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CELLULAR AND MOLECULAR

Isoform of Protein Kinase C Prevents Oxidant-Induced Nuclear Factor-B Activation and I-B Degradation: A Fundamental Mechanism for Epidermal Growth Factor Protection of the Microtubule Cytoskeleton and Intestinal Barrier Integrity

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

Received for publication May 2, 2003
Accepted June 11, 2003.

	Abstract

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Oxidant damage and gut barrier disruption contribute to the pathogenesis of a variety of inflammatory gastrointestinal disorders, including inflammatory bowel disease (IBD). In our studies using a model of the gastrointestinal (GI) epithelial barrier, monolayers of intestinal (Caco-2) cells, we investigated damage to and protection of the monolayer barrier. We reported that activation of nuclear factor-B (NF-B) via degradation of its endogenous inhibitor I-B is key to oxidant-induced disruption of barrier integrity and that growth factor (epidermal growth factor, EGF) protects against this injury by stabilizing the cytoskeletal filaments. Protein kinase C (PKC) activation seems to be required for monolayer maintenance, especially activation of the atypical isoform of PKC. In an attempt to investigate, at the molecular level, the fundamental events underlying EGF protection against oxidant disruption, we tested the intriguing hypothesis that EGF-induced activation of PKC- prevents oxidant-induced activation of NF-B and the consequences of NF-B activation, namely, cytoskeletal and barrier disruption. Monolayers of wild-type (WT) Caco-2 cells were incubated with oxidant (H₂O₂) with or without EGF or modulators. In other studies, we used the first gastrointestinal cell clones created by stable transfection of varying levels (1–5 µg) of cDNA to either overexpress PKC- or to inhibit its expression. Transfected cell clones were then pretreated with EGF or a PKC activator (diacylglycerol analog 1-oleoyl-2-acetyl-glycerol, OAG) before oxidant. We monitored the following endpoints: monolayer barrier integrity, stability of the microtubule cytoskeleton, subcellular distribution and activity of the PKC- isoform, intracellular levels and phosphorylation of the NF-B inhibitor I-B, and nuclear translocation and activity of NF-B subunits p65 and p50. Monolayers were also fractionated and processed to assess alterations in the structural protein of the microtubules, polymerized tubulin (S2), and monomeric tubulin (S1). Our data indicated that relative to WT monolayers exposed only to oxidant, pretreatment with EGF protected cell monolayers by 1) increasing native PKC- activity; 2) decreasing several variables related to NF-B activation [NF-B (both p50 and p65 subunits) nuclear translocation, NF-B subunits activity, I-B degradation, and phosphorylation]; 3) increasing stable tubulin (increased polymerized S2 tubulin and decreased monomeric S1 tubulin); 4) maintaining the cytoarchitectural integrity of microtubules; and 5) preventing hyperpermeability (barrier disruption). In addition, relative to WT cells exposed to oxidant, monolayers of transfected cells stably overexpressing PKC- (3.0-fold increase) were protected as indicated by decreases in all measures of NF-B activation as well as enhanced stability of microtubule cytoarchitecture and barrier function. Overexpression induced stabilization of I-B and inactivation of NF-B was OAG-independent, although EGF potentiated this protection. Approximately 90% of the overexpressed PKC- resided in particulate (membrane + cytoskeletal) fractions (with less than 10% in cytosolic fractions), indicating constitutive activation of the isoform of PKC. Furthermore, antisense transfection to stably inhibit native PKC- expression (–95%) and activation (–99%) prevented all measures of EGF-induced protection against NF-B activation and monolayer disruption. We conclude the following: 1) EGF protects against oxidant disruption of the intestinal barrier integrity, in large part, through the activation of PKC- and inactivation of NF-B (an inflammatory mediator); 2) activation of PKC- is by itself required for monolayer protection against oxidant stress of NF-B activation; 3) the mechanism underlying this novel biological effect of the atypical PKC isoform seems to involve suppression of phosphorylation and enhancement of stabilization of I-B; and 4) development of agents that can mimic or enhance PKC--induced suppression of NF-B activation may be a useful therapeutic strategy for preventing oxidant damage to GI mucosal epithelium in disorders such as IBD. To our knowledge, this is the first report that PKC- can inhibit the dynamics of NF-B and cytoskeletal disassembly in cells.

A key discovery in recent years in GI inflammation (IBD) research was recognition that a leaky and disrupted gut can cause intestinal inflammation and that maintaining a normal mucosal barrier is required for intestinal health. In animal models, for example, 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). But, the pathophysiology of mucosal barrier disruption in IBD remains poorly understood. Nonetheless, several studies, including our own, showed that chronic gut inflammation in IBD is associated with excessive amounts of oxidants (e.g., H₂O₂) and that a high level of these oxidants seems to be a key contributor to mucosal injury (Keshavarzian et al., 1992, 2003; McKenizie et al., 1996; Kimura et al., 1998; Banan et al., 2000a,d, 2001c). Oxidant-induced disruption is of clinical importance not only because oxidants are common in inflammation (e.g., they are elaborated by neutrophils that infiltrate the mucosa during inflammation) but also because they can lead to mucosal barrier dysfunction and, in turn, lead to the initiation and/or continuation of mucosal inflammation and injury (Hollander, 1992, 1998; Keshavarzian et al., 1992, 1999; Yamada et al., 1993; Hermiston and Gordon, 1995). Accordingly, understanding how gut barrier integrity can be protected against oxidative, proinflammatory conditions is of fundamental clinical and biological importance.

In our efforts to better understand endogenous defensive mechanisms, we have been investigating mechanisms underlying oxidant-induced mucosal injury and barrier disruption and protection against this injury by growth factor pathways. Our hope was to devise a rational basis for the development of potentially more effective treatment regimens for inflammatory disorders of the GI tract, especially IBD. For example, using monolayers of intestinal cells, we showed in a series of studies (Banan et al., 1999, 2000a,b, 2001a,b,d, 2002c) that cytoskeletal oxidation, disassembly, and disruption are key events in oxidant-induced barrier disruption and that growth factors (EGF or transforming growth-) prevent damage by stabilizing the cytoskeleton in large part through the activation of protein kinase C (PKC).

The PKC family, which includes at least 12 known isoenzymes, can be classified into three subfamilies (Ponzoni et al., 1993; Babich et al., 1997; Maruvada and Levine, 1999; Banan et al., 2001a,d, 2002c). The conventional PKC isoforms (, 1, 2, and ) require calcium, diacylglycerol (DAG), and phospholipid for activation, whereas the novel PKC isoforms (, , , , and µ) require only DAG and phospholipid. Activation of the third subfamily, atypical PKCs (, , and ), is independent of calcium and DAG (Cho et al., 1998; Banan et al., 2002c). As we and others have reported, intestinal epithelial cells (e.g., Caco-2) express at least 10 isoforms of PKC, including PKC-, PKC-1, PKC-2, PKC-, PKC-, PKC-, PKC-, PKC-, PKC-, and PKC- (McKenna et al., 1995; Wang et al., 1996; Banan et al., 2001a,d, 2002c). These isozymes differ in their tissue expression, intracellular distribution, substrate specificity, and activation, suggesting that each isoform may have a unique role in signal transduction (Melloni et al., 1990; Mischak et al., 1993; Persons et al., 1998; Banan et al., 2002b,c). For instance, we showed using wild-type (naive) intestinal Caco-2 cells that EGF induces the membrane association of the native isoform of PKC (PKC-) and thus considered it as a potential contributor to EGF protection of the GI epithelial barrier (Banan et al., 2001d). Using transfected clones, we then found that PKC-, an atypical (DAG)-independent isoform of PKC, is required for a substantial fraction of protection of the monolayer barrier function (Banan et al., 2002c). Despite the essential importance of the isoform of PKC to intestinal permeability, the fundamental mechanism for PKC--mediated, EGF-induced protection of monolayer barrier permeability remains poorly understood.

