INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE GASTROINTESTINAL (GI) epithelium is a highly selective permeability barrier that normally permits the absorption from the lumen of nutrients, water, and electrolytes but prevents the passage of harmful proinflammatory and toxic molecules into the mucosa and the systemic circulation. Disruption of GI barrier integrity, in contrast, can allow the penetration of normally excluded luminal substances (e.g., immunoreactive antigens, endotoxin) into the mucosa and can lead to the initiation or continuation of inflammatory processes and mucosal damage (4, 36, 43, 44). Indeed, loss of mucosal barrier integrity has been implicated in the pathogenesis of multiple organ system dysfunction, inflammatory bowel disease (IBD), necrotizing entercolitis, ethanol- and nonsteroidal anti-inflammatory drug-induced chemical injury, and a variety of other GI disorders as well as several systemic disorders (e.g., alcoholic liver disease) (4, 36, 43, 44). The underlying difficulty in managing these inflammatory disorders is due, in large part, to our limited understanding of their pathophysiology and lack of effective preventive strategies.

Although the pathophysiology of mucosal barrier dysfunction in these disorders remains poorly understood, several studies (4, 5, 10, 12, 17, 35, 36, 44, 45, 48), including our own, have shown that chronic gut inflammation is associated with oxidative stress and that this stress appears to be a key cause of injury. Oxidative stress is of substantial clinical importance not only because oxidants are common in inflammation, but also because they can lead to mucosal barrier hyperpermeability and, in turn, to the initiation and/or perpetuation of mucosal inflammation and injury (35, 36, 43, 44). A major advance in recent years in GI inflammation (IBD) research was recognition that a leaky gut can cause intestinal inflammation and that maintaining/protecting a normal barrier function is key to intestinal health. In animal models, for example, intestinal barrier hyperpermeability induced by the injection of bacterial endotoxin into the mucosa can elicit an oxidative and inflammatory condition similar to IBD (66). Moreover, transgenic mice with a leaky gut exhibit symptoms of intestinal inflammation (34). Accordingly, understanding how GI barrier integrity is protected under oxidative, proinflammatory conditions is of fundamental clinical and biologic importance.

We have been investigating the mechanisms underlying oxidant-induced injurious pathways and growth factor-mediated protective pathways against that injury (and the GI barrier dysfunction) to devise a rational basis for potentially more effective treatment regimens for inflammatory disorders of the GI tract. It was shown (4, 5, 7-11) that cytoskeletal disassembly and disruption are key events in this oxidative injury and that growth factors [epidermal growth factor (EGF) or transforming growth factor (TGF)-] prevent damage by stabilizing the cytoskeleton in large part through the activation of protein kinase C (PKC). For example, it was reported (5, 7-9, 11) that maintaining an intact microtubule cytoskeleton is required for protection of intestinal barrier integrity by EGF via PKC.

The PKC family, which includes at least 12 known isoenzymes, can be classified into three subfamilies based on differences in sequence homology and cofactor requirement (2, 7-9, 37, 47, 50, 52, 54, 55). The conventional (or classic) PKC isoforms (, ₁, ₂, ) require calcium, diacylglycerol (DAG), and phospholipid for their activation, whereas the novel PKC isoenzymes (, , , , µ) are calcium independent but require DAG and phospholipid. Activation of the third group, atypical PKC isoforms (, , ), is independent of both calcium and DAG (8, 25). Intestinal epithelial cells, including Caco-2 cells, express at least six isoforms of PKC: PKC-, -₁, -₂, -, -, and - as we and others have reported (1, 7-9, 18, 25, 49, 65). These isoforms differ in their activation, tissue expression, intracellular distribution, and substrate specificity, suggesting that each isozyme has a unique, nonredundant role in signal transduction (37, 47, 50, 52, 54, 55).

The involvement in protective mechanisms by PKC in the GI epithelium as it was originally reported was a novel finding (7, 9). Banan et al. (9) showed using wild-type (WT) Caco-2 intestinal cells that EGF induces the membrane translocation of the native PKC- isoform and therefore considered it as a possible contributor to EGF-mediated protection of the GI epithelial barrier. To address this possibility, we developed several unique cell lines, some clones stably overexpressing PKC-, the other clones underexpressing PKC-. Banan et al. (8) found that PKC-, an atypical, DAG-independent isoform of PKC, is required for a substantial fraction of EGF-mediated protection of the monolayer barrier function. Also, PKC--mediated protection was DAG independent, because overexpression by itself afforded substantial protection. Despite the critical importance of the -isoform of PKC, the fundamental mechanism for the protection afforded by PKC- (and EGF) remains unknown.

