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CELLULAR AND MOLECULAR

Inhibition of Oxidant-Induced Nuclear Factor-B Activation and Inhibitory-B Degradation and Instability of F-Actin Cytoskeletal Dynamics and Barrier Function by Epidermal Growth Factor: Key Role of Phospholipase- Isoform

Division of Digestive Diseases, Departments of Internal Medicine, Pharmacology, and Molecular Physiology, Rush University Medical Center, Chicago, Illinois

Received October 31, 2003; accepted December 10, 2003.

	Abstract

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Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-B (NF-B) activation is required for oxidant-induced disruption of cytoskeletal and barrier integrity. Epidermal growth factor (EGF) stabilizes the F-actin cytoskeleton and protects against oxidant damage, but the mechanism remains unclear. We hypothesized that the mechanism involves activation of phospholipase C- (PLC-), which prevents NF-B activation and the consequences of this activation, namely, cytoskeletal and barrier disruption. We studied wild-type and transfected cells. The latter were transfected with varying levels (1-5 µg) of cDNA to either stably overexpress PLC- or to inhibit its activation. Cells were pretreated with EGF before exposure to oxidant (H₂O₂). Stably overexpressing PLC- (+2.0-fold) or preincubating with EGF protected against oxidant injury as indicated by 1) decreases in several NF-B-related variables [NF-B (p50/p65 subunit) nuclear translocation, NF-B subunit activity, inhibitory-B (I-B) phosphorylation and degradation]; 2) increases in F-actin and decreases in G-actin; 3) stabilization of the actin cytoskeletal architecture; and 4) enhancement of barrier function. Overexpression induced inactivation of NF-B was potentiated by EGF. PLC- was found mostly in membrane and cytoskeletal fractions (<9% in the cytosolic fractions), indicating its activation. Dominant negative inhibition of endogenous PLC- (-99%) substantially prevented all measures of EGF protection against NF-B activation. We concluded 1) EGF protects against oxidant-induced barrier disruption through PLC- activation, which inactivates NF-B; 2) Activation of PLC- by itself is protective against NF-B activation; 3) the ability to modulate the dynamics of NF-B/I- B is a novel mechanism not previously attributed to the PLC family of isoforms in cells; and 4) development of PLC- mimetics represents a possible new therapeutic strategy for IBD.

Although the pathophysiology of mucosal barrier hyperpermeability in IBD remains unclear, it is known that chronic inflammation in IBD is dependent on high levels of oxidants such as H2O₂ (Keshavarzian et al., 1992, 2003; McKenizie et al., 1996; Banan et al., 2000a,c) as well as on activation of nuclear factor-B (NF-B) (Rogler et al., 1998; Shreiber et al., 1998; Neurath et al., 1999; Banan et al., 2003a,d,e). In fact, both oxidant stress and NF-B induction are important to the promotion of an inflammatory response and key in mucosal damage during IBD (Barnes and Karin, 1997; Shreiber et al., 1998; Jobin et al., 1999; Neurath et al., 1999; Banan et al., 2000a,c, 2003a,d,e; Keshavarzian et al., 2003). NF-B is typically composed of p50 and p65 subunits and is tightly modulated by an endogenous cytosolic inhibitor, I-B, which complexes with the p65 protein and traps NF-B in the cytosol in an inactive form. When activated NF-B modulates key cellular processes involved in an inflammatory response such as the up-regulation of inducible nitric-oxide synthase-driven oxidative processes (Barnes and Karin, 1997; Banan et al., 2003a). Using monolayers of intestinal cells, we reported a new mechanism that NF-B activation is crucial in oxidant-induced disruption of epithelial barrier function (Banan et al., 2003a). In view of these considerations, understanding how gut barrier function is protected against oxidative, proinflammatory conditions of NF-B activation is of substantial clinical and biological value.

In an effort to better understand key endogenous defensive mechanisms, we have been investigating events underlying oxidant-induced mucosal damage and barrier disruption and protection against this damage mediated by growth factor-triggered signaling processes. Our hope has been to devise new strategies for the development of more effective treatment regimens for inflammatory disorders of the GI tract, in general, and IBD, in particular. For instance, using monolayers of intestinal cells as a well established model of gut barrier function, we showed that cytoskeletal disassembly/instability is a key event in oxidant-induced injury (Banan et al., 2000a,b) and that growth factor (EGF or transforming growth factor-) prevents injury by stabilizing the cytoskeleton in large part through a signaling pathway mediated by EGF-receptor and then phospholipase C- (PLC-) (Banan et al., 2001c, 2003b). The involvement in protective mechanisms by PLC- in the GI epithelium as we originally reported was a novel concept (Banan et al., 2001c). We showed using wild-type (naive) intestinal Caco-2 cells that after activation of EGF receptor, EGF causes the distribution of the native PLC- isoform into the cell membrane and thus considered this PLC isoform as a potential contributor to EGF-mediated protection of the GI epithelial barrier function. Using targeted molecular interventions, we then found that maintaining an intact actin cytoskeleton is necessary for protection of intestinal barrier by EGF apparently through PLC- (Banan et al., 2002a). Despite the crucial importance of the isoform of PLC to intestinal barrier function, the molecular mechanisms underlying PLC--mediated, EGF-induced protection of monolayer barrier and actin cytoskeletal integrity are poorly understood.

Accordingly, in the current investigation, we tested the hypothesis that PLC- not only prevents oxidant-induced NF-B activation and I-B degradation but also that it is key to EGF-mediated protection against the damaging consequences of this activation through the stabilization of I-B. We used both pharmacological and targeted molecular interventions using several transfected intestinal cell lines. In several clones the full-length PLC- isoform was reliably overexpressed; in the other clones, endogenous PLC- activity was inhibited. We now report new mechanisms, i.e., suppression of the stress of NF-B activation and of cytoskeletal instability, by the isoform of PLC in epithelial monolayers. To the best of our knowledge, this is the first time that PLC- has been demonstrated to inhibit the dynamics of NF-B and I-B in cells.

	Materials and Methods

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Cell Culture. Caco-2 cells were obtained from American Type Culture Collection (Rockville, MD) at passage 15. This cell line was chosen because they form monolayers that morphologically resemble small intestinal cells, with defined apical brush borders and a highly organized actin network upon differentiation (Meunier et al., 1995; Banan et al., 2000b). 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 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. The utility and characterization of this cell line has been reported previously (Meunier et al., 1995).

Plasmids and Stable Transfection. The sense and dominant negative plasmids of PLC- were constructed and then stably transfected by Lipofectin reagent (Invitrogen, Carlsbad, CA) as we described previously (Banan et al., 2001c, 2002a). Expression was controlled by simian virus 40 early promoter present in pXf vector. The dominant negative PLC- 1 fragment from the Z region (designated as PLCz) of human PLC- 1, which covers the src homology (SH)2 and SH3 domains (amino acids 517-901), was isolated by reverse transcriptase/polymerase chain reaction and cloned into an eukaryotic expression vector, pXf (Chen et al., 1994; Turner et al., 1997). Control conditions included vector (pXf) alone. Multiple clones stably overexpressing PLC- or lacking PLC- activity were assessed by immunoblotting as well as tested for PLC- activity (see below). These cells were then plated on Biocoat Collagen I Cell Culture Inserts (BD Biosciences, Bedford, MA) and subsequently used for experiments.