In other studies, we reported on the importance of nuclear factor-B (NF-B)-dependent mechanisms in oxidant-induced cytoskeletal and barrier disruption (Banan et al., 2003a,b). Indeed, NF-B activation is essential to the promotion of an inflammatory response such as in IBD (Rogler et al., 1998; Schreiber et al., 1998; Neurath et al., 1999; Banan et al., 2003b). NF-B is composed of two subunits (p50 and p65), and its activation is tightly regulated by an endogenous cytoplasmic inhibitor, I-B, which complexes with NF-B and traps it in the cytoplasm in an inactive form (Jobin et al., 1999; Moon et al., 1999). Once activated, NF-B seems to regulate several important cellular processes involved in inflammatory responses such as the up-regulation of inducible nitric-oxide synthase. For example, the amount of NF-B activation has been shown to correlate with the degree of mucosal inflammation and disease activity index in the intestinal mucosa of patients with IBD (Rogler et al., 1998; Schreiber et al., 1998; Neurath et al., 1999). Interestingly, other tissue studies in IBD patients demonstrate the presence of induced NF-B in intestinal mucosal epithelial cells (IECs) (Rogler et al., 1998). We and others also observed that a number of these NF-B-dependent events also occur in intestinal mucosa from patients with IBD (Barnes and Karin, 1997; Rogler et al., 1998; Schreiber et al., 1998; Banan et al., 2000d, 2003a,b; Keshavarzian et al., 2003). Thus, activation of NF-B is highly relevant to oxidative stress and inflammation and seems to be an important factor in tissue damage during IBD.

Accordingly, we tested the hypothesis that PKC- not only prevents oxidant-induced NF-B activation and I-B degradation but also that it is key to EGF-mediated protection against the stress of this activation through the stabilization of I-B. To this end, we used both pharmacological and targeted molecular interventions using several transfected intestinal cell lines that we recently developed. In several clones, the atypical PKC- isoform was reliably overexpressed; in the other clones, PKC- expression and activity were severely inhibited. Herein, we report novel pathophysiological mechanisms, prevention of the stress of NF-B activation and of cytoskeletal disruption under oxidant conditions, by the atypical isoform of PKC in epithelial monolayers.

	Materials and Methods

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Cell Culture. Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA) at passage 15. The utility and characterization of this cell line have been reported previously (Gilbert et al., 1991; Meunier et al., 1995; Banan et al., 1998a).

Plasmids and Stable Transfection. The sense and antisense plasmids of PKC- were constructed and then stably transfected by Lipofectin reagent (Invitrogen, Carlsbad, CA) (Banan et al., 2002a). Expression was controlled by -actin promoter.

Cultures of Caco-2 cells grown to 50 to 60% confluence were cotransfected with G-418 resistance plasmid (for colony picking) and expression plasmids encoding either sense PKC- or anti-sense PKC- by Lipofectin. Control conditions included vector alone. After transfection, cells were subjected to G-418 selection (0.6 mg/ml) over 4 weeks. Thus, colony picking (i.e., selection) was performed by G-418. Resistant cells were maintained in culture media/fetal bovine serum and 0.2 mg/ml G-418 (selection medium). Multiple clones stably over-expressing PKC- or lacking PKC- were assessed by immunoblotting and then used for experiments.

Experimental Design. First, postconfluent 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 showed previously, H₂O₂ at 0.1 to 0.5 mM disrupts microtubules and barrier integrity and activates NF-B (Banan et al., 1998a, 1999, 2000a,c, 2001a,b,d, 2003a). EGF at 10 ng/ml (but not 1 ng/ml) prevents both microtubule and barrier disruption. These experiments were then repeated using stably transfected cells. In all experiments, we assessed microtubule cytoskeletal stability (cytoarchitecture and assembly), tubulin levels, I-B distribution (cytosolic expression and degradation), I-B phosphorylation, NF-B p65 and p50 subunit activity (cytosolic levels, nuclear translocation, and activity), barrier integrity (permeability), and PKC- subcellular distribution and activity (immunoblotting and in vitro kinase assay).

Second, cell clone monolayers that were stably overexpressing PKC- were preincubated (10 min) with EGF (1 and 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.

Third, monolayers of antisense-transfected cells stably lacking PKC- protein and activity were treated with high (protective) doses of EGF and then oxidant. In corollary series of experiments, we investigated the effects of PKC- under- or overexpression on the state of 1) I-B degradation, phosphorylation, or stabilization; 2) NF-B activation or inactivation; 3) tubulin assembly and disassembly; and 4) the stability of the cytoarchitecture of the microtubule cytoskeleton.

Fractionation and Immunoblotting of PKC. Differentiated cell monolayers grown in 75-cm² flasks were processed for the isolation of the cytosolic, membrane, and cytoskeletal fractions (Banan et al., 2002a). Protein content of the various cell fractions was assessed by the method of Bradford (1976).

For immunoblotting, samples (25 µg of protein/lane) were added to a standard SDS buffer, boiled, and then separated on 7.5% SDS-PAGE (Banan et al., 2002a). The immunoblotted proteins were incubated with the primary mouse monoclonal anti-PKC- (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:1,000 dilution. A horseradish peroxidase-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR) was used as a secondary antibody at 1:4,000 dilution. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences Inc., Piscataway, NJ) and autoradiography, and subsequently analyzed by densitometry. The identity of the PKC- bands was confirmed as we described previously (Banan et al., 2002a).

Immunoprecipitation and PKC- Activity Assay. Immunoprecipitated PKC- was collected and processed for its ability to phosphorylate a synthetic peptide (Banan et al., 2002c). Briefly, after treatments, confluent cell monolayers were lysed by incubation for 20 min in 500 µl of a standard cold-lysis buffer and then immunoprecipitated by monoclonal anti-PKC- (1:2,000 dilution, in excess). The immunocomplexes were collected by centrifugation (2,500g, 5 min) in a microfuge tube and washed three times with a standard immunoprecipitation buffer. They were then washed one time with kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgCl₂, 2 mM MnCl₂, and 20 µM ATP) and resuspended in 20 µl of kinase buffer and 5 µl of 5x reaction buffer (0.25 mg/ml L--phophotidyl-L-serine and 1 mg/ml histone H1 or -peptide) plus 5 µCi of [-³²P]ATP, and subsequently incubated for 5 min at 30°C. Reactions were then stopped by the addition of 8 µlof5x sample buffer, and the samples were boiled at 95°C for 5 min before separation by 7.5% PAGE. The extent of histone H1 or -peptide phosphorylation was determined by scintillation counting of excised Coomassie Blue-stained polypeptide bands. Counts for blanks were subtracted from the sample activity. Sample activity was also corrected for protein concentration (Bradford method), and PKC- 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 (Renard et al., 2001; Banan et al., 2003a). 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 binding of NF-B to its consensus sequence was then detected using a primary anti-NF-B (p65 and p50) antibody (Santa Cruz Biotechnology, Inc.), 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 from naive and transfected cells were prepared as described above. 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%) (Jobin et al., 1999).

