of parental Caco-2 cells for 30 min and then outcomes were assessed over time from 0 to 8 h. Outcomes measured are described below. In the second series of experiments, monolayers were preincubated with EGF (1–100 ng/ml) or saline for 10 min and then exposed to oxidant (H₂O₂). As we showed previously (Banan et al., 1999, 2000a, 2001a,b), H₂O₂ at 0.5 mM disrupts microtubules and barrier integrity; EGF at 10 ng/ml (but not 1 ng/ml) prevents this disruption. These experiments were then repeated using monolayers composed of stably transfected cells expressing the I-B mutant. Reagents were applied on the apical side of monolayers unless otherwise indicated. Because our previous studies (Banan et al., 2000a,d) showed that regardless of whether apical or basolateral exposure of oxidants was used the results were qualitatively similar, all current studies used apical application. In all experiments, microtubule cytoskeletal stability (cytoarchitecture, assembly, and disassembly), tubulin levels, I-B distribution (cytosolic expression and degradation), NF-B p65 subunit activity (cytosolic levels, nuclear translocation, and activity), NF-B p50 subunit activity (cytosolic levels, nuclear translocation, and activity), and barrier integrity (permeability) were assessed.

In the third series of experiments, to ascertain the specificity of EGF's action on NF-B, monoclonal anti-EGF receptor antibody (anti-EGF-R, 1 µg/ml) was preadministered with the growth factor. To further show the specificity and role of EGF-R in such protection, we used EGF-R-specific tyrosine kinase inhibitors tyrophostin-25 (25 µM) or AG-1478 (150 nM), and as a control the inactive analog tyrophostin-A1 (150 nM) (Banan et al., 2000a, 2001c), which were added to the monolayers 30 min before EGF.

In the fourth series of experiments, we further investigated the potential importance of the I-B/NF-B pathway in oxidant-mediated damage and in growth factor protection by using several other pharmacological experiments (all 30-min preincubations). Monolayers of parental cells were preincubated with different I-B/NF-B pathway inhibitors and then incubated with or without EGF before exposure to oxidant. These inhibitors included the following: known inhibitors of the degradation of I-B: 1) curcumin (20 µM) and 2) 1-pyrrolidine dithiocarbamate (PDTC, 20 µM; Jobin et al., 1999; Todisco et al., 1999), alone or in combination with H₂O₂. Other chemical inhibitors of the degradation of I-B, 3) carbobenzyloxyleu-leu-leucinol (MG-132, 10 µM; Jobin et al., 1999; Tang and Leppla, 1999), and 4) lactacystin (10 µM; Tang and Leppla, 1999; Todisco et al., 1999). Controls were treated with vehicle. We confirmed that these doses of inhibitors were not toxic to cells. Outcomes measured were as described above.

In a fifth series of experiments, monolayers of I-B dominant negative transfected cells were incubated with oxidant (H₂O₂) ± EGF or vehicle. In all experiments, I-B levels and NF-B activity were determined (see below). Other outcomes measured were as described above. In corollary experiments, we investigated the effects of I-B degradation or stabilization and NF-B activation or inactivation on the state of tubulin assembly and disassembly and on stability of the cytoarchitecture of the microtubule cytoskeleton. Monomeric and polymerized fractions of tubulin (the structural protein subunit of microtubules) were isolated and then analyzed by quantitative immunoblotting (Banan et al., 1999, 2000a). Microtubule integrity was assessed by 1) immunofluorescent labeling and fluorescence microscopy to determine the percentage of cells with normal microtubules, 2) detailed analysis by high-resolution laser scanning confocal microscopy (LSCM), and 3) immunoblot analysis of monomeric (S1) and polymerized (S2) tubulin pools.

Analysis of NF-B Activation. NF-B (p65 and p50 subunit) activation was assessed by a novel ELISA procedure (Renard et al., 2001). The ELISA kits (Trans-AM NF-B) used were obtained from a commercial vendor (Active Motif of North America, Carlsbad, CA). Cell monolayers grown in 25-cm² flasks were processed for the isolation of the cytosolic and nuclear fractions. Protein content of these cell fractions was assessed by the Bradford method (Bradford, 1976). Subsequently, cells fractions were added to a commercially prepared 96-well plate to which oligonucleotides containing a consensus-binding site for the NF-B had been immobilized (Trans-AM; Active Motif of North America). The NF-B activity test is based on a validated ELISA principle in which NF-B is captured by a double-stranded oligonucleotide probe containing the consensus binding sequence for either NF-B p65 or p50 subunits (Renard et al., 2001). Consequently, the NF-B contained in the cell extracts (e.g., nuclear fractions) is specifically captured by the probe bound in microwell plates. The binding of NF-B to its consensus sequence (i.e., activation) was then detected using a primary anti-NF-B (p65 or p50) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by a secondary antibody conjugated to horseradish peroxidase. The results were quantitated by a chromogenic reaction (Renard et al., 2001), which was then read for absorbance at 450 nm by an NOA 280 microplate analyzer (Sievers Instruments, Inc., Boulder, CO).

Western Blot Analysis of Changes in NF-B Subunit Levels and Nuclear Translocation. Cellular nuclear and cytosolic extracts were prepared as described above. Protein content of the cell fractions was assessed by the Bradford method (Bradford, 1976). Briefly, NF-B nuclear translocation was determined by comparing the levels of NF-B protein (e.g., p65 subunit) expression in the cytosolic versus nuclear extracts by anti-p65 (or anti-p50) antibody using a nondenaturing gel (6%) (Jobin et al., 1999). For immunoblotting, samples (20 µg of protein/lane) were placed in a standard sample buffer, boiled, and then subjected to PAGE. Proteins were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Inc., Piscataway, NJ) and autoradiography.

Western Blot Analysis of I-B Degradation and Expression Levels. I-B levels of expression in the cytosolic extracts as well as its degradation (i.e., disappearance from the cytosolic fractions) were assessed by anti-I-B antibody (Santa Cruz Biotechnology) using a Western blot protocol (10% gel) (Moon et al., 1999). Briefly, samples (20 µg of protein/lane) were added to a standard SDS buffer, boiled, and then separated on SDS-PAGE. As for NF-B, proteins were visualized by enhanced chemiluminescence and subsequently autoradiographed.

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 described previously (Banan et al., 1998b, 1999, 2000a, 2001b). Cells were then processed for incubation with a primary antibody, monoclonal mouse anti--tubulin (Sigma-Aldrich, St. Louis, MO), 1:200 dilution for1hat37°C, and then incubated with a secondary antibody (fluorescein isothiocyanate-conjugated goat anti-mouse; Sigma-Aldrich), 1:50 dilution for 1 h at room temperature. Slides were washed thrice in Dulbecco's phosphate-buffered saline and subsequently mounted in Aquamount. After staining, cells were observed with an argon laser ( = 488 nm) using a 63x oil immersion plan-apochromat objective, numerical aperture 1.4 (Carl Zeiss, Jena, Germany). Cells from desired areas of monolayers were processed using the image processing software on a ultrahigh-resolution LSCM (Carl Zeiss). The cytoskeletal elements were examined in a blinded manner for their overall morphology, orientation, and disruption as we have described previously (Banan et al., 1998b, 1999, 2000a, 2001b). At least 1200 cells/group (200 x six slides) were examined in four different fields by LSCM, and the percentage of cells displaying normal microtubules was determined. The identity of the treatment groups for all slides was decoded only after examination was complete.