Other studies (10, 12) reported on the importance of the inducible nitric oxide (NO) synthase (NOS; iNOS)-dependent mechanisms in the underlying cause of oxidant-induced intestinal cytoskeletal and barrier disruption. The original concept was demonstrated that specific iNOS and NO/ONOO-mediated nitration and oxidation of key cytoskeletal protein subunits and the disruption that is caused to cytoskeletal networks lead to the loss of GI barrier integrity. Indeed, overproduction and uncontrolled generation of iNOS-derived reactive nitrogen metabolites (e.g., NO, ONOO) have been proposed by several recent studies (16, 39, 42, 56, 60), including our own, to be an important factor in tissue damage during inflammation, including IBD. For example, Banan et al. (16) and Keshavarzian et al. (42) showed that a number of these oxidative reactions, including cytoskeletal nitration and oxidation, also occurs in intestinal mucosa from patients with IBD.

In view of the aforementioned considerations, we hypothesized that PKC- not only prevents oxidant-induced iNOS upregulation and its injurious consequences, but also it is key to EGF-mediated protection of microtubule cytoskeleton and intestinal barrier integrity against the oxidative stress of this upregulation. To this end, we used both pharmacological and targeted molecular interventions employing several unique transfected intestinal cell lines we have developed. In several clones, the atypical isoform PKC- was reliably overexpressed; in the other clones, PKC- expression was inhibited. Herein, we report novel biological functions, i.e., prevention of the oxidative stress of iNOS induction and of cytoskeletal protein oxidation, by the atypical -isoform of PKC.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell culture. Caco-2 cells were obtained from ATCC (Rockville, MD) at passage 15. This cell line was chosen for our studies because they form monolayers that morphologically resemble small intestinal cells, with defined apical brush borders, tight junctions, and a highly organized microtubule network on differentiation (14, 29, 51). Caco-2 cells also form monolayers that can be studied for weeks rather than just days, as is typical of most fresh in vitro preparations (38, 41, 63). This allowed us to measure alterations in intestinal barrier integrity. In addition, Caco-2 cells closely resemble normal intestinal cells in that they express intestinal hydrolases such as sucrase-isomaltase and alkaline phosphatase. Furthermore, these cells are similar to native intestinal epithelial cells in that they have receptors for prostaglandins, growth factors, vasoactive intestinal peptide, low-density lipoprotein, insulin, and specific substrates such as dipeptides, fructose, glucose, hexoses, and vitamin B₁₂ (14, 29, 51). Accordingly, this cell line provides a suitable in vitro model for our studies. The utility and characterization of this cell line has been previously reported (51).

Plasmids and stable transfection. The sense and antisense plasmids of PKC- were constructed and then stably transfected by Lipofectin reagent (GIBCO-BRL) as previously described (7, 8, 25). Expression was controlled by -actin promoter. The antisense PKC- plasmid (p-actin SP-As-PKC-) was constructed by ligating the 2.3-kb EcoRI fragment of PKC- cDNA from pJ6-PKC- into the unique EcoRI sites of the p-actin SP vector (8, 25). The antisense orientation of the plasmid was confirmed by SamI restriction digestion. Multiple clones stably overexpressing PKC- or lacking PKC- were assessed by immunoblotting and were plated on Biocoat Collagen I cell culture inserts (Becton Dickinson, Bedford, MA) and subsequently used for experiments.

Experimental design. First, postconfluent monolayers of WT cells were preincubated with EGF (1-10 ng/ml) or isotonic saline for 10 min and then exposed to oxidant (H₂O₂, 0.5 mM) or vehicle (saline) for 30 min. As previously shown (4, 5, 7, 9, 12, 14, 17), H₂O₂ at 0.5 mM disrupts microtubules and barrier integrity and upregulates iNOS. EGF at 10 ng/ml (but not 1 ng/ml) prevents both microtubule and barrier disruption. These experiments were then repeated using transfected cells. In all experiments, microtubule stability (cytoarchitecture, tubulin assembly/disassembly), barrier integrity, PKC- subcellular distribution, iNOS activity, NO levels, reactive nitrogen metabolites (RNM)/RNM levels (e.g., ONOO), oxidative stress dichlorofluorescein fluorescence (DCF), tubulin nitration (nitrotyrosination), and tubulin oxidation (carbonylation) were assessed.