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 standard Western blot protocol (10% gel) (Moon et al., 1999). I-B phosphorylation was assessed by an anti-phospho-I-B (Ser 32/36). Proteins were visualized by enhanced chemiluminescence and subsequently autoradiographed.

Determination of Cell Oxidative Stress. Oxidative stress was assessed by measuring the conversion of a nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (Molecular Probes) into a fluorescent dye, dichlorofluorescein (DCF) (Banan et al., 1999, 2000b, 2001d). Briefly, monolayers grown in 96-well plates were preincubated with the membrane-permeable 2',7'-dichlorofluorescein diacetate (10 µg/ml for 30 min) before the subsequent treatments. Fluorescent signals from samples were quantitated using a fluorescence multiplate reader set at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

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 (Banan et al., 1996, 1998a,b, 1999,2000a, 2001a). Cells were subsequently processed for incubation with a primary antibody, monoclonal mouse anti--tubulin (Sigma-Aldrich, St. Louis, MO), and then with a secondary antibody (fluorescein isothiocyanate-conjugated goat anti-mouse; Sigma-Aldrich). 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., 1998a,b, 1999, 2000a, 2001a).

Microtubule (Tubulin) Fractionation and Quantitative Immunoblotting of Tubulin Assembly and Disassembly. Polymerized (S2) and monomeric (S1) fractions of tubulin were isolated using a method we described previously (Banan et al., 1998a, 1999, 2000a, 2001a). Isolated tubulin samples were flash frozen in liquid N₂ and stored at –70°C until immunoblotting. For immunoblotting, samples (5 µg of protein/lane) were placed in a standard SDS sample buffer, boiled, and then subjected to PAGE on 7.5% gels. Standard (purified) tubulin controls (5 µg/lane) were run concurrently with each run. To quantify the relative levels of tubulin, the optical density of the bands corresponding to immunolabeled tubulin were measured with a laser densitometer.

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 (Sanders et al., 1995; Kennedy et al., 1998; Banan et al., 1999, 2000a,b, 2001a,b,c). 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 and 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). Correlation 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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We initially confirmed our previous findings that intestinal cells cotransfected with cDNA encoding both G-418 resistance (for selection or colony picking) and PKC- sense stably overexpress the (72-kDa) isoform of PKC (3-fold compared with wild-type cells) and that this overexpression protects monolayer barrier integrity against exposure to oxidant challenge (Banan et al., 2002a).

Stable Overexpression of PKC- Isoform Protects against Oxidant-Induced NF-B Activation: Inhibition of the Nuclear Translocation and the Activation of Both NF-B Subunits. Because PKC- protects against oxidant-induced disruption, we surmised that this protection may be due to the suppression of oxidant-activated pathways such as the one triggered by NF-B. Accordingly, using our wild-type and transfected cell clones, we measured the nuclear translocation of NF-B subunits p65 and p50 under conditions of oxidant challenge. We also measured the nuclear activation of these subunits by a unique ELISA assessment of the cellular nuclear fractions. In wild-type cells (those not overexpressing PKC-), oxidant H₂O₂ alone resulted in a substantial activation of both p50 and p65 subunits of the NF-B (Fig. 1, A and B). The p65 subunit protein of NF-B as well as its p50 subunit was activated to an almost identical degree. Overexpression of PKC- (Z) by itself afforded protection against oxidant-induced NF-B subunit activation. Indeed, only cells stably overexpressing PKC- showed substantial suppression of NF-B activity under oxidant challenge. Such inactivation did not require the presence of growth factor EGF in the cell culture media. Although 1 ng/ml EGF did not afford significant protection against NF-B activation (of both p50 and p65 subunits) in wild-type cells, this concentration of growth factor did potentiate the NF-B inactivation observed in cells overexpressing PKC-. In wild-type cells, higher doses of EGF (10 ng/ml) were required for NF-B inactivation (Fig. 1, A and B). As might be expected, transfection of the empty vector alone did not prevent oxidant-induced NF-B activation. For example, the level of p65 subunit that was activated was 0.045 ± 0.01 (O.D. 450 nm) for vector-transfected cells exposed to vehicle, 1.21 ± 0.12% for vector-transfected cells exposed to H₂O₂ alone, and 0.49 ± 0.05% for PKC- sense-transfected cells incubated in H₂O₂. Similarly, the level of p50 subunit that was activated was 0.041 ± 0.02 for vector-transfected cells exposed to vehicle, 1.15 ± 0.09% for vector-transfected cells exposed to H₂O₂ alone, and 0.41 ± 0.08% for PKC- sense-transfected cells incubated in H₂O₂. These alterations did not seem to be caused by 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, exhibiting comparable NF-B activation.

View larger version (27K): [in this window] [in a new window]   Fig. 1. A and B, overexpression of PKC- isoform protects against oxidant-induced activation of both NF-B subunits, p50 subunit (A) and p65 subunit (B), in Caco-2 cells as determined by a unique ELISA-based assay. A novel cell clone recently developed in our laboratory (see Materials and Methods) that overexpresses PKC- by approximately 3-fold was used. Intestinal monolayers transfected to stably overexpress PKC- [Z] were incubated with growth factor (EGF) before exposure to oxidant (H2O2). Wild-type [WT] monolayers, those not overexpressing isoform, were treated in a similar manner. Transfected cells overexpressing PKC- show substantial suppression of NF-B subunit activities (p50 and p65) induced by oxidant insult. NF-B in wild-type monolayers was inactivated only by a high dose of EGF (10 ng/ml). NF-B subunit activity was assessed by a highly sensitive ELISA assay of nuclear extracts in especially coated multiwell plates that contained specific oligonucleotides containing a consensus-binding site for either the p50 subunit or the p65 subunit of the NF-B. *, p < 0.05 versus vehicle. +, p < 0.05 versus H2O2 in wild-type cells. &, p < 0.05 versus PKC- overexpressing [Z] 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. n = 6/group in all figures unless otherwise indicated.

Multiple clones of intestinal cells transfected with 1, 2, 3, or 5 µg of PKC- sense cDNA showed (Table 1) a dose-dependent suppression of NF-B activation against oxidant (H₂O₂)-induced challenge. The clone transfected with 3 µg of PKC- sense ( 3) provided maximum inhibition of NF-B activation against oxidative insult, without any loss of cell viability (0% cell death assessed by ethidium homodimer probe). Thus, we used this stable clone for overexpressing PKC- in subsequent experiments.

View this table: [in this window] [in a new window]   TABLE 1 Effects of transfection of varying amounts of PKC- sense or antisense cDNA on both the NF-B activity and I-B- levels in intestinal Caco-2 monolayers Values are means ± S.E.M. after treatments. Cells stably transfected with varying amounts of PKC- 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 PKC- antisense (1, 2, 3, or 5 µg) were treated with EGF (10 ng/ml) before exposure to oxidant. Select treatments from wild-type (WT, untransfected) cell monolayers are also shown. Monolayer NF-B activity and I-B- levels were assessed as described under Materials and Methods.

Figure 2, A and B, show representative Western blots of the alterations in NF-B subunit translocation into the cell nuclear fractions, confirming that NF-B is inactivated. For example, PKC- overexpression substantially inhibits oxidant-induced translocation of the NF-B p50 subunit (Fig. 2A) as well as its p65 subunit (Fig. 2B) into the cell nuclear fractions. This is shown by band (lane) densities that were reduced to a level close to that of the controls, indicating suppression of NF-B in cells overexpressing PKC-. As described above, only high doses of EGF (e.g., 10 ng/ml) prevented NF-B nuclear translocation in wild-type cells. In contrast, oxidant caused the translocation or shift of NF-B subunits to the nucleus in these wild-type cells, paralleling NF-B activation.