Microtubule (Tubulin) Fractionation and Quantitative Immunoblotting of Tubulin Assembly and Disassembly. Polymerized (S2) and monomeric (S1) fractions of tubulin were isolated using a unique method we described previously (Banan et al., 1998b, 1999, 2000a, 2001b). 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% dimethyl sulfoxide, 1 mM MgSO₄, anti-protease cocktail (10 µg/ml), 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 105,000g for 45 min at 4°C, and the supernatant containing the soluble monomeric pool of tubulin (S1) 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, anti-protease cocktail (10 µg/ml), and 10 mM CaCl₂] and incubated on ice for 60 min. Subsequently, samples were centrifuged at 48,000g for 15 min at 4°C, and the supernatant (S2 fraction or cold/Ca²⁺-soluble fraction) was removed. To ensure the complete removal of the S2 fraction, the remaining pellet was treated with the Ca²⁺-containing depolymerization buffer twice more by resuspension and centrifugation. The "microtubules" were recovered by separately incubating (at 37°C for 30 min) the S1 and S2 fractions with stabilizing agents, taxol (10 µM) and GTP (1 mM), in microtubule stabilization buffer [0.1 M PIPES, pH 6.9, 30% glycerol, 5% dimethyl sulfoxide, anti-protease cocktail (10 µg/ml), 1 mM EGTA, 1 mM MgCl₂, and 1 mM GTP] to promote polymerization of tubulin. Tubulin was then recovered by centrifugation and resuspended in the above-described stabilization buffer (microtubule stabilization buffer). 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 described previously (Banan et al., 1998b, 1999, 2000a, 2001b). To quantify the relative levels of tubulin, the optical density of the bands corresponding to immunoradiolabeled tubulin were measured with a laser densitometer.

Determination of Barrier Permeability by Fluorometry. Status of the integrity of monolayer barrier function was assessed by a widely used and validated technique that measures the apical-tobasolateral paracellular flux of fluorescent markers such as fluorescein sulfonic acid (FSA; 200 µg/ml, 0.478 kDa) as we (Banan et al., 1999, 2000a,b,c,d, 2001a,b,c,d, 2002a,b,c,d) and others (Hurani et al., 1993; Sanders et al., 1995; Menconi et al., 1997; Unno et al., 1997; Kennedy et al., 1998) have described. Briefly, fresh phenol-free DMEM (800 µl) was placed into the lower (basolateral) chamber and phenol-free DMEM (300 µl) containing probe (FSA) was placed in the upper (apical) chamber. Aliquots (50 µl) were obtained from the upper and lower chambers at zero time and at subsequent time points and transferred into clear 96-well plates (clear bottom; Costar, Cambridge, MA). Fluorescent signals from samples were quantitated using a fluorescence multiplate reader (FL 600, Bio-Tek Instruments, Winooski, VT). The excitation and emission spectra for FSA were as follows: excitation = 485 nm; emission = 530 nm. Clearance was calculated using the following formula: Cl (nanoliters per hour per square centimeter) = Fab/([FSA]a x S), where Fab is the apicalto-basolateral flux of FSA (light units per hour), [FSA]a is the concentration at baseline (light units per nanoliter), and S is the surface area (0.3 cm²). Simultaneous controls were performed with each experiment.

Statistical Analysis. Data are presented as mean ± S.E.M. All experiments were carried out with a sample size of at least six observations per treatment group that were run in triplicate on different days. Statistical analysis comparing treatment groups was performed using analysis of variance followed by Dunnett's multiple range test (Harter 1960). 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 considered statistically significant.

	Results

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We initially confirmed our previous findings (Banan et al., 2000a,d) that incubation of parental Caco-2 monolayers with oxidant (e.g., 0.5 mM H₂O₂) causes substantial barrier hyperpermeability (increases in FSA clearance) and cytoskeletal instability (Figs. 1B and 2B, respectively). Pretreatment of monolayers with growth factor (e.g., 10 ng/ml EGF) almost completely protected against these oxidant effects (Figs. 1B and 2B). These effects were specific to EGF as well as EGFR-mediated (Banan et al., 2000a, 2001c). In the current investigation, using both pharmacological and targeted molecular interventions (transfection), the possible role of NF-B/I-B in the underlying cause of oxidant disruption and in EGF protection were investigated.

View larger version (21K): [in this window] [in a new window]   Fig. 1. A and B, time course for oxidant (H2O2)-induced loss of Caco-2 cell monolayer barrier integrity (A, 0–8 h after). Note greater disruption (i.e., hyperpermeability) of barrier integrity induced by oxidant overtime. Panel B shows that a 30-min preincubation with agents that inhibit NF-B and I-B protect the epithelial barrier integrity against oxidant exposure (1–2 h after oxidant shown). NF-B inhibitors (curcumin or PDTC) or I-B inhibitors (lactacystin or MG-132) were added to the monolayers before subsequent exposure to oxidant (H2O2) or vehicle. In select experiments, monolayers were pretreated with a known positive control, growth factor (EGF), which was run concurrently. Barrier integrity is substantially protected against oxidant-induced disruption in either the inhibitors or EGF-pretreated monolayers. Barrier integrity was expressed as flux of the fluorescent probe FSA from the apical-to-basolateral compartment of cell culture Transwell inserts divided by the concentration of probe in the apical chamber. When normalized for the surface area of the monolayer, this expression has units of clearance. *, p < 0.05 versus vehicle; +, p < 0.05 versus H2O2; n = 6/group.