Experimental Design. In the first series of experiments, post-confluent monolayers of wild (naive)-type cells were preincubated with EGF (1-10 ng/ml) or isotonic saline for 10 min and then exposed to oxidant (H₂O₂, 0-0.5 mM) or vehicle (saline) for 30 min. As we previously showed, H₂O₂ at 0.1 to 0.5 mM disrupts F-actin and barrier integrity and activates NF-B (Banan et al., 2000a,b, 2003a). EGF at 10 ng/ml (but not 1 ng/ml) prevents both actin and barrier disruption. These experiments were then repeated using stably transfected cells. In all experiments, we assessed actin cytoskeletal stability (cytoarchitecture, assembly, and disassembly), actin molecular dynamics (F- and G-actin pools), I-B distribution (cytosolic expression and degradation), I-B phosphorylation, NF-B p65 subunit activity (cytosolic levels, both nuclear translocation and activity), NF-B p50 subunit activity (cytosolic levels, both nuclear translocation and activity), barrier integrity (permeability), and PLC- subcellular distribution as well as activity (immunoblotting, immunoprecipitation and in vitro assay).

In the second series of experiments, cell clone monolayers that were stably overexpressing PLC- were preincubated (10 min) with EGF (1 or 10 ng/ml) or vehicle before exposure (30 min) to damaging concentrations of oxidant (H₂O₂, 0.5 mM) or vehicle. Outcomes measured were as described above.

In the third series of experiments, monolayers of dominant negative, PLCz transfected cells stably lacking PLC- activity were treated with high (protective) doses of EGF and then oxidant. In corollary series of experiments, we investigated the effects of PLC- activation or inactivation on the state of 1) I-B degradation, phosphorylation, and stabilization; 2) NF-B activation and inactivation; 3) actin assembly and disassembly; and 4) cytoarchitecture of the actin cytoskeleton.

Fractionation and Immunoblotting of PLC- Cell monolayers grown in 75-cm² flasks were processed for the isolation of the cytosolic, membrane, and cytoskeletal fractions (Banan et al., 2002b, 2003b). Protein content of the various cell fractions was assessed by the Bradford method (Bradford, 1976). For immunoblotting, samples (25 µg protein/lane) were added to a standard SDS buffer, boiled, and then separated on 7.5% SDS-PAGE. The immunoblotted proteins were incubated with the primary mouse monoclonal anti-PLC- (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:1000 dilution. A horseradish peroxidase-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR) was used as a secondary antibody at 1:4000 dilution. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences Inc., Piscataway, NJ) and autoradiography and subsequently analyzed. The identity of the PLC- bands were confirmed (Banan et al., 2001c) by 1) using a PLC- blocking peptide in combination with the anti-PLC- antibody that prevents the appearance of the corresponding "major" band in Western blots. 2) Additionally, in the absence of the primary antibody to PLC-, no corresponding band for PLC- was observed. 3) The PLC- band ran at the expected molecular mass of 145 kDa as confirmed by a known positive control for PLC- (from rat brain lysates). 4) Prestained molecular weight markers (M_r 34,900 and 205,000) were run in adjacent lanes. We also confirmed (Banan et al., 2001c, 2003b) that overexpression of PLC- or dominant negative inhibition of PLC- did not affect the relative expression levels of other PLC isoforms nor did it injure the Caco-2 cells.

Immunoprecipitation and PLC- Activity. Immunoprecipitated PLC- was collected and processed for its ability to form [lH]inositol phosphates (Banan et al., 2001c). Briefly, after treatments, confluent cell monolayers were lysed by incubation for 20 min in 500 µl of cold-lysis buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, antiprotease cocktail (10 µg/ml), 10% glycerol, 1 mM sodium orthovanadate, 5 mM NaF, and 1% Triton X-100]. The lysates were clarified by centrifugation at 14,000g for 10 min at 4°C. For immunoprecipitation, the lysates were incubated for 2 h at 4°C with monoclonal anti-PLC- (1:2000 dilution, in excess). The extracts were then incubated with protein G-Sepharose for 1 h at 4°C. The immunocomplexes were collected by centrifugation (2500g, 5 min) in a Microfuge tube, washed three times with immunoprecipitation buffer containing 5 mM Tris-HCl, pH 7.4, and 0.2% Triton X-100. They were then washed one time with sample buffer (20 mM HEPES, pH 7.5) and resuspended in 20 µl of buffer and 5 µl of reaction buffer [5 µCi/ml [lH]myoinositol plus LiCl (10 mM, which inhibits inositol phosphate hydrolysis)] and subsequently incubated for 5 min at 30°C. Reactions were then stopped by the addition of 8 µl of 5x sample buffer and the [lH]inositol phosphates (IP) were recovered in the supernatant after centrifugation (16,000g, 5 min). The extracts were separated on Dowex formate ion-exchange mini-columns (Bio-Rad, Hercules, CA). Radioactivity present (IP content) in samples was quantified by scintillation counting with aqueous counting scintillant. Counts for blanks were subtracted from the sample activity. Sample activity was also corrected for protein concentration (Bradford method), and PLC- activity was reported as picomoles per minute per milligram of protein.

Analysis of NF-B Activation. NF-B (p65 and p50 subunit) activation was assessed by a unique ELISA procedure (Banan et al., 2003a). Monolayers of naive and transfected cells grown in 25-cm² flasks were processed for the isolation of the cytosolic and nuclear fractions. Cells fractions were added to a 96-well plate to which oligonucleotides containing a consensus-binding site for the NF-B had been immobilized (Trans-Am; Active Motif, Carlsbad, CA). 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. Consequently, only the activated NF-B is captured by the probe bound in microwell plates. The binding of NF-B to its consensus sequence was then detected using a primary anti-NF-B (p65 or p50) antibody (Santa Cruz Biotechnology, Inc.), followed by a secondary antibody conjugated to horseradish peroxidase. The results were quantitated by a chromogenic reaction, which was then read for absorbance at 450 nm by a Seivers NOA 280 microplate analyzer (Sievers, Boulder, CO).

Western Blot Analysis of Changes in NF-B Subunit Levels and Nuclear Translocation. Cellular nuclear and cytosolic extracts from naive and transfected cells were prepared as described above. Briefly, NF-B nuclear translocation was determined by comparing the levels of NF-B protein expression in the cytosolic versus nuclear extracts by anti-p65 and anti-p50 antibodies using a nondenaturing gel (6%) (Banan et al., 2003a). For immunoblotting, samples (20 µg protein/lane) were placed in a standard sample buffer, boiled, and then subjected to PAGE. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences Inc.) and autoradiography. For comparison of different blots, standard (positive) loading controls (20 µg of HeLa cell extracts/lane) for NF-B were run concurrently with each run.