View larger version (12K): [in this window] [in a new window]   Fig. 2. A and B, representative immunoblots of NF-B subunits, both p50 (A) and p65 (B), translocation into the nuclear fractions in the intestinal cells of either wild-type or transfected origin after treatments. The p50 protein bands (A) or p65 protein bands (B) from left to right correspond to wild-type cells exposed to vehicle (a); PKC--overexpressing cells exposed to vehicle (b); wild-type cells exposed to 0.5 mM H2O2 (c); PKC--overexpressing cells exposed to 0.5 mM H2O2 (d); wild-type cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (e); PKC--overexpressing cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (f); wild-type cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (g); and PKC--overexpressing cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (h). PKC- overexpression in transfected cells by itself suppresses NF-B subunits nuclear translocation induced by oxidant challenge (lane d in both A and B). In wild-type cells, only a high dose of EGF (10 ng/ml, lane g in both A and B) inhibits NF-B nuclear translocation. The region of gel shown was between the Mr 43,000 and 75,000 prestained molecular weights, which were run in adjacent lanes. Shown is a representative blot.

Figure 3 shows the time course for changes in NF-B activation in wild-type cells under oxidative condition and its prevention in transfected cells. PKC- overexpression substantially prevented the effects of H₂O₂ on NF-B. Maximal fold increase under H₂O₂ alone is 19 for NF-B activation; this increase is largely prevented by PKC- overexpression.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Time course for the prevention of the activation of NF-B (p65 subunit) in PKC--overexpressing cells. For comparison, wild-type cells are also shown where an increase in NF-B activation is seen after exposure to H2O2. Cells were exposed to 0.5 mM H2O2 at zero time.

PKC--Induced Protection Involves Stabilization of Cytosolic I-B: Inhibition of I-B Degradation. We next probed possible mechanisms by which PKC- expression reduces oxidant-induced NF-B activation. Because oxidants such as H₂O₂ increase I-B degradation and disrupt monolayer barrier permeability (i.e., hyperpermeability) (Banan et al., 2003a), we hypothesized that inhibition of the degradation of I-B, a 37-kDa endogenous inhibitor of NF-B, might be a key mechanism for PKC--induced effects.

To this end, multiple clones of intestinal cells, which were transfected with 1, 2, 3, or 5 µg of PKC- sense plasmid, demonstrated a dose-dependent stabilization (absence of degradation) of cytosolic I-B against H₂O₂ insult (Table 1). As for NF-B inactivation, the 3-µg stable clone of PKC- sense ( 3) provided the highest protection against I-B degradation. This stabilizing clone was subsequently used.

Figure 4A shows that PKC- overexpression using the 3-µg sense-transfected clone, which protects gut barrier integrity (Banan et al., 2002a), also causes a substantial reduction in I-B degradation (67–70% less I-B degradation). This level of I-B stability is almost comparable with controls (displaying steady-state levels of I-B). These assessments were done in cytosolic fractions of both transfected and untransfected Caco-2 monolayers. In wild-type cells, this same dose of H₂O₂ causes both hyperpermeability and increases in I-B degradation. PKC--induced inhibition of I-B degradation did not require EGF. However, a low EGF concentration, 1 ng/ml, which did not by itself afford stabilization of I-B in wild-type cells, potentiated PKC--induced I-B stabilization in transfected cells. Wild-type cells, which have native levels of PKC-, required a higher dose of EGF (10 ng/ml; Fig. 4A). As expected, transfection of only the empty vector did not confer protection against oxidant-induced I-B degradation [I-B levels were 100 ± 1% (arbitrary units) for vector-transfected cells exposed to vehicle, 7.1 ± 1.2% for vector-transfected cells exposed to H₂O₂ alone, and 67 ± 3% for PKC- sense-transfected cells incubated in H₂O₂]. This also did not seem to be caused by changes in the ability of oxidants to cause I-B degradation because vector-transfected and wild-type cells responded in a similar manner to H₂O₂. Indeed, both cell types exhibited comparable I-B degradation.

View larger version (52K): [in this window] [in a new window]   Fig. 4. A, protective/stabilizing effects of PKC--overexpression against degradation of cytosolic I-B induced by H2O2 in Caco-2 monolayers after treatment regimes. In parallel, its suppressive effects on NF-B activation (Figs. 1 and 2), PKC- overexpression stabilized I-B, a 37-kDa endogenous modulator of NF-B, against oxidant challenge. In contrast, oxidant caused degradation of the levels of I-B in wild-type cells [WT]. In these same cells, EGF (10 ng/ml) stabilized the cytosolic I-B. *, p < 0.05 versus vehicle. +, p < 0.05 versus H2O2 in wild-type cells. &, p < 0.05 versus PKC- overexpressing [Z] cells exposed to H2O2 or EGF before H2O2 in wild-type cells. #, p < 0.05 versus EGF (10 ng/ml) before H2O2 in wild-type cells. B, representative Western blot for the protective effects of PKC- overexpression on stabilization of I-B levels in the cytosolic fraction of Caco-2 cell monolayers. The treatment regimes for lanes a to h were described in Fig. 2, A and B. In wild-type cells, H2O2 resulted in a large increase in the degradation (i.e., disappearance) of I-B protein (37 kDa) (lane c), whereas in PKC--overexpressing cells, this degradation is prevented (lane d). The region of gel shown was between the Mr 34,000 and 44,000 prestained molecular weights, which were run in adjacent lanes.

Figure 4B depicts a representative Western blot showing that H₂O₂ substantially increases I-B degradation levels in wild-type cells, whereas transfected cells overexpressing PKC- exhibit near steady-state levels of I-B. For example, the corresponding optical density values for control was 5,100 ± 112; for 0.5 mM H₂O₂ was 385 ± 41; and for PKC- sense-transfected cells incubated in H₂O₂ was 3,610 ± 131, indicating stabilization of I-B against oxidant-induced degradation in these -expressing cells. Transfection of only the vector, similar to its lack of effects on NF-B, was ineffective in stabilizing I-B (not shown).

PKC--Induced Stabilization of Cytosolic I-B Involves Suppression of Ser 32/36 Phosphorylation of I-B. We then probed potential mechanisms by which PKC- enhances I-B stabilization. Because phosphorylation is a mechanism for modulating protein activity and stability, we surmised that inhibition of I-B phosphorylation might be an underlying mechanism for PKC--induced I-B stability (and subsequently NF-B inactivation).

Figure 5 shows I-B phosphorylation (i.e., phospho-I-B) levels in both transfected monolayers and in wild-type monolayers exposed to H₂O₂. PKC- overexpression markedly decreases I-B phosphorylation (Ser32/36 phospho-I-B). In wild-type cells, such suppression of I-B phosphorylation was promoted only by high doses (e.g., 10 ng/ml) of EGF. Oxidant, on the other hand, causes substantial I-B phosphorylation in these wild-type cells. As expected, transfection of vector alone did not affect phosphorylation levels (not shown).

View larger version (7K): [in this window] [in a new window]   Fig. 5. Phosphorylation of I-B (Ser 32/36) in the intestinal cells of both transfected and wild-type origin assessed by immunoblotting. The treatment regimes for lanes a to h were described in Fig. 2, A and B. In parallel with its stabilizing effects on I-B (Fig. 4), PKC- overexpression suppresses phosphorylation of the I-B (lane d). EGF (10 ng/ml), which stabilizes I-B, also inhibits phosphorylation of I-B in wild-type cells (lane g). Wild-type cells exposed to H2O2 alone show increased phosphorylation of I-B (lane c). Shown is a representative blot.