View larger version (70K): [in this window] [in a new window]   Fig. 2. A and B, time course for oxidant-induced reduction in the percentage of Caco-2 cells displaying a normal microtubule cytoskeleton (A, 0–8 h). Fewer cells displayed normal microtubules overtime. Panel B shows the protective effects of the inhibitors of NF-B (curcumin or PDTC) and I-B (lactacystin or MG-132) on the percentage of intestinal cells displaying a normal microtubule cytoskeleton. Treatments and conditions were as explained in Fig. 1. Cell monolayers grown on coverslips were processed for immunofluorescent staining with a primary monoclonal anti--tubulin antibody, and subsequently the microtubule elements were examined in a blinded manner for their overall morphology, reported as percentage of cells with normal microtubules. Note that microtubules are disrupted in oxidant-treated cells (B, 1–2 h after oxidant shown). In contrast, growth factor EGF and NF-B or I-B inhibitors protect the microtubules against this disruption. *, p < 0.05 versus vehicle; +, p < 0.05 versus H2O2; n = 6/group. C, intracellular distribution of the microtubule cytoskeleton as captured by ultrahigh-resolution LSCM in intestinal cell monolayers. Monolayers of Caco-2 cells were incubated with vehicle (a), 0.5 mM H2O2 (b), NF-B inhibitor PDTC and then with 0.5 mM H2O2 (c), or I-B inhibitor MG-132 plus 0.5 mM H2O2 (d), or growth factor EGF followed by 0.5 mM H2O2 (e). Treatments and conditions were as in Figs. 1B and 2B. In cells exposed to oxidant H2O2 (b), the microtubules exhibit a clear collapse, disorganization, fragmentation, and disruption of their architecture. In contrast, in the presence of the inhibitors PDTC (c) or MG-132 (d), microtubule cytoarchitecture is highly preserved and protected. Similarly, microtubules in the EGF-pretreated (e) monolayers seem to be normal microfilamentous structures, which disperse throughout the cytosol in an intact manner. This appearance is indistinguishable from the normal microtubules in control (vehicle-treated) monolayers (a). Bar, 25 µm. Shown is a representative photomicrograph; n = 6/group.

Disruptive Effects of Oxidants on Barrier Integrity Are Prevented by the Pharmacological Inhibitors of NF-B Activation or I-B Degradation. Oxidant induced loss of Caco-2 monolayer barrier integrity over time (Fig. 1A, 0–8 h). Pretreatment of these monolayers for 30 min with inhibitors of NF-B activation (curcumin or PDTC) substantially attenuated H₂O₂-induced loss of barrier integrity (66–68%) as demonstrated by decreased clearance of FSA (Fig. 1B, 1–2 h after oxidant shown). Preincubation with specific inhibitors of I-B degradation (lactacystin or MG-132) also protected against oxidant disruption of barrier (by approximately 70–72%). In the absence of oxidants, these inhibitors themselves did not injure monolayer barrier integrity (data not shown).

Oxidant Disruption of the Microtubule Cytoskeleton Is Abolished by the Inhibitors of NF-B Activation or I-B Degradation. Oxidant over time (0–8 h) reduced the percentage of cells displaying normal microtubules (Fig. 2A). Similar to their protective effects on barrier integrity, preincubation with inhibitors of NF-B activation (curcumin or PDTC) or I-B degradation (lactacystin or MG-132) markedly blunted oxidant-induced microtubule instability (Fig. 2B, 1–2 h after oxidant shown). This protection was similar to that of EGF, which also maintained microtubule integrity against oxidative insult.

Figure 2C reveals images (a–e) obtained from high-resolution laser scanning confocal microscopy of immunofluorescently stained monolayers. Panel a shows that control (vehicle-exposed) cells exhibit a normal and stellate distribution of the microtubule network. After exposure to oxidant (b), fragmentation, collapse, and disruption of the microtubules is seen. Preincubation with either the antioxidant inhibitor of NF-B PDTC (c) or the proteasome inhibitor of I-B MG-132 (d) protected the appearance of the microtubules against oxidant exposure. The appearance of the microtubules in these latter groups was indistinguishable from that of EGF-pretreated cells exposed to oxidant, which also showed a preserved microtubule cytoarchitecture (e).

To determine the possible protective effects of inhibitors of the NF-B activation or the I-B degradation on polymerization and depolymerization states of the microtubule cytoskeleton, we performed immunoblotting analysis of tubulin, the structural protein of microtubules. To this end, the polymerized tubulin fraction (S2) and the monomeric tubulin (S1) were isolated and analyzed by a SDS-PAGE fractionation technique we developed for this purpose. Figure 3A shows that H₂O₂ elicited a significant reduction in the stable polymerize tubulin fraction while increasing the unstable monomeric tubulin fraction, together indicating microtubule disassembly. In contrast, preincubation with either EGF or the aforementioned NF-B and I-B inhibitors protected tubulin from the disassembling effects of oxidant. This enhanced level of assembly was toward that seen in the vehicle group.

View larger version (62K): [in this window] [in a new window]   Fig. 3. A, immunoblotting analysis of the intracellular tubulin pools in Caco-2 cell monolayers after treatment regimes shown. Conditions were as in Figs. 1B and 2B. Normal tubulin assembly is protected/preserved in the growth factor EGF or the NF-B and I-B inhibitor groups, whereas in oxidant alone-exposed cells tubulin assembly is reduced. Tubulin fractions were extracted from intestinal cells and processed for a unique SDS-PAGE fractionation and immunoblotting using monoclonal anti--tubulin antibody followed by horseradish peroxidase-conjugated secondary antibody, and subsequently autoradiographed. To quantify the relative levels of tubulin bands, the optical density of the bands corresponding to immunoradiolabeled tubulin were measured with a laser densitometer. Percentage of polymerized tubulin = [(S2)/(S2 + S1)], where S2 + S1 is the total cellular tubulin pool. Polymerized tubulin (S2) and the monomeric tubulin (S1). *, p < 0.05 versus vehicle; +, p < 0.05 versus H2O2; n = 6/group. B, representative Western immunoblot of the tubulin fractions from intestinal Caco-2 monolayers. Tubulin extracted from intestinal cells was subjected to immunoblotting protocols as described in A. Immunoblotted tubulin on nitrocellulose (NTC) membranes was visualized by ECL and autoradiography. The tubulin bands from left to right correspond to vehicle (a), 0.5 mM H2O2 challenge (b), EGF + 0.5 mM H2O2 (c), curcumin + 0.5 mM H2O2 (d), PDTC + 0.5 mM H2O2 (e), lactacystin + 0.5 mM H2O2 (f), MG-132 + 0.5 mM H2O2 (g), and tubulin standard (50 kDa) (h). Oxidant disrupts dynamics of tubulin polymerization (lane b). This disassembly is shown by a tubulin band density that is reduced to a level well below that of the vehicle-exposed cells (lane a). In EGF or the NF-B or I-B inhibitor groups (lanes c–g), normal tubulin assembly is enhanced to the levels comparable with that of vehicle (control) cells. Shown is a representative blot. n = 6/group.

Figure 3B shows, using representative Western blots, how the same agents alter tubulin assembly, again demonstrating that these NF-B modulators/inhibitors protect the assembly of tubulin. This is shown by increased band density toward that of vehicle-exposed cells. These findings on the protection against tubulin disassembly by NF-B/I-B inhibitors parallel our findings on the protective effects of these same agents on microtubule cytoarchitecture as well as on the monolayer barrier integrity against oxidant challenge.