Western Blot Analysis of I-B Degradation, Expression Levels, and Phosphorylation. 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, Inc.) using a Western blot protocol (10% gel) (Banan et al., 2003a). Samples (20 µg protein/lane) were added to a standard SDS buffer, boiled, and then separated on SDS-PAGE. For assessment of the I-B phosphorylation levels an anti-phospho-I-B (Ser 32/36) was used. Separate blots were done for I-B phosphorylation and I-B expression levels. As for NF-B, proteins were visualized by enhanced chemiluminescence and subsequently autoradiographed. Standard (positive) loading controls (20 µg of HeLa cell extracts/lane) for I-B were included in each run. To quantify the relative levels of I-B (e.g., to monitor its degradation), the optical density of the bands corresponding to immunolabeled I-B were measured with a laser densitometer.

Immunofluorescent Staining and High-Resolution Laser Scanning Confocal Microscopy of Actin Cytoskeleton. Cells from monolayers were fixed in cytoskeletal stabilization buffer and then postfixed in 95% ethanol at -20°C (Banan et al., 2000b). Cells were subsequently processed for incubation with fluorescein isothiocyanate-phalloidin (specific for F-actin staining; Sigma-Aldrich, St. Louis, MO), 1:40 dilution for 1 h at 37°C. 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). The cytoskeletal elements were examined in a blinded manner for their overall morphology, orientation, and disruption (Banan et al., 2000a,b, 2001a). The identity of the treatment groups for all slides was decoded only after examination was complete.

Actin Fractionation and Quantitative Western Immunoblotting of F-Actin and G-Actin. Polymerized (F-) actin and monomeric (G-) actin were isolated using a especially developed series of extraction and ultracentrifugation steps (Banan et al., 2000b). Cells were gently scraped and pelleted with centrifugation at low speed (700 rpm, 7 min, 4°C) and resuspended in actin stabilization-extraction buffer [0.1 M PIPES, pH 6.9, 30% glycerol, 5% dimethyl sulfoxide, 1 mM MgSO₄, antiprotease cocktail (10 µg/ml), 1 mM EGTA, and 1% Triton X-100] at room temperature for 20 min. Actin fractions were separated after a series of centrifugation and extraction steps. Cell lysates were centrifuged at 105,000g for 45 min at 4°C, and the supernatant containing the soluble monomeric pool of G-actin (or 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, antiprotease 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 or F-fraction or cold/Ca²⁺-soluble fraction) was removed. To ensure the complete removal of the F-fraction, the remaining pellet was treated with the Ca²⁺-containing depolymerization buffer twice more by resuspension and centrifugation. The "actin" was recovered by separately incubating (at 37°C for 30 min) the S1 and S2 fractions with stabilizing agents, phalloidin (1 µM) and ATP (0.1 mM), in actin stabilization buffer [0.1 M PIPES, pH 6.9, 30% glycerol, 5% dimethyl sulfoxide, antiprotease cocktail (10 µg/ml), 1 mM EGTA, 1 mM MgCl₂, and 0.1 mM ATP] to promote polymerization of actin. Actin was then recovered by centrifugation and resuspended in the above-described 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, and then subjected to PAGE on 7.5% gels. Standard (purified) actin controls (5 µg/lane) were run concurrently with each run. To quantify the relative levels of actin, the optical density of the bands corresponding to immunolabeled actin were measured with a laser densitometer (Banan et al., 2000b).

Determination of Barrier Permeability by Fluorometry. Status of the integrity of monolayer barrier function was confirmed by a widely used and validated technique that measures the apical to basolateral paracellular flux of fluorescent markers such as fluorescein sulfonic acid (FSA, 200 µg/ml; 0.478 kDa) as described previously (Banan et al., 2001a,b,c, 2000a,b, 2003a,b). 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 excitation, 485 nm; emission, 530 nm. Clearance (Cl) was calculated using the following formula: Cl (nanoliters per hour per square centimeter) = Fab/([FSA]a x S), where Fab is the apical to 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. 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 deemed statistically significant.

	Results

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Our earlier findings (Banan et al., 2003b) showed that intestinal cells transfected with PLC- sense stably overexpress the (145-kDa) isoform of phospholipase C (2.3-fold compared with wild-type cells) and that this overexpression either by itself or in synergy with added growth factor (EGF) protects monolayer barrier integrity against oxidative stress. In the present investigation, using pharmacological and molecular biological interventions, we have investigated the molecular mechanisms underlying PLC--mediated protection.

Importance of PLC- Isoform in Protection against Oxidant-Induced NF-B Activation: Inhibition of Both Nuclear Distribution and Activation of NF-B Subunits p65 and p50. We surmised that PLC- protection could be due to the suppression of oxidant triggered pathways such as the proinflammatory NF-B. Using our wild-type and transfected clones, we measured both the nuclear distribution of NF-B subunits (p65 and p50) as well as their activation under conditions of oxidant challenge. In wild-type cells (those not overexpressing PLC-), oxidant H₂O₂ alone induced substantial activation of both NF-B subunits (Fig. 1, A and B). On the other hand, overexpression of PLC- [PLC-] by itself afforded substantial protection against NF-B subunits activation. This inactivation did not necessitate the presence of growth factor EGF. Although a low (nonprotective) dose of EGF (e.g., 1 ng/ml) did not afford significant protection against NF-B activation in wild-type cells, this concentration of growth factor did potentiate NF-B inactivation in PLC- overexpressing clones. In wild-type cells, higher (protective) doses of EGF (e.g., 10 ng/ml) were required for NF-B inactivation. Transfection of only the empty vector, as might be expected, did not suppress NF-B activation. For instance, the level of p65 subunit that was activated was 0.09 ± 0.02 (OD, 450 nm) for vector-transfected cells exposed to vehicle, 1.56 ± 0.20 for vector-transfected cells exposed to H₂O₂ alone and 0.27 ± 0.05 for PLC- sense-transfected cells incubated in H₂O₂. Similarly, the level of p50 subunit that was activated was 0.10 ± 0.03 for vector-transfected cells exposed to vehicle, 1.46 ± 0.15 for vector-transfected cells exposed to H₂O₂ alone and 0.32 ± 0.06 for PLC- sense-transfected cells incubated in H₂O₂. These alterations did not seem to be due to changes in the ability of oxidants to cause NF-B activation because empty vector-transfected cells and wild-type cells responded in a similar manner to H₂O₂, both exhibiting comparable NF-B activation.