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 as previously described (7-9). Protein content of the various cell fractions was assessed by the Bradford method (21). For immunoblotting, samples (75 µg protein/lane) were added to a standard SDS buffer, boiled, and then separated on 7.5% SDS-PAGE (7, 8). The immunoblotted proteins were incubated with the primary mouse monoclonal anti-PKC- (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:2,000 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 (ECL; Amersham, IL) and autoradiography (e.g., 1 h at 20°C) and subsequently analyzed by densitometry. The exposure times were adjusted to ensure linear responses. Under these immunological detection conditions, the chemiluminescence assay was linear between 25 and 100 µg of total protein. The identity of the PKC- band was confirmed as previously described (8). We also confirmed that overexpression of PKC- or antisense inhibition of PKC- expression did not affect the relative expression levels of other PKC isoforms, nor did it injure the Caco-2 cells.

Assay of NOS activity. Conversion of L-[³H]arginine (Amersham, Arlington Heights, IL) to L-[³H]citrulline was measured in the cell homogenates by scintillation counting. Experiments in the presence of NADPH, without Ca²⁺ and with 5 mM EGTA, determined Ca²⁺-independent NOS (iNOS) activity (10, 12).

Western blot of the level of iNOS. After treatments, the cells were washed once with cold PBS, scraped into 1 ml of cold PBS, and harvested in a standard antiprotease cocktail. For immunoblotting, samples (25 µg protein/lane) were separated on 7.5% SDS-PAGE. Membranes were visualized by ECL and autoradiography (10, 12).

Chemiluminescence Analysis of NO. NO production was assessed by a unique chemiluminescence procedure (10, 12). Briefly, cells were homogenized and the endogenous nitrate (NO) and nitrite (NO), the metabolic degradation products of NO, were then reduced to NO using vanadium (III; Sigma, St. Louis, MO) and HCl at 90°C before the measurement of NO concentration by a Seivers NOA 280 analyzer (Sievers, Boulder, CO). NO was expressed in micromoles per liter and calculated by comparison to the chemiluminescence of a standard solution of NaNO₂. The absolute NO values were reported as micromoles per 1 × 10⁶ cells.

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 (4, 10, 12). Fluorescent signals from samples were quantitated using a fluorescence multiplate (excitation = 485 nm, emission = 530 nm). The dependence of the assay on ROM production (e.g., H₂O₂ or · O generation) was shown as previously reported by adding either an active H₂O₂ oxidant scavenger, catalase, or active superoxide radical scavenger [superoxide dismutase (SOD)] or as control conditions either an inactive H₂O₂ or superoxide scavenger (heat-inactivated catalase or SOD, respectively) (4, 10, 12).

Immunofluorescent staining and high-resolution laser scanning confocal microscopy of microtubules. Cells from monolayers were fixed in cytoskeletal stabilization buffer and then postfixed in 95% ethanol at 20°C as previously described (4, 5, 9, 13-15). Cells were subsequently processed for incubation with a primary antibody, monoclonal mouse anti--tubulin (Sigma) and then with a secondary antibody (FITC-conjugated goat anti-mouse; Sigma). After being stained, cells were observed with an argon laser ( = 488 nm) using a ×63 oil immersion plan-apochromat objective, numerical aperature 1.4 (Zeiss, Germany). The cytoskeletal elements were examined in a blinded fashion for their overall morphology, orientation, and disruption (4, 5, 9, 13).

Microtubule (tubulin) fractionation and quantitative immunoblotting of tubulin assembly and disassembly. Polymerized (S2) and monomeric (S1) fractions of tubulin were isolated using a series of extraction and ultracentrifugation steps as described (4, 5, 9, 14). 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) tubulin loading controls (5 µg/lane) were run concurrently with each run. To additionally verify equal loading, blots were routinely stained with 0.1% India ink in Tris-buffered saline-Tween 20 (TBST) buffer. To quantify the relative levels of tubulin, the optical density of the bands corresponding to immunoradiolabeled tubulin were measured with a laser densitometer.