Intracellular Translocation and Activation of NF-B and the Distribution of Its Endogenous Inhibitor I-B in Intestinal Cell Monolayers: Oxidant-Induced Activation of NF-B and Its Inactivation by EGF. To further investigate whether NF-B-dependent mechanisms are essential in oxidant-induced disruption of monolayers and protection by EGF, we used a highly sensitive ELISA to assess cellular nuclear fractions. We show (Fig. 4A) that NF-B is dose dependently activated after exposure to a range of concentrations of H₂O₂. Equally important, this is essentially the same range of concentrations of H₂O₂ (EC₅₀ 0.5 mM), which we previously showed to cause Caco-2 barrier hyperpermeability (Banan et al., 2000a,d). The p65 subunit protein of NF-B as well as its p50 subunit was activated to an almost identical degree (p65 activation shown in Fig. 4A). Figure 4B is the time course for the affect of oxidant (0.5 mM) on NF-B (p65 subunit) activity, paralleling time-course findings on barrier integrity and microtubule cytoskeleton (see above). Figure 4C shows that in the presence of a 20-fold excess of the wild-type (WT) consensus oligonucleotide probe, which competes for NF-B binding, NF-B activation is almost completely prevented. On the other hand, the mutated consensus oligonucleotide had no effect on NF-B activation, further indicating assay specificity.

View larger version (30K): [in this window] [in a new window]   Fig. 4. A–C, inducing affects of varying concentrations (0–5 mM) of oxidant (H2O2) on NF-B (p65 subunit) activity in Caco-2 cells determined by a unique ELISA-based assay (A). H2O2 in a dose-dependent manner activates NF-B. Panel B shows the activity time course (0–8 h) for NF-B (p65 subunit) after exposure to 0.5 mM concentration of H2O2. In separate studies (C), the specificity of this assay was demonstrated in the presence or absence of wild-type or mutated NF-B consensus probes. For example, NF-B binding is competed by an excess WT probe in which 20-fold (20 pmol/ml) of this WT probe almost completely abrogates NF-B binding activity. In contrast, the mutated probe excess does not do so. NF-B activity was assessed by a novel ELISA assay of nuclear extracts in multiwell plates that contained specific oligonucleotides containing a consensus-binding site for the p65 subunit of the NF-B. *, p < 0.05 versus vehicle; n = 6/group.

Figure 5A shows that preincubation of monolayers with EGF before H₂O₂ dose dependently attenuated NF-B activation (decreases in optical density at 450 nM). The 10-ng/ml dose of EGF provided almost complete protection against NF-B activation. The protective ability of this same dose paralleled the protective effects of EGF in the assays described previously [e.g., percentage of cells with normal microtubule cytoskeleton (Fig. 2), permeability of monolayer barrier integrity (Fig. 1)]. Also, EGF by itself (in the absence of oxidant) can completely suppress even the "basal" level of NF-B activity (100% inhibition by either 100 or 10 ng/ml EGF alone compared with 95% inhibition with these same doses of EGF plus added oxidant).

View larger version (27K): [in this window] [in a new window]   Fig. 5. A and B, suppressive (protective) effects of varying doses of growth factor (EGF) on NF-B (p65 subunit) activity in intestinal cells (A). Monolayers were exposed to oxidant (H2O2, 0.5 or 5 mM) or were pretreated with varying doses of EGF (1, 5, 10, or 100 ng/ml) before the subsequent exposure to oxidant. EGF substantially and dose dependently inactivates NF-B. Panel B shows the specificity of EGF-induced NF-B inactivation in intestinal cells using either anti-EGF-receptor antibody or EGF-receptor tyrosine kinase inhibitors. Prevention of EGF (10 ng/ml)induced NF-B inactivation is seen by pretreatment of monolayers with monoclonal anti-EGF receptor antibody (anti-EGF-R, 1 µg/ml) or with specific EGF-receptor tyrosine kinase inhibitors [tyrphostin-25 (25 µM) and AG-1478 (150 nM)]. In contrast, the inactive analog, tyrphostin-A1 (ityrphostin-A1, 150 nM) was ineffective. NF-B activity was assessed as described in Fig. 4. *, p < 0.05 versus vehicle; +, p < 0.05 versus H2O2; &, p < 0.05 versus lower doses of EGF (1 or 5 ng/ml) + H2O2; #, p < 0.05 versus the corresponding active analog of inhibitor (tyrphostin-25) plus EGF and then H2O2; n = 6/group.

Figure 5B shows that the effects of EGF on NF-B are specific to this growth factor and are EGF-R-mediated because monoclonal anti-EGF-R antibody (anti-EGF-R) almost completely abolished EGF-mediated NF-B inactivation. To further show specificity, pretreatment with EGF-R-specific tyrosine kinase inhibitors tyrphostin-25 or AG-1478 completely prevented EGF-afforded NF-B inactivation. As might be expected, the inactive analog, tyrphostin-A1, was ineffective.

Because the previously noted pharmacological modulators of NF-B or I-B (e.g., PDTC or MG-132) were protective against the various measures of oxidant disruption, we assessed their effects on the state of NF-B activation as well. Figure 6 shows that preincubation with EGF or the various inhibitors of either NF-B or I-B almost completely abrogated NF-B activation.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Inhibitory effects of several different pharmacological inhibitors on NF-B activity in intestinal cells. Monolayers were exposed to oxidant (H2O2, 0.5 mM) or were pretreated with EGF or NF-B inhibitors (curcumin or PDTC) and I-B inhibitors (lactacystin or MG-132) before the subsequent exposure to oxidant. Conditions were as in Figs. 1B and 2B. NF-B (p65 subunit) activity of the nuclear extracts was determined as described previously. *, p < 0.05 versus vehicle; +, p < 0.05 versus H2O2; n = 6/group.

Oxidant-Induced Degradation of I-B and Its Stabilization by EGF. Assessment of the cytosolic and nuclear associated fractions (Fig. 7, A–D) from cells using Western immunoblotting further corroborated the findings from the NF-B activation studies noted above. For example, Fig. 7B, which is an immunoblot of the nuclear fractions, indicates that oxidant causes the translocation or shift of NF-B p65 subunit to the nucleus. Figure 7A is an immunoblot of the cytosolic fractions of the same H₂O₂-exposed monolayers, showing that oxidant leads to the disappearance of I-B (37-kDa endogenous inhibitor of NF-B) from the cytosol. Figure 7C shows H₂O₂-induced effects on both NF-B (translocation) and I-B (degradation) during the first 30 min. The results indicate that I-B is rapidly degraded in the cell cytosol before the NF-B distribution into the nucleus (normalized to 30-min data, which is arbitrarily assigned a value of 100).

View larger version (17K): [in this window] [in a new window]   Fig. 7. A–D, immunoblotting analysis of both NF-B and its endogenous modulator I-B in intestinal cells after shown treatment regimes (A and B). EGF (10 ng/ml) pretreatment protects against oxidant (0.5 mM H2O2)induced I-B degradation in cell cytosolic fractions (A). While rapidly stabilizing I-B in cytosol, EGF also prevents NF-B translocation to cell nuclear fractions (B). Shown is a representative blot from n = 6/group. C, densitometric analysis of nuclear NF-B and cytosolic I-B in these same H2O2-exposed cells, indicating that I-B degradation in cytosol occurs rapidly and precedes the NF-B nuclear translocation. D, densitometric analysis of the nuclear translocation of NF-B in growth factor EGF-pretreated monolayers, indicating that EGF rapidly prevents H2O2-induced NF-B nuclear translocation. n = 6/group.