View larger version (34K): [in this window] [in a new window]   Fig. 1. A and B, protective effects of PLC- isoform expression against oxidant-induced activation of NF-B subunits p50 (A) and p65 (B) in intestinal cells. A unique transfected intestinal cell clone we have developed (see Materials and Methods) that stably overexpresses the PLC- by 2.3-fold was used. Intestinal monolayers overexpressing PLC- (3-µg clone) or wild-type cells (not overexpressing PLC-) were incubated with growth factor (EGF, 10 min) before exposure to oxidant (H2O2, 30 min). Clones overexpressing "[PLC-]" show substantial inhibition of both NF-B p50 and p65 subunit activities that was induced by oxidant. In wild-type monolayers, NF-B was inactivated only by high (protective) doses of EGF (e.g., 10 ng/ml). NF-B subunit activity was determined by an ELISA of nuclear extracts in especially coated multiwell plates, which contained specific oligonucleotides containing a consensus-binding site for either the p50 or p65 subunit of NF-B. *, p < 0.05 versus vehicle. +, p < 0.05 versus H2O2 in wild-type. &, p < 0.05 versus. PLC- overexpressing PLC- cells exposed to H2O2 or pretreated with EGF before H2O2 in wild-type cells. #, p < 0.05 versus EGF (10 ng/ml) before H2O2 in wild-type cells. WT, wild-type cells; PLC-, cells transfected with PLC- sense. n = 6 per group in all figures, including subsequent ones, unless otherwise indicated. C and D, representative blots of NF-B protein subunits p50 (C) and p65 (D) distribution in the nuclear fractions of the intestinal cells of wild-type or transfected origin. The p50 or p65 protein bands are from the same treatments as described in A and B. In stably transfected clones (3-µg sense clone), PLC- expression prevents NF-B subunits nuclear distribution against exposure to oxidant (H2O2, 30 min). This levels of distribution is comparable with the control (vehicle-treated) cells that exhibit basal levels of NF-B (p50 and p65) in the nuclear extracts. In wild-type cells, only a high dose of EGF (10 ng/ml) suppresses NF-B nuclear distribution. The region of gel shown was between the Mr 43,000 and 75,000 prestained molecular weights, which were run in adjacent lanes. WT, wild-type cells; PLC-, cells transfected with PLC- sense. Shown is a representative blot.

Multiple clones of intestinal cells transfected with 1 to 5 µg of PLC- sense plasmid demonstrated (Table 1) a dose-dependent inhibition of oxidant (H₂O₂)-induced NF-B activation. The cell clone transfected with 3 µg of PLC- sense (+ 3) provided maximum (85%) suppression of NF-B activity and thus was used in subsequent experiments.

View this table: [in this window] [in a new window]   TABLE 1 Effects of transfection of varying amounts of PLC- sense or dominant negative mutant DNA on both NF-B activity and I-B levels in intestinal Caco-2 monolayers Intestinal cells stably transfected with varying amounts of PLC- sense DNA (1, 2, 3, or 5 µg) were exposed to oxidant (H2O2, 0.5 mM). In separate studies, cells transfected with varying amounts of a PLC- dominant negative DNA, namely, PLCz (1, 2, 3, or 5 µg), were treated with EGF (10 ng/ml) before exposure to oxidant. Wild-type (WT) cells, which are not transfected, are also shown. Monolayer NF-B (p65) activity and I-B levels were assessed as described under Materials and Methods.

Representative Western blots of the changes in NF-B subunit distribution in the cell nuclear fractions are shown in Fig. 1, C and D. In transfected clones, PLC- overexpression inhibits oxidant-induced distribution of the p50 (C) and p65 (D) subunits of NF-B into the cell nuclear fractions as shown by band densities that were reduced to a level comparable with the controls. In wild-type cells, as for NF-B inactivation, only high (protective) doses of EGF (e.g., 10 ng/ml) prevented NF-B nuclear distribution. In these same cells, oxidant caused the distribution of NF-B subunits to the nucleus, paralleling findings on NF-B activation.

Figure 2 shows a time course for changes in NF-B activation in wild-type cells and its suppression in PLC- overexpressing clones. These data further corroborate the aforementioned findings. Maximal fold increase in NF-B activation by H₂O₂ alone is 20; this increase is suppressed by PLC- overexpression.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Time course for the inhibition of the activation of NF-B (p65 subunit) in 3-µg transfected PLC- overexpressing clone (PLC-). Wild-type (WT) cells are also shown where NF-B is activated after H2O2 exposure. Cells were exposed to 0.5 mM H2O2 at zero time.

Key Role of PLC- in Stabilization of Cytosolic I-B: Inhibition of I-B Degradation. Because oxidants increase both I-B degradation and NF-B activation as well as disrupt monolayer barrier permeability (Banan et al., 2003a), we surmised that decreases in degradation of I-B (a 37-kDa endogenous inhibitor of NF-B) is a critical mechanism underlying PLC--induced inactivation of NF-B under conditions of oxidant challenge.

To this end, multiple clones of intestinal cells transfected with 1 to 5 µg of PLC- sense cDNA demonstrated (Table 1) a dose-dependent stabilization (absence of degradation) of cytosolic I-B against H₂O₂ exposure. As for NF-B inactivation, the 3-µg clone of PLC- (+ 3) led to the highest level of protection against I-B degradation. For example, there was a substantial decrease in oxidant-induced I-B degradation (70% less degradation) in the 3-µg PLC- overexpressing clone, which is similar to the steady-state levels of I-B seen in the controls. PLC- expression induced stabilization of I-B did not require EGF. But, a low concentration (1 ng/ml) of EGF, which by itself did not afford stabilization of I-B in wild-type cells, potentiated I-B stability in clones overexpressing PLC- (not shown). In wild-type cells, similar to NF-B inactivation, I-B stabilization required a higher dose of EGF (10 ng/ml; Table 1).

Figure 3A is a representative immunoblot showing the stabilization of cytosolic I-B levels by PLC- overexpression using the 3-µg sense transfected clone (a clone that also protects barrier function; Banan et al., 2003b). H₂O₂ induces I-B degradation in wild-type cells, whereas PLC- overexpressing clones show almost steady-state levels of I-B. The corresponding OD for control was 5000 ± 32; 0.5 mM H₂O₂, 690 ± 175; and PLC- sense-transfected clones incubated in H₂O₂, 4187 ± 124, indicating I-B stabilization in the PLC- clones. Transfection of the empty vector alone, as expected, did not confer protection to I-B (not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 3. A, representative immunoblot for the protective (stabilizing) effects of PLC- overexpression on I-B levels in the cytosolic fractions of intestinal cells. The I-B bands are after the treatment regimes shown. Paralleling its inhibitory effects on NF-B, PLC- overexpression (3-µg clone) stabilizes the 37-kDa I-B (an endogenous modulator of NF-B) against oxidant insult (H2O2, 30 min). On the other hand, oxidant alone degrades I-B in wild-type cells. In these wild-type cells, EGF (10 ng/ml) protects the I-B. The region of gel shown was between the Mr 34,000 and 44,000 prestained molecular weights, which were run in adjacent lanes. WT, wild-type cells; PLC-, cells transfected with PLC- sense. B, phosphorylation of I-B in the intestinal cells of either transfected or wild-type origin assessed by immunoblotting. The I-B phosphorylation (Ser 32/36) bands are from the same treatment regimes as described in A. As for its protective effects on the I-B, PLC- overexpression in transfected cells (3-µg sense clone) prevents the phosphorylation of I-B. EGF (10 ng/ml), which stabilizes I-B (A), also suppresses I-B phosphorylation in wild-type cells. Wild-type cells when exposed only to oxidant (H2O2, 30 min) exhibit increased phosphorylation of I-B. Here, separate blots were run for assessment of I-B phosphorylation than those for I-B expression levels. WT, wild-type cells; PLC-, cells transfected with PLC- sense. Shown is a representative blot.