Using immunoprecipitation analysis (Fig. 6, A and B), we further investigated the fundamental events underlying the protective/stabilizing affect of PKC- on I-B, directly examining whether this PKC isoform physically associates with I-B. Intestinal Caco-2 cells were immunoprecipitated with a monoclonal PKC- antibody and then these immunoprecipitates were analyzed for the presence of I-B. Compared with the resting (wild-type) vehicle-treated cells, which did not show any association between these proteins, a small amount of I-B coprecipitated with PKC- in EGF-pretreated wild-type cells (Fig. 6A). The amount of I-B coprecipitation was dramatically increased in transfected cells overexpressing PKC-, indicating increased formation of a PKC-/I-B complex. In a reverse protocol (Fig. 6B), anti-I-B antibody was used and immune complexes were analyzed for the presence of PKC-. As expected, PKC- was not detectable in the complex in wild-type vehicle-treated cells (i.e., no coprecipitation with I-B). In contrast, stable transfection resulted in an accumulation of I-B /PKC- complex, confirming the coassociation findings in Fig. 6A. To further show specificity of the formation of the PKC-/I-B complex, we also probed cell lysates from another PKC isoform clone, the classical PKC--transfected clone, which showed no such physical association (not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 6. A and B, PKC- overexpression results in coassociation (complex formation) of the atypical PKC- isoform and the I-B in intestinal cells. Coimmunoprecipitation of the PKC-/I-B complexes can be seen in transfected cells overexpressing (both A and B). A, cleared cell lysates were incubated with excess (1:2,000 dilution) anti-PKC- monoclonal antibody bound to protein A beads for 3 h and then the immune complexes were resolved by SDS-PAGE using the corresponding anti-PKC- or anti-I-B primary antibody, followed by horseradish peroxidase-conjugated secondary antibody. Note that wild-type (resting) vehicle-treated cells show no PKC-/I-B complex (lane b), whereas the PKC--overexpressing cells (or the EGF-treated cells) show the PKC-/I-B complexes (indicated by *). B, a reverse protocol using anti-I-B antibody was used for immunoprecipitation. Here, anti-I-B complexes were immunoprecipitated with anti-I-B before immunoblotting with appropriate antibodies. An identical pattern of coassociation is seen between I-B/PKC-. The bands are from cell lysate (a), wild-type cells exposed to vehicle (b), PKC--overexpressing cells exposed to vehicle (c), wild-type cells exposed to 0.5 mM H2O2 (d), PKC--overexpressing cells exposed to 0.5 mM H2O2 (e), wild-type cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (f), and PKC--overexpressing cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (g). WB, Western blot; IP, immunoprecipitated with the shown antibody.

In parallel with the suppression of oxidant-induced affects, PKC- overexpression inhibited oxidative stress as determined by reduction in the fluorescence of DCF (Fig. 7). In wild-type cells, where H₂O₂ substantially increased DCF fluorescence, oxidative stress was suppressed only by high doses (10 ng/ml) of EGF. In the absence of oxidant, we observed significantly lower levels of oxidative stress. Transfection of vector alone did not suppress oxidative stress (not shown).

View larger version (56K): [in this window] [in a new window]   Fig. 7. Protective (suppressive) effects of PKC- overexpression against the oxidative stress in cell monolayers induced by oxidant exposure assessed by the changes in DCF fluorescence intensity. In wild-type cells [WT], EGF (10 ng/ml) by itself is also capable of suppressing this oxidative stress. *, p < 0.05 versus vehicle. +, p < 0.05 versus H2O2 in wild-type cells. &, p < 0.05 versus PKC--overexpressing [Z] cells exposed to H2O2 or EGF before H2O2 in wild-type cells. #, p < 0.05 versus EGF (10 ng/ml) before H2O2 in wild-type cells.

Suppression of NF-B Activation in Transfected Cells Protects Tubulin Assembly and the Architecture of the Microtubule Cytoskeleton. Because it is known that oxidants in this intestinal model disrupt the cytoskeleton, we assessed the state of tubulin polymerization as well as microtubule cytoarchitecture. PKC- overexpression confers protection to the assembly of tubulin (Fig. 8) as well as the cytoarchitecture of the microtubule cytoskeleton (Fig. 9, a–c). For instance, confocal microscopy reveals that intestinal cells over-expressing PKC- show a normal and stellate architecture of the microtubules even after exposure to oxidant (Fig. 9c). This preserved appearance is indistinguishable from that of control (and untreated) cells (Fig. 9a), which also show an intact organization of the microtubules. In contrast, wild-type cells (not overexpressing PKC-) that are challenged with H₂O₂ exhibit instability, fragmentation, and collapse of the microtubule cytoskeleton (Fig. 9b). This protection of both the assembly and structure of microtubule (tubulin-based) cytoskeleton by PKC- overexpression parallels the protective effects of PKC- overexpression against oxidant-induced NF-B activation and I-B degradation.

View larger version (6K): [in this window] [in a new window]   Fig. 8. Representative immunoblot of the tubulin assembly in Caco-2 cell monolayers overexpressing the atypical PKC- isoform. Tubulin cytoskeleton was extracted from both wild-type and transfected monolayers and then fractionated by SDS-PAGE using monoclonal anti--tubulin antibody followed by horseradish-conjugated secondary antibody, and subsequently autoradiographed using the enhanced chemiluminescence system. The tubulin polymerization bands from left to right correspond to wild-type cells exposed to vehicle (a), PKC--overexpressing cells exposed to vehicle (b); wild-type cells exposed to 0.5 mM H2O2 (c) PKC--overexpressing cells exposed to 0.5 mM H2O2 (d), wild-type cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (e), PKC--overexpressing cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (f), wild-type cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (g), PKC--overexpressing cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (h), and tubulin standard (50 kDa) (i). PKC- overexpression in transfected cells protects normal tubulin assembly against oxidant exposure. This protection is shown by a tubulin polymerization band density that is almost comparable with that of the control (vehicle). In wild-type cells, a high dose of EGF (10 ng/ml), but not a low dose (1 ng/ml), promotes normal tubulin assembly. Shown is a representative blot.

View larger version (78K): [in this window] [in a new window]   Fig. 9. High-resolution laser scanning confocal microscopy reveals the intracellular organization of the microtubule (tubulin-based) cytoskeleton in intestinal cell monolayers. Untreated (control) cells were exposed to vehicle (a). Wild type Caco-2 cells were exposed to 0.5 mM H2O2 (b). PKC--overexpressing monolayers were also exposed to 0.5 mM H2O2 (c). Microtubules in control cells look like normal and radially dispersed structures throughout the cytosol (a), whereas wild-type cells exposed to H2O2 (b) show clear fragmentation and collapse of the microtubules. Only in cells overexpressing PKC- (c) is normal microtubule cytoarchitecture preserved against oxidant-induced damage. This normal appearance is indistinguishable from that of the control monolayers. Bar, 25 µm. Shown is a representative photomicrograph.