In contrast, EGF (Fig. 7B) suppressed oxidant-induced translocation of NF-B into the cell nuclear fractions. Figure 7D shows an early time course (0–30 min) for this protective effect of EGF, further indicating that EGF rapidly prevents NF-B translocation. EGF (Fig. 7A) also prevented degradation of cytosolic I-B that is induced by oxidants. Similar to EGF, known inhibitors of I-B degradation (e.g., MG-132) prevented both I-B degradation and NF-B translocation (data not shown). These rapid protective effects of EGF (i.e., I-B stabilization and NF-B inactivation) are consistent with its known protective effects on the microtubule cytoskeleton and on barrier integrity.

Intracellular Stabilization of I-B and Inactivation of the NF-B in Intestinal Cells Correlate with Different Indices of Monolayer Barrier and Cytoskeletal Protection. Using data across all experimental conditions, we found significant (p < 0.05 for each) correlations (Table 1) between 1) I-B levels and barrier integrity as well as between 2) NF-B inactivation and barrier integrity. This is consistent with the idea that degradation of I-B and activation of NF-B may causally contribute to oxidant-induced disruption and that stabilization of I-B and suppression of NF-B activation may contribute to protection of barrier integrity. Robust correlations were also found when either microtubule integrity or tubulin assembly (S2 pool) was correlated with NF-B inactivation. When another marker of protection, reduced tubulin disassembly (S1 pool) was used against NF-B, an additional robust correlation was observed. When these same three markers of cytoskeletal integrity (e.g., cytoarchitecture) were plotted against I-B stabilization, other robust correlations were observed, further suggesting that stabilization of I-B is key in protection of cytoskeletal and barrier integrity against oxidative stress. Furthermore, the similar EC₅₀ values for EGF protection provides additional evidence that EGF inactivation of NF-B is key in protection of cytoskeleton and barrier. For example, the EC₅₀ value for EGF-induced NF-B inactivation is approximately 5 ng/ml, which is similar to our reported EC₅₀ value for EGF-mediated cytoskeletal and barrier protection, 5 ng/ml (Banan et al., 2000a, 2001c). Not surprisingly, we note similar EC₅₀ values for oxidant-induced NF-B activation (0.5 mM; Fig. 4A) and cytoskeletal and barrier disruption (0.5 mM; Banan et al., 2000a).

View this table: [in this window] [in a new window]   TABLE 1 Correlations of several indices of barrier and cytoskeletal integrity with I-B and NF-B

Stable Overexpression of a Dominant Negative Mutant (Super-Repressor) of I-B after Transfection of Intestinal Cells. To further demonstrate that NF-B and I-B contribute to oxidant-induced loss of barrier permeability and to EGF-mediated protection of barrier and cytoskeletal integrity, we used a dominant negative approach to stabilize (i.e., prevent degradation of) the I-B protein and thus prevent NF-B activation and its injurious consequences. Initially, intestinal cells were stably transfected with plasmid DNA encoding both neomycin resistance (for colony selection) and varying amounts (1, 2, 3, or 4 µg) of I-B mutant. Western immunoblotting analysis of cell lysates of these stably transfected cells from confluent monolayers shows (Fig. 8A) the levels of super-repressor expression among these clones. There is a dose-dependent effect of these varying amounts (1–4 µg) of I-B mutant transfection on the super-repressor protein levels.

View larger version (27K): [in this window] [in a new window]   Fig. 8. A–C, stable expression of a dominant negative for I-B (superrepressor) in intestinal cells transfected with varying amounts (1, 2, 3, or 4 µg) of plasmid encoding mutant I-B (A). The levels of super-repressor protein expression are dose dependently enhanced by increasing the amount of plasmid transfected. Cell lysates from the neomycin-resistant confluent clones were used for immunoblotting. Panel B shows the beneficial effects of the stable dominant negative inhibition of I-B on both the NF-B (C) and I-B (B) in the 3-µg mutant I-B clone. In B, note the prevention of H2O2-induced I-B cytosolic degradation (i.e., I-B stabilization) in this clone stably transfected with the 3-µg mutant for I-B (bottom). Also, note (C) the expected and almost complete suppression of the NF-B nuclear translocation in this same mutant clone. On the other hand, in parental cells (not transfected), H2O2 causes not only the degradation (disappearance) of cytosolic I-B (B, top) but also the nuclear translocation of NF-B (C, top). As expected, control vector alone (CMV) is ineffective (middle panels of both B and C). Cytosolic and nuclear cell extracts from Caco-2 monolayers were subjected to PAGE by the appropriate monoclonal anti-I-B or anti-NF-B antibody and then processed for autoradiography by ECL reagent. Representative blots from n = 6/group.

Figure 8B shows an immunoblot of cell cytosolic fractions of parental (wild-type) Caco-2 cells that were stably transfected with the 3-µg plasmid for the mutant I-B (3-µg clone). Data from these H₂O₂-exposed monolayers shows that this I-B dominant negative clone effectively prevents I-B degradation, thereby stabilizing I-B levels. This is indicated by the presence of the 37-kDa I-B in the cytosol. In contrast, parental cells (those not transfected) exhibited an almost complete disappearance (degradation) of I-B within 10 min. As expected, transfection of vector alone (CMV) was ineffective in stabilizing/protecting cytosolic I-B.

Prevention of Oxidant-Induced NF-B activation in clones transfected with mutant I-B Immunoblotting assessment of NF-B nuclear translocation (Fig. 8C) further shows that in the same stable clone, which was transfected with the 3-µg I-B dominant mutant, the nuclear translocation of NF-B is effectively prevented. This is exhibited by the absence of NF-B (p65 subunit) in the nuclear fractions after H₂O₂ exposure. In contrast, parental cells (not transfeted) show oxidant-induced translocation of NF-B to the nucleus within 10 min. Transfection of vector (CMV) alone, as expected, did not inhibit NF-B nuclear translocation. Indeed, both vector-transfected cells and parental cells responded in a similar fashion to H₂O₂.

ELISA analysis of NF-B activation (Fig. 9A) further corroborated the above-mentioned findings. The same I-B negative mutant also effectively prevented NF-B activation in the nucleus (tantamount to its inactivation). Figure 9B is an early time course (0–30 min) for NF-B inactivation in this I-B mutant clone. This rapid inactivation is indistinguishable from that occurring in parental cells pretreated with EGF.

View larger version (27K): [in this window] [in a new window]   Fig. 9. A and B, protective effects of the stable dominant negative mutant of I-B (3-µg clone) against oxidant-induced NF-B activity in Caco-2 cell as shown by ELISA assessment (A). I-B mutant completely suppresses NF-B activity induced by oxidants in these stably transfected cells, whereas vector alone (CMV) does not do so. B, an analysis of NF-B (p65 subunit) activity in intestinal cell monolayers showing that this mutant I-B, similar to EGF, rapidly suppresses (inactivates) NF-B. Note an almost complete absence of NF-B activity in this dominant mutant clone under oxidative insult. Nontransfected cells exposed to oxidant H2O2, in contrast, show substantially enhanced levels of NF-B activity. n = 6/group.