Stabilization of I-B by PLC- Isoform Involves Prevention of I-B Phosphorylation (ser 32/36). We then determined the mechanism underlying PLC--induced I-B stabilization (i.e., decreased I-B degradation) and consequent NF-B inactivation. Because alteration in phosphorylation is a key mechanism for modulating I-B protein stability and function (Jobin et al., 1999), we surmised that reduction of I-B phosphorylation is a key mechanism for PLC--induced decreases in I-B degradation and NF-B inactivation.

I-B phosphorylation levels from both transfected and wild-type monolayers exposed to H₂O₂ are shown in Fig. 3B. PLC- expression reduced the I-B phosphorylation (Ser32/36 phospho-I-B) in transfected clones. In wild-type cells, inhibition of I-B phosphorylation was seen only by high doses (e.g., 10 ng/ml) of EGF. Oxidant, in contrast, markedly increased I-B phosphorylation in these wild-type cells. As before, transfection of empty vector was ineffective (not shown).

To further study the mechanism underlying the protective affect of PLC- on the I-B, we used immunoprecipitation analysis (Fig. 4, A and B). Thus, cells were initially immunoprecipitated with an anti-PLC- antibody and then the immune complexes were analyzed for the presence of I-B (to see whether this PLC isoform physically associates with I-B). Wild-type (resting) vehicle-treated cells did not show any association between these proteins (Fig. 4A), whereas a small amount of I-B coprecipitated with PLC- in EGF-pretreated wild-type cells. The amount of I-B coprecipitation was markedly increased in transfected clones overexpressing PLC-, demonstrating enhanced formation of a PLC-/I-B complex. Using an opposite protocol (Fig. 4B), we further corroborated the aforementioned coassociation findings. Here, an anti-I-B antibody was used and immune complexes were then analyzed for the presence of PLC-. Not surprisingly, PLC- was not detectable in the complex in wild-type (vehicle)-treated cells (indicating no coprecipitation with I-B). Stable overexpression of PLC- led to an accumulation of I-B /PLC- complex, paralleling findings in Fig. 4A. In a third protocol, we further assessed the specificity of the formation of the PLC-/I-B complex. We thus probed lysates from another PLC isoform clone, namely, the PLC-ß that exhibited no physical association with I-B (not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 4. A and B, PLC- overexpression leads to coassociation of the 145-kDa PLC- isoform and the 37-kDa I-B in intestinal cells. Note the coimmunoprecipitation of the PLC-/I-B complexes in transfected cells (3-µg sense clone) overexpressing isoform of PLC (A and B) For A, cleared cell lysates were incubated with excess (1:2000 dilution) of monoclonal anti-PLC- antibody bound to protein A beads and the immune complexes were then resolved by PAGE using the corresponding anti-PLC- or anti-I- B antibodies. Wild-type (vehicle-treated) cells display no PLC-/I-B complex. PLC- overexpressing clones or the EGF-treated cells, in contrast, exhibit the PLC-/I-B complexes (see *). For B, an opposite protocol using anti-I-B antibody was performed for immunoprecipitation, and the complexes formed were then examined. Thus, complexes seen were immunoprecipitated with anti-I-B before PAGE with the appropriate antibodies. As might be expected, a similar pattern of coassociation is found between I-B/PLC-. WT, wild-type cells; PLC-, cells transfected with PLC- sense; WB, Western blot; IP, immunoprecipitated with the shown antibody.

NF-B Inactivation in PLC--Transfected Clones Stabilizes Both the Assembly and Cytoarchitecture of F-Actin Cytoskeleton. In parallel with the suppression of oxidant-induced affects, PLC- expression conferred protection to the assembly of actin pool (Fig. 5A, percentage of polymerized actin fraction) and the architecture of actin cytoskeleton (Fig. 5B, a-c, laser confocal microscopy). Fluorescent images in Fig. 5B show that clones overexpressing PLC- (c) exhibit a preserved and smooth cytoarchitecture of the actin cytoskeleton under H₂O₂ exposure, which is comparable with the normal and intact organization of actin in control (untreated) cells (a). Wild-type cells, which were challenged with H₂O₂, show instability, beading, and collapse of the actin cytoskeleton (b). These findings parallel the stabilizing effects of PLC- against NF-B activation and I-B degradation.

View larger version (71K): [in this window] [in a new window]   Fig. 5. A, analysis of the polymerized F-actin pool (an index of actin integrity) from the PLC- overexpressing clones compared with wild-type cells. PLC- overexpression (3-µg clone) enhances F-actin assembly against oxidant (H2O2,30 min) challenge as shown by an increase in F-actin, which is comparable with the control (vehicle). In wild-type cells, in contrast, oxidant results in a large decrease in the assembly F-actin. This is prevented by EGF (10 ng/ml). F-Actin (Triton-insoluble) extracts were fractionated and processed for PAGE and subsequently analyzed for percentage of polymerized F-actin by laser densitometry. Percentage of polymerized F-actin = [(F)/(F + G)], where F + G is the total intracellular actin pool. *, p < 0.05 versus vehicle. +, p < 0.05 versus H2O2 in wild-type. &, p < 0.05 versus PLC- overexpressing PLC- cells exposed to H2O2 or pretreated with EGF before H2O2 in wild-type cells. #, p < 0.05 versus EGF (10 ng/ml) before H2O2 in wild-type cells. WT, wild-type cells; PLC-, cells transfected with PLC- sense. B, cytoarchitecture of the F-actin cytoskeleton as revealed by laser scanning confocal microscopy (LSCM) of intestinal monolayers. a, from untreated (control) cells exposed to vehicle. b, from wild-type Caco-2 cells exposed to 0.5 mM H2O2 (30-min incubation). c, from PLC- overexpressing monolayers (3-µg clone) exposed to 0.5 mM H2O2 (30-min incubation). Only in clones overexpressing PLC- (c) normal actin organization is preserved against oxidant challenge, which is indistinguishable from the control monolayers (a). F-Actin cytoskeleton in these control cells is intact and smoothly distributed at areas of cell-to-cell contact (a), whereas wild-type cells exhibit clear disruption and instability of F-actin "ring" under oxidant insult (b). Scale bar, 25 µm. Shown are representative photos.