Constitutive Activation of the Overexpressed PKC- in Transfected Intestinal Cells Correlates with Several Different Indices of NF-B in Monolayers. Overexpressing the 72-kDa PKC- in intestinal cells leads to its distribution into mostly the particulate fractions (particulate = membrane + cytoskeletal fractions) with a much smaller distribution in the cytosolic fractions (Banan et al., 2002a), indicating the constitutive activation of the isoform (Table 2). Overexpressed PKC- isoform is "constitutively active" because achieving this intracellular distribution did not require EGF or even a PKC activator such as OAG. Pretreatment of these cells with EGF, however, enhanced the fraction of PKC- isoform in the membrane and cytoskeletal fractions, reaching near total levels for PKC-. On the other hand, in wild-type cells PKC- is found in a mostly cytosolic distribution (suggesting inactivity) with smaller pools in the membrane and cytoskeletal (particulate) fractions. As might be expected, wild-type cells incubated with EGF also show increased membrane and cytoskeletal distribution of native PKC-.

View this table: [in this window] [in a new window]   TABLE 2 Analysis of the subcellular distribution of PKC- isoform in cell fractions from both stably transfected and wild type intestinal cells Intestinal (Caco-2) cell monolayers were pretreated with epidural growth factor (EGF) followed by exposure to oxidant (H2O2) and subsequently processed for the isolation of cytosolic, membrane, and cytoskeletal (Triton X-100-insoluble) cell fractions. Constitutive activation of the overexpressed PKC- [Z] is seen in transfected cells as indicated by distribution mostly in membrane plus cytoskeletal (i.e., "particulate") cell fractions. Wild-type [WT] cells show a mainly cytosolic pool of native PKC- isoform, indicating its inactivity. In WT cells, native PKC- is shifted into the particulate fractions after high doses of EGF (e.g., 10 ng/ml). Relative subcellular levels of PKC- in various cell fractions were quantitated by measuring the anti-PKC- immunoreactivity with a laser densitometer. The density number for the membrane pool in the corresponding vehicle-treated cells was assigned an arbitrary value of 100, and all other densities were normalized to that value, reported in arbitrary units. Values are means ± S.E.M.

Figure 10 shows the activity levels of PKC- isoform (determined by in vitro kinase assay) from immunoprecipitated particulate cell fractions of Caco-2 cells, which were stably transfected with PKC- cDNA to overexpress this isoform. There is a substantial increase in the activity levels of the PKC- isoform in these transfected (vehicle-exposed) cells. As might be expected, EGF further activates PKC- in these transfected cells, reaching near maximal activation levels for this isoform. Wild-type cells exposed to vehicle, in contrast, show basal activity levels for PKC- in the particulate cell fractions. As expected in these wild-type cells, EGF further activates native PKC-, but at much lower levels compared with that of transfected cells under similar condition.

View larger version (51K): [in this window] [in a new window]   Fig. 10. Increases in PKC- activity in differentiated intestinal cells that were stably transfected with a plasmid (3 µg) encoding the atypical isoform as determined by in vitro kinase assay. Note the constitutive activation of the PKC- in these transfected cells [Z] as indicated by its high activation levels. EGF further activates PKC- in these transfected cells, reaching near maximal activation levels for this isoform. Also, note the near complete suppression of PKC- activity in Caco-2 cells that were transfected with an antisense plasmid (3 µg) to inhibit the native PKC- isoform. In these antisense-transfected cells [AS], almost complete (–99.5%) suppression of native PKC- activity is achieved. In these same cells even the addition of EGF cannot increase the atypical isoform activity. *, p < 0.05 versus corresponding vehicle. +, p < 0.05 versus H2O2 in wild-type cells. &, p < 0.05 versus PKC--overexpressing [Z] cells exposed to H2O2 or EGF before H2O2 in wild-type cells or antisense cells. #, p < 0.05 versus EGF (10 ng/ml) before H2O2 in wild-type cells or antisense cells.

Using data across all experimental conditions, we found significant inverse correlations (e.g., r = –0.90; p < 0.05) between PKC- activity (in vitro kinase assay or optical density from the particulate fraction) and NF-B inactivation, further suggesting that activation of is key in protection against oxidant-induced NF-B activation. Other robust correlations were seen when either NF-B nuclear translocation or I-B degradation were correlated with the PKC- levels (r = –0.89, –0.92, respectively; p < 0.05 for each). When other markers of stability such as either microtubule integrity or tubulin assembly were correlated with the PKC- levels, additional robust correlations were observed (r = 0.89, 0.92, respectively; p < 0.05 for each). Similarly, we found other robust correlations such as those between I-B phosphorylation or I-B stabilization and PKC- activation (r = –0.86, 0.91, respectively; p < 0.05 for each), furthermore indicating that activation of isoform is key in NF-B inactivation through stabilization of I-B.

Stable Antisense Inhibition of PKC- to Inactivate Native Isoform and Its Prevention of EGF-Induced NF-B Inactivation and I-B Stabilization. The above-mentioned findings indicate that PKC- may play an essential intracellular role in protection against NF-B activation. To independently investigate a possible role for PKC- in EGF-mediated NF-B inactivation, we used stable antisense (AS)-transfected PKC- clones of Caco-2 cells that we have recently developed. Using this unique antisense approach for PKC-, we are capable of substantially reducing the steady-state activity levels for native isoform by approximately 99.5% (Fig. 10, 3-µg clone). In these antisense cells, EGF cannot increase the native PKC- isoform activity.

Table 1 demonstrates the dose-dependent effects of varying amounts (1, 2, 3, or 5 µg) of PKC- antisense plasmid on suppression of both EGF-induced NF-B inactivation and I-B stabilization in intestinal cells. The cell clone that was stably transfected with 3 µg of antisense plasmid resulted in maximum inability of EGF to prevent oxidant-induced NF-B activation or I-B degradation. Thus, this clone was used for other inhibition experiments.

We showed that stable antisense inhibition of native PKC- activity substantially prevents the protection afforded by 10 ng/ml EGF against NF-B activation (Fig. 11, p65 subunit activity levels shown). In wild-type (naive) cells, on the other hand, this same concentration of EGF almost completely prevents oxidant-induced NF-B activation. A large percentage (65%) of EGF-induced NF-B inactivation is PKC--dependent. PKC- isoform inactivation by itself did not affect basal NF-B activity.

View larger version (48K): [in this window] [in a new window]   Fig. 11. Stable antisense inactivation of native PKC- isoform inhibits the protective (suppressive) effects of growth factor (EGF) against oxidant-induced NF-B activation. A unique antisense-transfected cell clone recently developed in our laboratory (see Materials and Methods), which almost completely lacks native PKC- activity, was grown as monolayers and subsequently pretreated with a high dose of EGF (10 ng/ml) and then exposed to 0.5 mM H2O2. Monolayer of wild-type Caco-2 cells was also treated in a similar manner. NF-B subunit (p65) activity was assessed by an ELISA-based assay of nuclear extracts using a specific consensus binding site for the p65 subunit of NF-B. *, p < 0.05 versus corresponding vehicle. +, p < 0.05 versus corresponding H2O2. &, p < 0.05 versus EGF + H2O2 in wild-type cells.

Analysis of both the I-B levels (Fig. 12A) and I-B phosphorylation (Fig. 12B) from these antisense-transfected cells additionally demonstrates that inactivation of native PKC- isoform substantially attenuates both EGF's enhancement of I-B stabilization (Fig. 12A) and reduction of I-B phosphorylation (Fig. 12B). As for NF-B inactivation, a large percentage (65%) of EGF-induced I-B stabilization seems to be PKC--dependent in intestinal monolayers.