Protective Effects of Mutant I-B on Monolayer Barrier and Microtubule Integrity. In the absence of oxidant, I-B stabilization (which is associated with NF-B inactivation) did not affect Caco-2 monolayer barrier integrity (FSA clearance; Fig. 10). Stable I-B mutant suppression of NF-B activity did, however, substantially attenuated oxidant-induced barrier hyperpermeability. Indeed, a large percentage (70%) of oxidant-induced monolayer hyperpermeation seems to be I-B (NF-B)-sensitive. In other inhibition studies (Table 2), multiple clones of intestinal Caco-2 cells stably transfected with 1, 2, 3, or 4 of I-B mutant DNA showed a dose-dependent protection of monolayer barrier integrity against oxidant-induced injury. Interestingly, as for the stabilization of I-B and suppression of the NF-B activity (see above), stable clone transfected with 3-µg plasmid for I-B dominant negative provided maximum protection against oxidant-induced barrier dysfunction (Fig. 10; Table 2), and it was thus used for subsequent inhibition studies.

View larger version (32K): [in this window] [in a new window]   Fig. 10. Prevention of the disruptive effects of oxidant (H2O2) on monolayer barrier integrity of intestinal cells using the stable dominant negative stabilization of I-B (3-µg clone). This dominant inhibition is protective against oxidant-induced loss of barrier function as demonstrated by reduced FSA clearance. Transfected cells [I-B mutant or vector alone (CMV)] or nontransfected cells (WT) were incubated with H2O2 (0.5 mM) or vehicle. FSA clearance was determined as described in Fig. 1. Mutant, dominant negative mutant stabilization of I-B (3-µg clone. Vector, vector alone transfected. *, p < 0.05 versus vehicle; +, p < 0.05 versus H2O2 in WT cells; &, p < 0.05 versus H2O2 in mutant cells; n = 6/group.

View this table: [in this window] [in a new window]   TABLE 2 Effects of transfection of varying amounts of mutant I-B on barrier integrity and microtubule cytoskeleton following treatment regimes Intestinal Caco-2 cells stably transfected with varying amounts (1, 2, 3, or 4 µg) of an I-B dominant-negative (Ser 32/36 mutant) were exposed to oxidant (H2O2, 0.5 mM). In separate studies, cells stably transfected with these varying amounts of mutant I-B were preincubated either with a low dose of EGF (1 ng/mL, known to be nonprotective in wild-type cells) or with a high dose of EGF (10 ng/ml, known to be protective in wild-type cells) prior to oxidant exposure. Select treatments from wild-type (WT) cell monolayers are also shown. Monolayer barrier function (fluorescein sulfonic acid, FSA clearance) and microtubule cytoskeletal integrity (%normal) were then assessed.

The combination of fully protective doses of EGF (10 ng/ml) and I-B mutant (3-µg plasmid transfected) elicited no additional effects on barrier integrity (Table 2) compared with those evoked by either one alone under oxidant challenge. Indeed, the extent of such protection was similar to protection induced by either one alone (i.e., no potentiation), suggesting, once again, that EGF works through the NF-B-related mechanisms.

In parallel, analysis of the percentage of dominant negative transfected cells with a normal microtubule cytoskeleton demonstrates (Fig. 11A; Table 2) that dominant negative inhibition of I-B degradation (i.e., stabilizing I-B) attenuated injury to microtubules caused by oxidant. The presence of a dominant mutant by itself did not injure the microtubules. As for barrier integrity, EGF (10 ng/ml) and I-B mutant (3 µg) showed no potentiating effect on protection of microtubules (Table 2) compared with those elicited by either one, further indicating that they are mechanistically related.

View larger version (38K): [in this window] [in a new window]   Fig. 11. A, stable dominant mutant stabilization of I-B (3-µg clone) protects against the damaging effects of oxidant (H2O2) on the microtubule cytoskeleton. The percentage of Caco-2 cells in monolayers with normal microtubule cytoskeleton was assessed in the dominant negative transfected or vector alone transfected cells treated with H2O2 (0.5 mM) or vehicle. Mutant, dominant negative mutant stabilization of I-B (3-µg clone). Vector, vector alone transfected. *, p < 0.05 versus vehicle; +, p < 0.05 versus H2O2 in WT cells; &, p < 0.05 versus H2O2 in mutant cells; n = 6/group. B, stable dominant negative stabilization of I-B also protects against the reduction of tubulin assembly by oxidant H2O2, as determined by immunoblotting analysis of tubulin pools from Caco-2 cells. The polymerized tubulin (S2, index of assembly) and monomeric tubulin (S1, index of disruption) were extracted and processed for analysis. Conditions were similar to those in A. Percentage of polymerized tubulin = [(S2)/(S2 + S1)]. Mutant, dominant negative mutant stabilization of I-B (3-µg clone). Vector, vector alone transfected. *, p < 0.05 versus vehicle; +, p < 0.05 versus H2O2 in WT cells; &, p < 0.05 versus H2O2 in mutant cells; n = 6/group.

Immunoblotting analysis of tubulin from these dominant negative transfected cells further demonstrates (Fig. 11B) that in the presence of I-B stabilization (or NF-B inactivation), oxidant does not elicit any decreases in the stable S2 tubulin fraction (nor any increases in monomeric S1 tubulin). This indicates prevention of (or protection against) microtubule disassembly and disruption. In all cases, the data obtained using transfected cells were consistent with the data obtained using pharmacological agents.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Investigating the fundamental mechanisms through which the intestinal cytoskeleton and permeability barrier are protected against oxidant-induced disruption is of significant biological and clinical importance because oxidative damage results in a "leaky gut", and a hyperpermeable gut is thought to be one of the underlying mechanisms in the pathogenesis of IBD (Hollander 1992, 1998; Keshavarzian et al., 1992; Hermiston and Gordon 1995; McKenizie et al., 1996). In the current study, using monolayers of intestinal epithelial cells, we demonstrate that activation of NF-B is required for the deleterious effects induced by oxidants on microtubule cytoskeletal assembly and cytoarchitecture and on barrier integrity. A second conclusion is that the rapid stabilization of I-B and subsequent inactivation of NF-B seem to be essential to EGF-mediated protection of the cytoskeleton and barrier integrity. These data suggest that NF-B, which can be regulated in opposite directions, is a key modulator of epithelial cytoskeletal and barrier dynamics under pathophysiological conditions. To the best of our knowledge, this is the first time this mechanism has been ascribed to the disruption and protection of epithelial cytoskeletal and barrier permeability. We feel particularly confident in drawing these conclusions because they are supported by several independent lines of evidence as discussed below.