Immunoblotting determination of protein tubulin oxidation and tubulin nitration. Oxidation and nitration of the microtubule (tubulin) cytoskeleton were assessed, respectively, by measuring protein carbonyl and nitrotyrosine formation using a unique method (5, 10, 12). To avoid unwanted oxidation of tubulin samples, all buffers contained 0.5 mM DTT and 20 mM 4,5-dihydroxy-1,3-benzene sulfonic acid (Sigma). Processing and film exposure were as in a standard Western blot protocol (5, 10, 12). The relative levels of oxidized or nitrated tubulin were then quantified by measuring, with a laser densitometer, the optical density (OD) of the bands corresponding to anti-DNP (carbonylation) or antinitrotyrosine (nitration) immunoreactivity. Immunoreactivity was expressed as the percentage of carbonyl or nitrotyrosine formation (OD) in the treatment group compared with the maximally oxidized or nitrated tubulin standards, which also served as loading controls. These tubulin loading controls (5 µg/lane) were run concurrently with corresponding treatment groups. To further verify equal loading of lanes, blots were routinely stained with 0.1% India ink in TBST buffer.

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 we and others have described (4, 5, 7, 9-11, 38, 41, 58, 63). After treatments, fluorescent signals from samples were quantitated by a fluorescence multiplate reader (FL 600, BIO-TEK Instruments).

Statistical analysis. Data are presented as means ± SE. All experiments were carried out with a sample size of at least six observations per treatment group. Statistical analysis comparing treatment groups was performed using analysis of variance followed by Dunnett's multiple-range test (32). Correlational analyses were done using the Pearson test for parametric analysis or, when applicable, the Spearman test for nonparametric analysis. P values <0.05 were deemed statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We initially confirmed our earlier preliminary finding that intestinal cells cotransfected with cDNA encoding both G-418 resistance (for selection) and PKC- sense stably overexpress the -isoform (72 kDa) of PKC (~2.9-fold compared with WT cells) and that this overexpression protects monolayer barrier integrity against exposure to oxidant challenge (8). Overexpression of PKC- at these levels caused no cellular toxicity (0% cell death assessed by ethidium homodimer probe) (8). In the current investigation, using both pharmacological and molecular biological interventions, we studied the underlying mechanism by which PKC- protects.