PLC- Subcellular Distribution and Activation Correlates with Multiple Indices of NF-B Inactivation. After overexpression of the PLC- isoform (145 kDa), it is mostly distributed into the particulate cell fractions (particulate = membrane + cytoskeletal fractions) with only a small distribution in the cytosolic cell fractions (Banan et al., 2003c), indicating the activation of this isoform. Pretreatment with EGF enhances the fraction of PLC- found in the particulate fractions. In wild-type cells, PLC- is present in a mostly cytosolic distribution with a smaller pool in the particulate fractions, indicating its inactivity. EGF also increased membrane and cytoskeletal distribution of native PLC- in these wild-type cells.

Table 2 depicts the activity measurements of PLC- isoform (by in vitro assay) from immunoprecipitated particulate cell fractions. In stably transfected clones exposed to vehicle, PLC- overexpression markedly increases the activity of isoform. EGF additionally activates PLC- in these transfected clones, climbing to near maximal activation levels. Basal PLC- activity levels are seen in wild-type cells exposed to vehicle. In these wild-type cells, EGF further activates native PLC-; however, as might be expected, at lower levels compared with the transfected clones under similar conditions.

View this table: [in this window] [in a new window]   TABLE 2 Activity levels of PLC- in intestinal cells of transfected and wild-type origin Intestinal cells stably transfected with PLC- sense DNA (3 µg) were exposed to oxidant (H2O2, 0.5 mM). In corollary studies, cells transfected with a PLC- dominant negative DNA, PLCz (3 µg) were incubated with EGF before oxidant challenge. Wild-type (WT) cells (not transfected) are also shown. PLC- isoform activity was assessed in vitro as described under Materials and Methods.

Using data across all-experimental conditions, we report significant inverse correlations (e.g., r = -0.92; p < 0.05) between PLC- activity (in vitro kinase assay or optical density from the particulate fraction) and NF-B inactivation, further suggesting that activation of PLC- is critical in protection against NF-B activation. Additional robust correlations were found between either NF-B nuclear distribution or I-B degradation and the PLC- levels (r = -0.91, -0.95, respectively; p < 0.05 for each). Similarly, when markers of monolayer stability such as either actin integrity or actin assembly were correlated with the PLC- levels, other robust correlations were seen (r = 0.90, 0.89, respectively; p < 0.05 for each). We found still other supporting correlations such as those between I-B phosphorylation or I-B stabilization and PLC- activation (r = -0.88, 0.93, respectively; p < 0.05 for each), furthermore indicating that activation of PLC- isoform is important in NF-B inactivation via stabilization of I-B.

Inactivation of Native PLC- Isoform by Dominant Negative Inhibition: Prevention of EGF-Induced NF-B Inactivation and I-B Stabilization. The aforementioned findings together indicate that PLC- could play a key intracellular function in protection against oxidant-induced NF-B activation. To further assess the possible role of PLC- in EGF-mediated suppression of NF-B activation, we used stable dominant negative transfected "PLCz mutant" clones of Caco-2 cells we developed (Banan et al., 2001c). To this end, cDNA encoding a PLCz dominant negative fragment from the Z region of human PLC- 1 was used. Using this targeted molecular approach, we are capable of substantially reducing the steady-state activity levels for native PLC- isoform by approximately 99% (Table 2, 3-µg dominant clone/- 3). Indeed, EGF can no longer enhance the native PLC- isoform activity in these dominant negative PLCz mutants.

Table 1 also demonstrates the dose-dependent effects of varying amounts (1-5 µg) of PLC- dominant negative plasmid (i.e., PLCz) on prevention of EGF-induced NF-B inactivation and I-B stabilization in intestinal cells. Clone that was stably transfected with 3 µg of PLCz plasmid led to maximum suppression of EGF's effects and thus was subsequently used for other inhibition experiments.

Figure 6 shows that stable dominant negative inhibition of native PLC- activity largely prevents the protection afforded by high (protective) doses (e.g., 10 ng/ml) of EGF against NF-B subunit activation, p50 (Fig. 6A) and p65 (Fig. 6B). In contrast, this same dose of EGF completely abrogates oxidant-induced NF-B subunit activation in wild-type cells. Indeed, a large proportion of EGF-induced NF-B inactivation (89%) is dependent on the PLC-. Inactivation of PLC- by itself had no affect on basal NF-B activity.

View larger version (32K): [in this window] [in a new window]   Fig. 6. A and B, stable dominant negative inactivation of the native PLC- isoform by the "PLCz" fragment inhibits the protective (suppressive) effects of growth factor (EGF) against oxidant-induced activation of NF-B subunits p50 (A) and p65 (B). A unique PLCz mutant cell line (3-µg dominant negative clone) that almost completely lacks native PLC- activity (Table 2) was used. Monolayers were preincubated for 10 min with a high (protective) dose of EGF (10 ng/ml) and then exposed for 30 min to H2O2 (0.5 mM). Wild-type Caco-2 cells are also shown. EGF cannot inactivate NF-B subunits in the PLCz dominant negative mutants, but it does suppress NF-B in the wild-type cells. NF-B subunit p65 and p50 activities were assessed by an ELISA of nuclear extracts using a specific consensus binding site for either the p65 or p50 subunit of NF-B. *, p < 0.05 versus vehicle. +, p < 0.05 versus H2O2. &, p < 0.05 versus EGF + H2O2 in wild-type cells. WT, wild-type cells; PLCz mutant, dominant negative inhibition of native PLC- activity.

Assessment of the I-B levels (Table 1) and I-B phosphorylation (Fig. 7) from these dominant-negative clones additionally demonstrates that inactivation of native PLC- isoform largely attenuates both EGF's enhancement of I-B stabilization and reduction of I-B phosphorylation. A large proportion (80%) of EGF-induced I- B stabilization is PLC--dependent.

View larger version (48K): [in this window] [in a new window]   Fig. 7. Dominant negative inactivation of native PLC- isoform attenuates EGF's suppression of I-B phosphorylation in intestinal cells. Cell monolayers either lacking PLC- activity (3-µg PLCz mutant) or expressing native PLC- activity (wild type) were incubated with EGF before H2O2 as described in Fig. 6. The I-B phosphorylation is reduced by EGF in wild-type cells, whereas this protective effect is substantially prevented in dominant negative PLCz clones. *, p < 0.05 versus vehicle. +, p < 0.05 versus H2O2. &, p < 0.05 versus EGF + H2O2 in wild-type cells. WT, wild-type cells; PLCz mutant, dominant negative inhibition of native PLC- activity.