First, exposure of parental intestinal monolayers to oxidant activates NF-B and evokes a cascade of changes that are consistent with the proposed mechanism. Oxidant degrades I-B, translocates NF-B subunits (e.g., p65) to the cell nucleus, and then activates it. Oxidant then reduces the level of stable polymerized tubulin pool (while increasing the size of unstable monomeric tubulin pool), decreases the number of Caco-2 cells with normal microtubule architecture, and disrupts monolayer barrier integrity. Thus, this disruption of both barrier and cytoskeletal integrity requires degradation of I-B and subsequent translocation and activation of NF-B. Second, pretreatment of intestinal monolayers with EGF, which prevents oxidant-induced hyperpermeability, simultaneously stabilizes I-B and then suppresses NF-B activity and evokes an inverse and consistent cascade of alterations that are further consistent with the proposed mechanism. Equally important, these EGF effects on NF-B are specific to this growth factor as well as being EGF-R-mediated because several anti-EGF-R inhibitors effectively suppressed this action. In fact, EGF was a powerful inactivator of NF-B, even suppressing basal NF-B activity. Third, these effects of EGF are selectively mimicked by several different I-B inhibitors (e.g., MG-132) and agents known to modulate NF-B (e.g., PDTC). These known inhibitors of NF-B pathway substantially prevented oxidant-induced injury. Fourth, cells that are stably transfected with dominant negative to I-B and that show both stabilization of I-B and inactivation of NF-B were rendered less sensitive to all the injurious effects of oxidant. Indeed, this dominant negative inhibition of I-B (e.g., 3-µg mutant clone) induces an EGF-like protection. As in EGF-pretreated parental cells, the reduced sensitivity to oxidant in mutant Caco-2 clones requires stabilization of I-B and subsequent suppression of NF-B. It was thus not surprising that the mutant clones were protected, in a dose-dependent manner, against oxidant-induced disruption. Furthermore, the I- B mutant clones were not any more sensitive to protection by EGF (e.g., 3-µg mutant clone + 10-ng/ml dose of EGF; Table 2). In this mutant clone, which by itself was fully protected, stabilization of I-B did not potentiate the protective effects of the exogenously added EGF, further indicating that EGF and NF-B are mechanistically related. The concordance of our findings using both pharmacological inhibition and molecular targeting further supports a novel role for I-B/NF-B in these barrier permeationand cytoskeletal-related processes.

Finally, robust and statistically significant correlations between several different outcome measures (Table 1) and similar EC₅₀ values for EGF-mediated protection, as well as similar EC₅₀ values for oxidant-induced injury (e.g., NF-B, microtubules, and barrier) further support our conclusions. For example, the high strength of these correlations, which explains 90 to 95% of the variance, is consistent with the idea that stabilization of endogenous I-B and suppression of NF-B activation are critical to protection of the intestinal microtubules and barrier integrity against oxidative insult. In this view, modulation of this same system by oxidant challenge in the opposite direction, namely, degradation of I-B and activation of NF-B, leads to the instability of the microtubule (tubulin-based) cytoskeleton and barrier function. Our studies on NF-B are consistent with two intracellular cascades that modulate the I-B/NF-B system in opposite directions. In one the proinflammatory conditions of oxidative stress initiates a sequence that results in increased translocation and activation of NF-B, and in turn, leads to increased disassembly of polymerized tubulin pools and subsequently leads to instability of the microtubule cytoskeleton and monolayer barrier integrity. In the other, EGF initiates a sequence that flows in the opposite direction, attenuating oxidative disruption.

The findings of this report using both pharmacology and targeted molecular interventions are consistent not only with known properties of NF-B but also with other published reports. NF-B is a key factor in the immune response triggered by an array of other molecules such as phorbol esters, other inflammatory cytokines (e.g., IL-1, IL-6, and IL-12), double-stranded RNA, cAMP, and some viral transactivators (Baeuerle and Henkle, 1994; Barnes and Karin 1997; Renard et al., 2001). In previous studies, cytokine-induced nuclear translocation and activation of NF-B seemed to be essential in the promotion of an inflammatory response in non-GI cell models such as macrophages (Menon et al., 1993; Rogler et al., 1998; Tang and Leppla, 1999) as well as in GI models such as intestinal cells (Jobin et al., 1999; Moon et al., 1999). For example, inflammatory stimuli (e.g., lipopolysaccharide) can induce degradation of I-B leading to the translocation of "free" NF-B into the nucleus and, in turn, its induction (Baeuerle and Henkle, 1994; McKenizie et al., 1996). Furthermore, NF-B activation (as indicated by p65 nuclear translocation) has been shown in the intestinal mucosa of patients with ulcerative colitis and Crohn's disease (Rogler et al., 1998; Schreiber et al., 1998; Neurath et al., 1999), where high levels of oxidants, including H₂O_2, as well as loss of mucosal barrier integrity have been reported (Hollander 1992, 1998; Keshavarzian et al., 1992, 2003; McKenizie et al., 1996). There, the amount of NF-B activation was shown to correlate with the degree of mucosal inflammation and disease activity index. Interestingly, other tissue studies in IBD patients demonstrate the presence of induced NF-B in intestinal mucosal epithelial cells (Rogler et al., 1998). Our current study now suggests a unique role for NF-B under pathophysiological conditions of oxidant challenge, namely, disruptive effects on the cytoskeletal assembly and barrier permeability in intestinal cell monolayers.

Depending on the cellular model and experimental conditions used, growth factors such as EGF can have different effects on NF-B. For example, EGF can activate NF-B in several non-GI models such as breast cancer cells and fibroblasts among others (Biswas et al., 2000; Hirota et al., 2001). Hirota et al. (2001) showed that TNF- transactivates overexpressed EGF-R in a redox-sensitive manner, and this activation enhanced NF-B activation in fibroblasts. Biswas et al. (2000) showed that the basal level of active NF-B in estrogen negative breast cancer cells was elevated by EGF and inhibited by the anti-EGF-R antibody, suggesting that EGF acts as a NF-B activation factor. In contrast, other studies using non-GI models, similar to our current GI study, have shown that growth factors (e.g., EGF) effects can be mediated through the interruption of NF-B pathway (Conner et al., 1999; Ohtsubo et al., 2000). For instance, inactivation of NF-B signaling mediates EGF-induced cell cycle arrest in A-431 cells (a cell line not unlike our Caco-2 cells, which is also of carcinoma origin) (Ohtsubo et al., 2000). Similarly, another growth factor, hepatocyte growth factor, decreases NF-B activity after HGF treatment (Conner et al., 1999). Furthermore, EGF does not enhance NF-B activity in human microvascular endothelial cells (Izumi et al., 1994). It is noteworthy that some of the reports showing EGF activation of NF-B (Hirota et al., 2001) have used an overexpression model of EGF-R, a model which has been linked with growth factors' nonprotective effects such as progression of neoplastic abnormalities. This model is not used in our studies of growth factors protective capabilities on intestinal barrier integrity.