Stable overexpression of PKC- isoform protects against oxidative damage to the cytoskeleton: inhibition of both tubulin nitration and oxidation. Because PKC- protects against oxidant-induced injury, we surmised that this protection may be due to the inhibition of oxidant-activated pathways. Thus, with the use of our WT and transfected cells, we measured the "footprints" of RNM formation, nitrotyrosine moieties, under conditions of oxidant challenge. We also simultaneously measured oxidation footprints by assessing the carbonylation levels. This was done by sequentially fractionating and purifying the 50-kDa tubulin molecule, the structural protein of the microtubule cytoskeleton, from cell monolayers and subsequently immunoblotting these fractions. In WT cells (those not overexpressing PKC-), oxidant H₂O₂ alone resulted in substantial levels of nitration and oxidation of the tubulin cytoskeleton (Fig. 1A). In contrast, overexpression of PKC- by itself afforded protection against oxidant-induced tubulin nitration and tubulin carbonylation compared with those in WT cells. Indeed, only cells stably overexpressing PKC- were protected against oxidant-induced nitration and oxidation injuries. Protection 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 tubulin nitration or oxidation in WT cells, this concentration did potentiate the protection observed in cells overexpressing PKC-. In WT cells, higher doses of EGF (10 ng/ml) were required for protection (Fig. 1A). As expected, transfection of only the vector alone did not confer protection against oxidation and nitration. For instance, the percentage of tubulin that was nitrated was 0% for vector-transfected cells exposed to vehicle, 0.72 ± 0.05% for vector-transfected cells exposed to H₂O₂ alone, and 0.06 ± 0.08% for PKC- sense transfected cells incubated in H₂O₂. Similarly, the percentage of tubulin that was carbonylated was 0% for vector-transfected cells exposed to vehicle, 0.64 ± 0.04% for vector-transfected cells exposed to H₂O₂ alone, and 0.11 ± 0.06% for PKC- sense transfected cells incubated in H₂O₂ (all ratios were normalized to a nitrated or oxidized tubulin standard loading control run concurrently). These oxidative alterations did not appear to be caused by changes in the ability of oxidants to cause oxidation/nitration as vector-transfected cells and WT cells responded in a similar fashion to H₂O₂, exhibiting comparable tubulin oxidation.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   A: overexpression of PKC- protects against both oxidant-induced nitration (nitrotyrosination) and oxidation (carbonylation) injury to the tubulin cytoskeleton of Caco-2 monolayers assessed by immunoblotting. A novel sense transfected cell line previously developed in our laboratory (see MATERIALS AND METHODS) that overexpresses PKC- by 2.9-fold was used. These intestinal monolayers stably overexpressing PKC- or wild-type (WT) cells were incubated with growth factor [epidermal growth factor (EGF)] before exposure to oxidant (H2O2). Transfected cells overexpressing PKC- [Z] show protection of tubulin-based cytoskeleton against oxidant-induced nitrotyrosination and carbonylation injury. Tubulin in WT monolayers was protected only by a high dose of EGF (10 ng/ml). Nitration or oxidation was normalized to a nitrated or oxidized purified tubulin standard, which also served as loading control (5 µg). *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2 in WT. &P < 0.05 vs. PKC--overexpressing cells transfected with PKC- sense (n = 6/group) {[Z]} cells exposed to H2O2 or pretreated with EGF before H2O2 in WT cells. #P < 0.05 vs. EGF (10 ng/ml) before H2O2 in WT cells. [WT], WT cells. B and C: representative immunoblot photomicrographs of the tubulin nitration (B) and tubulin oxidation (carbonylation; C) following treatments as in A. The tubulin nitration (antinitrotyrosine) bands (B) or tubulin carbonylation (antidinitrophenylhydrazone) bands (C) from left to right correspond to WT cells exposed to vehicle (a); PKC--overexpressing cells exposed to vehicle (b); WT cells exposed to 0.5 mM H2O2 (c); PKC--overexpressing cells exposed to 0.5 mM H2O2 (d); WT 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); WT 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); corresponding 5 µg/lane of nitrated or oxidized tubulin loading control (standard, 50 kDa; i). PKC--overexpression in transfected cells by itself protects tubulin cytoskeleton against nitration and oxidation damage by oxidant challenge (lane d in both figures). This is comparable with that of the control (vehicle treated) tubulin exhibiting no nitration or oxidation (corresponding lanes a and b). Note that in WT cells, only a high dose of EGF (10 ng/ml, lane g in both figures) prevented tubulin nitration and oxidation. Shown is a representative blot (n = 6/group).

PKC--induced protection involves downregulation of iNOS and iNOS-driven reactions: inhibition of iNOS, NO, ONOO, and oxidative stress. We next probed possible mechanisms by which PKC- overexpression reduces nitration and oxidation of cytoskeletal proteins. Because we already showed that oxidants such as H₂O₂ upregulate iNOS and increase the levels of RNM and of oxidation and nitration of cytoskeletal proteins (10, 12, 42), we hypothesized that inhibition of iNOS-driven pathways might be a key mechanism for PKC--induced protection.

View this table: [in this window] [in a new window]   Table 1.   Effects of transfection of varying amounts of PKC- sense or antisense DNA on both the iNOS activity and NO levels in intestinal Caco-2 monolayers

View larger version (29K): [in this window] [in a new window]   Fig. 2.   A: protective effects of PKC- overexpression against upregulation of inducible nitric oxide (NO) synthase (iNOS) activity induced by H2O2 in Caco-2 monolayers assessed by L-[3H]citrulline formation. *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2 in WT. &P < 0.05 vs. PKC- overexpressing Z cells exposed to H2O2 or EGF before H2O2 in WT cells. #P < 0.05 vs. EGF (10 ng/ml) before H2O2 in WT cells (n = 6/group). B: representative Western blot for the protective effects of PKC- overexpression on downregulating iNOS protein levels in Caco-2 cell monolayers. The iNOS bands are from WT cells exposed vehicle (a); PKC--overexpressing cells exposed to vehicle (b); WT cells exposed to 0.5 mM H2O2 (c); PKC--overexpressing cells exposed to 0.5 mM H2O2 (d); WT 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); WT 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). In WT cells, H2O2 resulted in a large increase in the levels of iNOS protein (~130 kDa), whereas in PKC--overexpressing cells, this upregulation is prevented. The region of gel shown was between the Mr 126,000 and 218,000 prestained molecular weights that were run in adjacent lanes.