View larger version (32K): [in this window] [in a new window]   Fig. 12. A, prevention of the protective effects of growth factor (EGF) on the stabilization of cytosolic I-B levels in intestinal cells by the stable antisense inhibition of native PKC- activity. Caco-2 cells almost totally lacking native PKC- activity were treated as described in Fig. 11. EGF cannot stabilize I-B in these antisense cells. *, p < 0.05 versus corresponding vehicle. +, p < 0.05 versus corresponding H2O2. , p < 0.05 versus EGF + H2O2 in wild-type cells. B, analysis of the suppressive effects of the antisense inactivation of PKC- isoform on EGF's prevention of I-B phosphorylation in intestinal monolayers. Conditions as in Fig. 12A. The I-B phosphorylation level is substantially diminished by EGF in wild-type cells. This protective effect is inhibited by the inactivation of native PKC- isoform using antisense inhibition. *, p < 0.05 versus corresponding vehicle. +, p < 0.05 versus corresponding H2O2. &, p < 0.05 versus EGF + H2O2 in wild-type cells.

Analysis of oxidative stress (Fig. 13) from these same antisense clones further shows that stable inactivation of the isoform prevents EGF-induced DCF fluorescence down-regulation. Furthermore, immunoblotting analysis of the state of tubulin assembly from the antisense-transfected cells demonstrates (Fig. 14) that antisense inhibition of PKC- attenuated protection against tubulin depolymerization by a high (protective) dose of EGF. Here, EGF can no longer prevent oxidant-induced tubulin disassembly. PKC- isoform inactivation by itself did not alter the assembly of tubulin.

View larger version (64K): [in this window] [in a new window]   Fig. 13. Antisense inhibition of PKC- activity prevents the protective effects of EGF on the down-regulation of oxidative stress (DCF fluorescence intensity) in intestinal cells. Conditions as in Fig. 11. *, p < 0.05 versus corresponding vehicle. +, p < 0.05 versus corresponding H2O2. &, p < 0.05 versus EGF + H2O2 in wild-type cells.

View larger version (77K): [in this window] [in a new window]   Fig. 14. Immunoblotting analysis showing the suppressive effects of the antisense inactivation of PKC- isoform on EGF's enhancement of tubulin assembly in Caco-2 cells. This stable inhibition of activity attenuates EGF-mediated enhancement of tubulin polymerization. Cells either lacking PKC- activity (antisense-transfected) or expressing native PKC- activity levels (wild type) were incubated with EGF before H2O2. Tubulin fractions were extracted from intestinal cells and then processed for immunoblotting and subsequently analyzed with a laser densitometer as described under Materials and Methods. Percentage of polymerization of tubulin = [(S2)/(S2 + S1)], where S2 + S1 is the total cellular tubulin pool. *, p < 0.05 versus corresponding vehicle. +, p < 0.05 versus corresponding H2O2. &, p < 0.05 versus EGF + H2O2 in wild-type cells.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
We believe our current investigation on the role of the atypical isoform of PKC in the suppression of NF-B and stabilization I-B in cells is both novel and significant because 1) it is of fundamental clinical and biological value to establish the idea that unique isoforms of PKC play key roles in endogenous protective mechanisms of cells against oxidant stress of NF-B activation; and 2) a greater understanding of how to prevent (e.g., by PKC-) the disruption of the intestinal mucosal barrier under pathophysiological conditions of NF-B induction should lead to the development of new therapeutics for inflammatory diseases of the GI tract that are due to oxidative injury after NF-B activation.

In the present study, using monolayers of intestinal epithelial cells as a well established model for gut barrier integrity, we demonstrated that the 72-kDa isoform of PKC is required for EGF-mediated protection against oxidant-induced NF-B activation and I-B degradation and the consequent injury to the integrity of the microtubule (tubulin-based) cytoskeleton and intestinal epithelial barrier. A second conclusion is that PKC- by itself seems to be key in protection of cell monolayer against the stress of NF-B activation. The mechanism for this novel effect of PKC- isoform seems to be the stabilization of the 37-kDa endogenous modulator of NF-B, namely, I-B. To the best of our knowledge, this is the first time this mechanism has been described in cells. These conclusions are based on several independent lines of evidence as discussed below.

First, overexpression and activation of PKC-, which we have reported to prevent oxidant-induced barrier hyperpermeability, induces an EGF-like protection against oxidant-induced NF-B activation. PKC- activation evokes a cascade of alterations that are consistent with the noted conclusions, including reduction of I-B phosphorylation, stabilization of I-B levels, and suppression of both NF-B (p65 and p50 subunits) nuclear translocation and activation. This protection seems to require expression and constitutive activation of the isoform. Second, overexpression of PKC- inhibits the oxidant injury to the tubulin (50-kDa) protein of the microtubule cytoskeleton. This expression of PKC- increases the stability of polymerized tubulin and preserves normal-looking microtubule cytoskeleton (a protective phenomenon known to be key in the maintenance of cell monolayer integrity). Third, a low, nonprotective concentration of EGF potentiates all measures of PKC--mediated protection against NF-B activation. For example, transfected cells that overexpress PKC- are more sensitive to the 1 ng/ml concentration of EGF. Fourth, antisense to PKC-, which causes almost complete inactivation of native PKC- (by 99.5%), substantially prevents the protective ability of EGF to suppress NF-B activation, tubulin instability, and microtubule disruption. EGF was also unable to reduce I-B phosphorylation, stabilize I-B levels, or decrease DCF fluorescence in these antisense-transfected cells. Fifth, PKC- activation quantitatively correlates with the changes in several different outcomes, indicating protection against NF-B activation.

Using both transfected and wild-type cells, we found correlations between PKC- isoform activation and protection against oxidant-induced NF-B activation (r = –0.90; p < 0.05) as well as several other key parameters. These included protection against oxidant-induced I-B degradation and PKC- activation (r = –0.92; p < 0.05), NF-B nuclear translocation and PKC- activation (r = –0.89; p < 0.05) and DCF fluorescence levels and PKC- activation (r = –0.90; p < 0.05). Similar correlations are reached when either tubulin assembly (increase S2 polymer pool) and PKC- activation (r = 0.92; p < 0.05) or percentage of normal microtubules and PKC- activation (r = 0.89; p < 0.05) are used. Furthermore, protection against oxidant-induced I-B phosphorylation and PKC- activation (r = –0.86; p < 0.05) as well as the I-B levels and PKC- activation (r = 0.91; p < 0.05) provide other consistent correlations. The high strength of these correlations indicates that PKC- activation is essential to protection against NF-B activation. In this view, activation of PKC- leads to the stabilization of I-B levels. Overall, our findings individually and together support our hypothesis that a crucial mechanism in the cascade of events that underlies EGF-induced protection of the GI tract against oxidant-induced damage involves direct interaction of PKC- with elements of another damaging cascade of events that is set in motion by oxidants. In the damaging cascade, oxidants cause I-B phosphorylation and degradation and activation of NF-B, an inflammatory mediator, which leads to cytoskeletal and barrier disruption. In the protective cascade, activation of PKC- prevents I-B degradation, thereby preventing NF-B activation and its damaging consequences. Other isoforms of PKC may also be involved in suppression of NF-B by EGF; however, our findings indicate that this suppression is mediated, in substantial part, through the atypical PKC- isoform.