Our findings are potentially relevant for developing new treatment and/or prevention strategies for IBD. They suggest a novel anti-inflammatory defensive mechanism that might, if it occurred in vivo, protect against the oxidative stress-induced I-B degradation and NF-B up-regulation and either prevent initiation or manifestation of the acute IBD attack. This defensive mechanism is seen in the ability of EGF to prevent oxidant-induced cellular injury and barrier disruption through stabilization of I-B and suppression of the activation of proinflammatory factor NF-B. The potential therapeutic use of modulating this anti-inflammatory mechanism would be consistent with current characterizations of the pathophysiology of IBD. Inflammation can be induced by proinflammatory agents (e.g., endotoxin) and this seems to be closely linked to the induction of NF-B (Barnes and Karin, 1997; Rogler et al., 1998; Schreiber et al., 1998; Yin et al., 1998; Neurath et al., 1999). Once up-regulated, NF-B seems to modulate key cellular events that may be essential in the initiation and perpetuation of inflammatory responses, including up-regulation of iNOS, IL-8, and cyclooxygenase-2. It is therefore not surprising that activation of NF-B is thought to be highly relevant to the promotion of inflammation such as occurs in IBD (Rogler et al., 1998; Schreiber et al., 1998; Neurath et al., 1999). This is thought to be especially possible for the transition from the inactive to active phases of inflammation in IBD (flare up) in which intestinal oxidants and proinflammatory molecules periodically create a vicious cycle that leads to sustained oxidative stress, hyperpermeability, inflammation, and tissue damage (Keshavarzian et al., 1992, 2003; McKenizie et al., 1996). The ability of endogenous protective agents (EGF) to stabilize I-B and prevent NF-B activation, such as we observed in intestinal Caco-2 cells, could play a key role in developing a new therapeutic modality to attenuate such a vicious cycle.

Previous studies showed that ROS and especially H₂O₂ can participate as second messengers in NF-B signaling pathway (Schreck et al., 1991; Kretz-Remy et al., 1996; Schmidt et al., 1996; Bonizzi et al., 2000). Treatment of cells with micromolar concentrations of H₂O₂ potently activates NF-B at levels even higher than that of TNF- (Schreck et al., 1991). This effect was prevented by antioxidant PDTC. Moreover, antioxidants such as PDTC and N-acetylcysteine block NF-B activation by a wide range of other stimuli, including IL-1, suggesting that ROS may act as signaling molecules in NF-B activation. A link between H₂O₂ (ROS) production and NF-B activation has been further established in studies in which cells overexpressing H₂O₂-degrading enzymes such as catalase (Schmidt et al., 1996) and glutathione peroxidase (Kretz-Remy et al., 1996) were deficient in activating NF-B in response to TNF-. In contrast, overexpression of superoxide dismutase, which enhances H₂O₂ production, potentiated NF-B activation (Schmidt et al., 1996). Thus, H₂O₂ can act as an intracellular messenger for NF--B induction by inflammatory stimuli.

Despite the essential importance of NF-B in inflammatory processes, the role of the NF-B in epithelial monolayer barrier function or dysfunction, especially in modulation of epithelial cytoskeletal cytoarchitecture as our new findings suggest, is not understood. Based on the known regulatory mechanisms for intestinal barrier permeability (Banan et al., 1999, 2000b, 2001d) in which iNOS up-regulation can lead to disruption of the cytoskeleton, whereas its prevention by EGF is key in protection of monolayers (Banan et al., 2002d), we propose that up-regulation or down-regulation of this mechanism can underlie I-B/NF-B's novel effects on monolayer cytoskeletal and barrier permeability under pathophysiological conditions of oxidant challenge. Specifically, first we showed that oxidants (e.g., H₂O₂ and HOCl) and oxidative stressors (e.g., alcohol) cause intestinal barrier and cytoskeletal disruption through the underlying up-regulation of iNOS pathway (Banan et al., 1999, 2000b, 2001d, 2002d). For example, pretreatment with either the iNOS inhibitor [L-NIL; L-N⁶-(1-iminoethyl)lysine] or the selective oxidant scavengers (urate, cysteine, and superoxide dismutase) protected against loss of barrier integrity induced by oxidative challenge (Banan et al., 1999). Our studies established that iNOS up-regulation and consequent overproduction of nitric oxide causes the cytoskeletal and barrier disruption. Second, interestingly, NF-B is a well known stimulator of the expression of several proinflammatory factors, including iNOS in non-GI and GI models (Baeuerle and Henkle, 1994; Jourd' heuil et al., 1997; Kaul et al., 1998; Jobin and Sartor, 2000). Indeed, activation of NF-B seems to be essential in iNOS up-regulation in various cellular models, including GI epithelium (Jobin and Sartor, 2000). Once activated, NF-B regulates several important cellular genes involved in oxidative response such as both TNF- and iNOS. For example, iNOS up-regulation, which was induced by inflammatory agents, including oxidative stress (H₂O₂), IL-1, TNF-, and lipopolysaccharide, was shown to be dependent upon the activation of NF-B (Barnes et al., 1997; Jourd' heuil et al., 1997; Kaul et al., 1998). Thus, NF-B activation is highly relevant to oxidative stress, iNOS up-regulation, and inflammation. Accordingly, based on this well characterized NF-B-dependent regulatory mechanism for iNOS as well as our aforementioned studies on iNOS and intestinal barrier disruption, it is possible that up-regulation of iNOS underlie NF-B's deleterious effects on monolayer cytoskeletal and barrier permeability by oxidants.

In summary, our findings demonstrate for the first time that modulation of the I-B/NF-B can influence the dynamics of microtubule cytoskeletal assembly and intestinal barrier integrity and that it is key for protection against oxidative disruption to occur under in vitro conditions in Caco-2 monolayers. This new knowledge may prove useful because suppressing the activation of the proinflammatory agent NF-B through activation of endogenous protective mechanisms by growth factors or growth factor mimetics may lead to unique therapeutic strategies for the treatment of a variety of oxidant-induced inflammatory disorders of the GI tract, including IBD.

	Footnotes

 
This work was supported in part by a grant from Rush University Medical Center, Department of Internal Medicine, and by a National Institutes of Health R01 Grant NIDDK60511 (to A.B.). Portions of this work might be presented in the abstract form during the annual meeting of the American Gastroenterological Association (Digestive Disease World), May 2003.

DOI: 10.1124/jpet.102.047415.

ABBREVIATIONS: GI, gastrointestinal; IBD, inflammatory bowel disease; NF-B, nuclear factor-B; iNOS, inducible nitric-oxide synthase; IL, interleukin; TNF-, tumor necrosis factor-; I-B, inhibitory factor-B; ROS, reactive oxygen species; EGF, epidermal growth factor; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; EGF-R, epidermal growth factor receptor; PDTC, 1-pyrrlolidine dithiocarbamate; MG-132, carbobenzyloxy-leu-leu-leucinol; LSCM, laser scanning confocal microscopy; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescence; FSA, fluorescein sulfonic acid; S2, polymerized tubulin; S1, monomeric tubulin; WT, wild type; AG-1478, tyrosine kinase inhibitor.

Address correspondence to: Dr. 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

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