In parallel, immunoblotting assessment of the molecular assembly of F-actin from these same dominant negative clones further demonstrates (Fig. 8) that suppression of native PLC- also prevents protection against actin disassembly by high (protective) doses of EGF. As might be expected, EGF cannot prevent oxidant-induced actin depolymerization in these mutant clones. PLC- inactivation by itself had no affect on actin assembly.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Inhibitory effects of the dominant negative inactivation of PLC- isoform on EGF's enhancement of the assembly of F-actin cytoskeletal pool in Caco-2 cells assessed by immunoblotting. F-Actin cytoskeletal extracts from Caco-2 cells were subjected to SDS-PAGE fractionation and immunoblotted using monoclonal anti-actin antibody followed by horseradish peroxidase-conjugated secondary antibody and then autoradiographed. The F-actin (43-kDa) polymerization bands are from the same treatment conditions as described in Fig. 6. In 3-µg PLCz mutant clones, inactivation of native PLC- largely attenuates EGF's protection of F-actin assembly against oxidant insult. This is shown by a reduction of the band (lane) density for polymerized actin. In the wild-type cells, in contrast, EGF preserves the polymerized actin pool. This level of assembly is comparable with the controls, which exhibit steady-state levels of the polymerized actin. WT, wild-type cells; PLCz mutant, dominant negative inhibition of native PLC- activity.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Using monolayers of intestinal epithelium as a widely used model for gut barrier integrity, we have shown in the current investigation that 1) the 145-kDa isoform of PLC is required for EGF-mediated protection against oxidant-induced activation of NF-B and degradation of I-B as well as the instability of the actin cytoskeleton and barrier function in epithelial cells. Moreover, 2) PLC- by itself is important in protection of monolayer integrity against oxidant-induced stress and NF-B activation. 3) The molecular mechanism underlying this unique effect of PLC- isoform seems to involve the stabilization of the 37-kDa I-B protein (an endogenous inhibitor of NF-B activity).

These three conclusions are supported by independent lines of evidence. Activation of PLC-, which we reported to prevent disruption of barrier function, causes an EGF-like protection against oxidant-induced NF-B stress. Specifically, PLC- expression, which leads to constitutive activation of this isoform in intestinal cells, induces a protective cascade of alterations:

Interestingly, we found a similar trend of changes when protein kinase C isoforms ( 1 and ) were expressed (Banan et al., 2003c,f). Additionally, PLC- activation promotes the stability of the molecular dynamics of the actin cytoskeleton (increased polymerized F-actin, reduced unstable G-actin) and protects monolayer integrity. Moreover, a nonprotective dose of EGF (1 ng/ml) potentiates all measures of PLC--mediated protection against NF-B stress and its consequences. Furthermore, dominant-negative inhibition of native PLC- (by PLCz) largely attenuates all of the stabilizing effects of EGF. In these PLCz mutant clones, EGF did not decrease I-B phosphorylation, enhance I-B stabilization, promote NF-B inactivation, or increase the cytoarchitectural stability of F-actin. Finally, the high strength of the correlations among different outcome measures for PLC- and NF-B/I-B further indicate that PLC- is key to protection against NF-B activation. Accordingly, our findings are consistent with our hypothesis that activation of PLC- inhibits I-B instability, thereby suppressing NF-B activation and its deleterious consequences in intestinal cell monolayers.

Our findings using molecular biological approaches are also consistent with other studies, including our previous reports. The PLC- isoform affects numerous cellular functions in both nonepithelial and epithelial cell models (Homma and Takenawa, 1992; Rotin et al., 1992; Chen et al., 1994, 1996; Goldschmidt-Clermont, 1996). For instance, modulation of the motogenic pathway in migrating intestinal cells that is mediated by growth factors is dependent on PLC- (Chen et al., 1994; Polk, 1998). Not surprisingly, PLC- is a key signal for reorganization of other cytoskeletal components, including tubulin-based microtubules (Banan et al., 2001c) and actin remodeler gelsolin (Janmey et al., 1992; Goldschmidt-Clermont, 1996). We previously showed that PLC- is membrane bound and phosphorylated by the EGF-receptor (EGF-R) via the SH2 or phosphotyrosine domains (Banan et al., 2001c). Indeed, epithelial PLC- is the only isoform of PLC that contains SH2 and SH3 domains and that is activated by growth factors (Chen et al., 1994; Polk, 1998; Banan et al., 2003b). The Z region of human PLC- 1 (amino acids 517-901), the so-called PLCz, which includes the SH2 and SH3 domains as well as the PLC-inhibitory domains, has been shown to specifically suppress PLC- 1 and not any other PLC isoform in epithelial cells (Chen et al., 1994, 1996; Polk, 1998). More specifically, PLCz contains the SH2-SH2-SH3 domains necessary for activation of PLC- 1 by EGF-R as we and others showed (Homma and Takenawa, 1992; Turner et al., 1992; Chen et al., 1994; Banan et al., 2001c, 2003b). In the current study using this specific dominant negative fragment for PLC-, PLCz mutant expression not only prevented the activation of native PLC- but also abrogated EGF protection. Thus, it seems that activating PLC will have distinct beneficial effects on the intestinal epithelium. Our findings on the 145-kDa isoform of PLC now suggest a novel role among the PLC isoforms, namely, modulation of NF-B function via stabilization of its regulator I-B.

In epithelial cells, PLC- signal activation produces inositol 1,4,5-trisphosphate and diacyl glycerol (DAG) (Homma and Takenawa, 1992; Reynolds et al., 1993; Chen et al., 1994; Polk, 1998). DAG is a key product of PLC--mediated phosphatidylinositol bisphosphate hydrolysis, which is known to induce the downstream activation of another key signal, namely, protein kinase C (PKC) (Nishizuka, 1992; Balogh et al., 1995; Banan et al., 2001a,b,c). Indeed, using the same intestinal cell model, we showed that PKC signaling is also critical to EGF protection of barrier and cytoskeletal integrity against oxidant challenge (Banan et al., 2001a,b, 2002b). That PKC is a downstream signal from PLC- isoform in the cellular protective processes is further indicated by our previous findings that PKC activators such as OAG (1-oleoyl-2-acetyl-sn-glycerol; a synthetic version of DAG) maintain cytoskeletal and barrier function while inhibiting NF-B even in the presence of PLC inactivation (Banan et al., 2001b, 2003c,f). PKC was also shown to be downstream of PLC in other systems as well (Nishizuka, 1992; Reynolds et al., 1993; Lindmark et al., 1998; Polk, 1998). Thus, it seems that EGF protection of cytoskeletal and barrier function is mediated by PLC- and then PKC signaling.

Consistent with our current findings, hydrogen peroxide has been shown to activate NF-B through degradation of I-B in a variety of cellular models. For example, activation of NF-B induced by H₂O₂ and tumor necrosis factor- (and its effects on intercellular adhesion molecule-1 expression) has been reported in endothelial cells (Barchowsky et al., 1995; True et al., 2000). Similarly, NF-B activation induced by oxidative stress was seen in colonic circular smooth muscle cells (Shi et al., 2003) as well as in Jurkat T-cells (Brennan and O'Neill, 1995). Nevertheless, our present and previous studies on damage and protection together support a new model that a fundamental mechanism in the cascade of events that underlies growth factor-mediated protection of the GI epithelial barrier and cytoskeletal integrity against oxidant-induced injury involves direct interaction of PLC- with key elements of another, injurious, cascade of events that is initiated by oxidants. In the injurious cascade, similar to previous studies (Barchowsky et al., 1995; Brennan and O'Neill, 1995; True et al., 2000), oxidants induce I-B phosphorylation and degradation and then activate NF-B, a crucial inflammatory mediator. We have expanded on these previous studies and shown that oxidants cause cytoskeletal and barrier instability through the activation of the NF-B pathway. Furthermore, in the protective cascade, activation of PLC- suppresses I-B degradation and, in turn, inhibits NF-B activation and its injurious consequences on cytoskeletal and barrier function in intestinal cells.