The findings of this report using targeted molecular interventions are consistent with known biochemical properties of PKC isoforms. For example, PKC- is known to be an "atypical" isoform of PKC that acts independently of calcium and PKC stimulators (such as OAG), and the actions of PKC- that we observed were consistent with its known biological properties. All PKCs consist of N-terminal regulatory domains and C-terminal catalytic domains (which are separated by a flexible hinge region) (Goodnight et al., 1995; Gopalakrishna and Jaken, 2000; Banan et al., 2001b, 2002a,c). In resting cells, most PKCs are mainly found in an inactive conformation. This is maintained by an intramolecular interaction between an autoinhibitory sequence (i.e., the pseudosubstrate on the N terminus) in the regulatory domain plus the substrate-binding region of the catalytic domain (Gopalakrishna and Jaken, 2000). In this inactive phase, PKC is mainly distributed in the soluble (cytosolic) fraction and only loosely bound to membrane components. Regulatory domains of PKC isoforms vary from one subfamily to the next as well as among individual isoforms within a given subfamily (Mullin et al., 1998; Gopalakrishna and Jaken, 2000; Banan et al., 2002a). For example, OAG (or DAG) binding sites are present in the regulatory ("zinc finger") domain of the classical PKC isoform (e.g., 1) but are absent from the regulatory domain of the atypical PKC isoforms such as (Banan et al., 2001b, 2002a). Not surprisingly, PKC- has very low affinity for OAG (DAG) at its zinc finger domains (Gopalakrishna and Jaken, 2000; Banan et al., 2002a). In fact, the atypical PKC- operates independently of traditional PKC activators (e.g., OAG or TPA) (Banan et al., 2002a). Similarly, atypical PKC isozymes and do not respond to phorbol esters or OAG (Gopalakrishna and Jaken, 2000). In contrast, OAG and TPA have been shown to induce activation of classical isoforms of PKC, such as 1 (Goodnight et al., 1995; Banan et al., 2001b, 2002b).

It is therefore not surprising that most cells express more than one type of PKC isoform, and thus differences among isozymes with respect to activation conditions, and subcellular locations suggest that individual PKC isoforms have distinct activation mechanisms as well as mediate distinct biological processes (Melloni et al., 1990; Mischak et al., 1993; Ponzoni et al., 1993; Housey et al., 1998; Banan et al., 2002a,b,c). For example, we recently reported that the "classical" 1 (78-kDa) isoform of PKC is required for protection through the normalization of Ca²⁺ homeostasis, indicating that this isoform performs a unique protective task in cells (Banan et al., 2001b, 2002b). In another study, we showed the disruptive effects of activation of the "novel" (75-kDa) isoform of PKC on the promotion of nitric oxide-mediated injury in colonocytes (Banan et al., 2002c). Also, a recent immunofluorescent study suggested that PKC- seems to be involved in tumor necrosis factor--induced injury in intestinal (IEC-18) cells (Chang and Tepperman, 2001). Thus, it seems that activating or mimicking different isoforms of PKC will have distinct beneficial (or damaging) effects on the epithelium. Our new findings on the 72-kDa isoform of PKC, we believe, suggests a new pathophysiological role among the atypical subfamily of PKC isoforms in intestinal cells, i.e., protection against oxidant stress of NF-B activation through the stabilization of its modulator I-B.

Although our data indicate that a significantly large portion of EGF protection against oxidant insult is mediated through PKC-, it should be noted that other PKC isoforms may also contribute to the protective effects of EGF. For example, we previously showed in Caco-2 intestinal cells (Banan et al., 2002a, 2001a,b) that EGF induces the activation of both PKC-1 and PKC- isoforms and therefore considered both isoforms as contributors to EGF-afforded protection. Subsequently, using transfected cells that either stably overexpressed PKC-1 or could not express PKC-1, we reported (Banan et al., 2001b) that PKC-1, a conventional or classical (DAG)-dependent isoform of PKC, was necessary for a substantial fraction (approximately 50%), but not all, of EGF protection. In other studies, we showed that EGF-induced protection against oxidant injury also depends on activation of the isoform of PKC (approximately 50%), but without the requirement for DAG (Banan et al., 2002a). Based on percentage of mediation of protection, it is reasonable to speculate that activation of both 1 and isoforms of PKC can account for 100% of EGF-induced protective effects (e.g., on NF-B) in our intestinal model.

Our findings and our conclusions are potentially relevant for developing new treatment strategies for IBD. They suggest a novel anti-NF-B defensive mechanism that might protect against stress of NF-B activation and either prevent initiation, continuation, or manifestation of the IBD attack. This defensive mechanism is seen in the ability of PKC- to prevent oxidant-induced disruption through suppression of NF-B activation and of I-B degradation. The potential therapeutic use of this anti-NF-B mechanism is consistent with the current characterizations of the pathophysiology of the inflammation, in general, and IBD, in particular. NF-B is a key factor in the inflammatory response triggered by an array of molecules such as phorbol esters, cytokines, double-stranded RNA, cAMP, oxidative stress, and some viral transactivators (Baeuerle and Henkle, 1994; Barne and Karin, 1997; Banan et al., 2003a). For instance, activation of NF-B by immune cytokines and oxidant stress seems to be essential in the promotion of an inflammatory response in non-GI cell models (Menon et al., 1993; Rogler et al., 1998; Tang and Leppla, 1999) as well as the GI models (Jobin et al., 1999; Moon et al., 1999; Banan et al., 2003a). 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; Neurath et al., 1999; Tang and Leppla, 1999), where high levels of oxidants, including H₂O₂, as well as loss of mucosal barrier integrity have been reported (Hollander 1992, 1998; Keshavarzian et al., 1992, 2003; McKenizie et al., 1996; Banan et al., 2000d). Here, the amount of NF-B activation correlated with the degree of mucosal inflammation and disease activity index. Interestingly, the presence of induced NF-B has also been shown in IECs from IBD patients (Rogler et al., 1998). We have also shown (Banan et al., 2000d, 2003b; Keshavarzian et al., 2003) that NF-B activation occurs in intestinal mucosa from patients with IBD. This activation is thought to be especially important in the transitions from the inactive to active (flare up) phases of inflammation in IBD in which intestinal oxidants and proinflammatory molecules periodically create a vicious cycle that can lead to sustained NF-B activation, oxidant stress, hyperpermeability, and inflammation, and consequently to mucosal tissue damage. The protective anti-NF-B effects of the PKC-, such as the ones we have found in cells, could play a pivotal role in preventing the establishment of such a vicious inflammatory cycle.

In summary, our findings using the first GI cells stably overexpressing or underexpressing "protective PKC isoform" demonstrate an original concept that PKC- seems to be responsible for a substantial portion of protection of the intestinal epithelium against oxidant stress induced by NF-B activation and perhaps is key to preventing amplification and perpetuation of an uncontrolled, oxidant-induced inflammatory cascade that can be ignited by free radicals and other oxidants present in the GI tract. This new knowledge may prove useful because enhancing the activity of protective PKC isoform through activation of specific PKCs or using PKC mimetics should lead to modern therapeutic avenues 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 two National Institutes of Health R01Grants (National Institute of Diabetes and Digestive and Kidney Diseases 60511 to A.B.; National Institute on Alcohol Abuse and Alcoholism 13745 to A.K.). Portions of this work were presented as an "abstract of distinction" during the annual meeting of the American Gastroenterological Association (Digestive Disease World), May 2003.

DOI: 10.1124/jpet.103.053835.

ABBREVIATIONS: GI, gastrointestinal; IBD, inflammatory bowel disease; EGF, epidermal growth factor; PKC, protein kinase C; DAG, diacylglycerol; NF-B, nuclear factor-B; I-B, inhibitor I-B; IEC, intestinal mucosal epithelial cell; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; DCF, dichlorofluorescein; FSA, fluorescein sulfonic acid; [Z], cells transfected with PKC- sense; OAG, 1-oleoyl-2-acetyl-glycerol.

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

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