Phosphorylation of I-B is generally induced by the redox-sensitive enzyme I-B kinase (I-K), which subsequently leads to the degradation of I-B and, in turn, results in activation of NF-B (Rogler et al., 1998; Jobin et al., 1999). A question that remains to be answered is how I-B phosphorylation and degradation might be decreased in our intestinal model. We now suggest two mechanisms by which the I-B phosphorylation might be decreased: 1) inactivation of I-K by increased PKC isoform activity; and 2) inactivation of I-K by increased PLC isoform activity and/or by PLC--induced PKC activation. These mechanisms require that increased PKC isoform activity leads to decreased I-B phosphorylation and its consequences on NF-B as was shown by two recent studies from our laboratory (Banan et al., 2003c,f). We showed that the classical 1 isoform of PKC (PKC- 1) is required for EGF-mediated NF-B inactivation through decreased I-B phosphorylation in intestinal cells. Similarly, the atypical isoform of PKC (PKC-) inactivates NF-B via reduced phosphorylation and degradation of I-B. Furthermore, these PKC isoforms, similar to PLC-, seem to be complexed (coassociated) with I-B, creating a novel "signalosome" modulating I-B (and NF-B) in intestinal cells. Consistent with these proposed mechanisms, we previously showed that PLC- is phosphorylated and activated after activation of EGF-R by exogenously added EGF (Banan et al., 2001c) and that EGF-R activity is required for EGF-induced I-B degradation and NF-B inactivation (Banan et al., 2003a). It remains to be seen whether I-K activity is attenuated after increased PKC (or PLC) activity and whether I-K complexes with PKC (or PLC) in this signalosome.

Our findings could be 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-induced stress is prevalent, and inactive (asymptomatic) phases when oxidative stress is low. Our series of findings suggest a new anti-NF-B defensive mechanism that could protect against the oxidative stress of NF-B and suppress initiation, perpetuation or manifestation of the IBD attacks. This concept is consistent with recent characterizations of the pathophysiology of IBD and of the proinflammatory nature of NF-B (Barnes and Karin, 1997; Schreiber et al., 1998; Banan et al., 2003a,d,e). NF-B activation is a crucial event in the inflammatory response triggered by an array of conditions, especially oxidative stress in both non-GI (Chen et al., 1995) and GI models (Rogler et al., 1998; Jobin et al., 1999; Banan et al., 2003a,c,f). NF-B activation occurs in the inflamed mucosa of patients with either ulcerative colitis or Crohn's disease (Rogler et al., 1998; Schreiber et al., 1998; Neurath et al., 1999; Banan et al., 2003d,e) where both oxidant (H₂O₂) stress and mucosal barrier hyperpermeability were also found (Keshavarzian et al., 1992, 2003; McKenizie et al., 1996; Hollander 1998; Banan et al., 2000c; Irvine and Marshall, 2000). We showed that the amount of oxidant stress and NF-B activation closely paralleled the degree of mucosal inflammation in patients with IBD (Banan et al., 2000c, 2003d,e; Keshavarzian et al., 2003). Interestingly, we also showed that the degree of mucosal cytoskeletal oxidation and instability correlated with the degree of inflammation and disease severity score in patients with IBD (Keshavarzian et al., 2003). The presence of activated NF-B has also been shown in intestinal mucosal epithelial cells from IBD patients (Rogler et al., 1998). Not surprisingly, induction of NF-B seems to be crucial to the perpetuation of the active, symptomatic phase of IBD when intestinal oxidative stress creates a vicious inflammatory cycle dependent on sustained NF-B activation, oxidant stress, cytoskeletal instability, and ultimately mucosal tissue damage. The defensive anti-NF-B effects mediated by PLC-, as we have seen in intestinal cells, could be pivotal in suppressing the continuation of inflammatory processes.

Accordingly, developing a means of restoring mucosal barrier function during conditions of oxidant stress using endogenous protective factors (e.g., EGF) or agents that trigger defensive signaling pathways (e.g., PKC and PLC) could be beneficial in the treatment of IBD. Treating with mimetics of growth factors such as EGF might be a simple and effective strategy for the treatment of IBD. For instance, mucosal barrier disruption induced by a variety of damaging conditions, including oxidant stress and NF-B activation, is prevented by EGF, independent of its known antisecretory properties (Wright et al., 1990; Bass and Luck, 1993; Banan et al., 2000a,b, 2003a). Not surprisingly, EGF is a crucial protective factor in the maintenance, growth, repair, and barrier function of gut mucosa. Previous studies have noted a marked enhancement in EGF-R immunoreactivity in the inflamed mucosa under IBD-like conditions (Wright et al., 1990). Indeed, pretreatment with EGF before the induction of IBD and daily thereafter accelerates healing of colonic mucosa in animals (Bass and Luck, 1993). Thus, EGF mimetics might synergize with the effects of agents triggering PLC- (or PKC) and/or the currently used antioxidants so that inflammatory processes are more effectively inhibited via the manipulation of both injurious and defensive intracellular mechanisms.

In summary, our findings using targeted molecular interventions to stably overexpress or inactivate PLC- demonstrate a new concept, that this "protective" PLC isoform seems to be crucial for a substantial portion of the endogenous protection of the intestinal epithelium against oxidant stress that is induced by NF-B activation. PLC- perhaps is also key to suppressing amplification and establishment of an uncontrolled, oxidant-initiated, inflammatory cascade that can be induced by free radicals and other oxidants found under pathophysiological conditions in the GI tract.

	Acknowledgements

 
We thank Dr. Allen Wells (Pittsburgh University Medical Center, Pittsburgh, PA) for generous help in providing the PLCz vector.

	Footnotes

 
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 RO1 grants (NIDDK 60511 to A.B. and NIAAA 13745 to A.K.).

DOI: 10.1124/jpet.103.062232.

ABBREVIATIONS: GI, gastrointestinal; IBD, inflammatory bowel disease; NF-B, nuclear factor-B; I-B, inhibitory-B; EGF, epidermal growth factor; PLC-, phospholipase C-; DMEM, Dulbecco's modified Eagle's medium; SH, src homology; OD, optical density; PAGE, polyacrylamide gel electrophoresis; IP, inositol phosphates; ELISA, enzyme-linked immunosorbent assay; FSA, fluorescein sulfonic acid; EGF-R, epidermal growth factor-receptor; DAG, diacyl glycerol; PKC, protein kinase C; PLCz, dominant negative inhibition of PLC-.

Address correspondence to: Dr. A. Banan, GI Physiology and Pharmacology, Section of Gastroenterology and Nutrition, Rush University of Chicago, School of Medicine, 1725 W. Harrison, Suite 206, Chicago, IL 60612. E-mail: ali_banan{at}rush.edu

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