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View larger version (37K): [in this window] [in a new window]   Fig. 3.   Concentrations of NO in the supernatant of homogenates of intestinal cells of transfected and WT origin assessed by chemiluminescence analysis. As for effects on iNOS (Fig. 2), PKC- overexpression prevents NO upregulation by oxidants. EGF (10 ng/ml), which downregulates iNOS, also suppresses NO overproduction in WT cells. *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2 in WT. &P < 0.05 vs. PKC--overexpressing [Z] cells exposed to H2O2 or pretreated with EGF before to H2O2 in WT cells. #P < 0.05 vs. EGF (10 ng/ml) before H2O2 in WT cells (n = 6/group).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Time course for the prevention of the induction of iNOS and increases in NO in PKC--overexpressing cells. Units are pmol · mg1 · protein1 for iNOS activity; 103 × optical density for iNOS protein levels; µmoles/106 cells for NO levels. Cells were exposed to 0.5 mM H2O2 at time 0.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Oxidative stress in cell monolayers that is induced by oxidant is attenuated by PKC- overexpression as determined by the changes in dichlorofluorescein (DCF) fluorescence intensity. Also, note that EGF (10 ng/ml) is by itself protective in WT cells. *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2 in WT. &P < 0.05 vs. PKC--overexpressing [Z] cells exposed to H2O2 or EGF before H2O2 in WT cells. #P < 0.05 vs. EGF (10 ng/ml) before H2O2 in WT cells (n = 6/group).

Suppression of oxidative stress in transfected cells protects the cytoarchitecture of the microtubule cytoskeleton. Because PKC- overexpression inhibited several measures of oxidative stress, including tubulin oxidation, in our intestinal model, we assessed microtubule cytoskeletal assembly. We initially confirmed the preliminary finding (8) that PKC- overexpression confers protection to both polymerized and monomeric forms of tubulin (not shown) as well as the cytoarchitecture of the microtubule cytoskeleton (Fig. 6, A-C). For example, Caco-2 cells overexpressing PKC- exhibit an intact cytoskeleton even after exposure to oxidant (Fig. 6C) as indicated by the normal appearance of a stellate architecture of the microtubule cytoskeleton originating from the perinuclear region. Without PKC- overexpression, WT cells challenged with H₂O₂ show instability, disruption, and collapse of the microtubules. Untreated (control) cells also exhibit a normal architecture of the microtubule cytoskeleton. The appearance of the microtubules in these untreated (and normal) cells was indistinguishable from that of transfected PKC--overexpressing cells exposed to oxidant that also showed a preserved microtubule cytoarchitecture. These findings on the protection of the microtubule cytoarchitecture by PKC- parallel our findings on the protective effects of PKC- overexpression against the injurious nitration and oxidation of the tubulin backbone of this structural element.

View larger version (62K): [in this window] [in a new window]   Fig. 6.   The intracellular distribution of the microtubule (tubulin based) cytoskeleton, as captured by high-resolution laser scanning confocal microscopy (LSCM) in intestinal cells from monolayers. WT Caco-2 cells were exposed to 0.5 mM H2O2. PKC--overexpressing monolayers were also exposed to 0.5 mM H2O2. Untreated (control) cells were exposed to vehicle (A). Microtubules in control cells appear as intact filamentous network, which course radially throughout the cytosol (A). Only in WT cells exposed to H2O2 (B), the microtubules appear disrupted and collapsed. In cells overexpressing PKC- (C) and incubated with oxidant, normal microtubule architecture is highly preserved and resembles the controls. Bar = 25 µm. Shown is a representative photomicrograph (n = 6/group).

Intracellular distribution and constitutive activation of the overexpressed PKC- in transfected intestinal cells correlates with five different indexes of oxidative stress in monolayers. We initially confirmed our preliminary findings in transfected, PKC--overexpressing, intestinal cells (Table 2) that the -isoform (72 kDa) is found mostly in the particulate fractions of these transfected cells with only a minor distribution to the cytosolic fractions, indicating its constitutive activation (8). Finding PKC- in particulate pools (particulate = membrane + cytoskeletal fractions) indicates that the overexpressed PKC- isoform is "constitutively active," because achieving this intracellular PKC- distribution did not require EGF or OAG (a PKC activator). Pretreatment of these transfected cells with EGF, nonetheless, further increased the fraction of PKC- isoform into the membrane and cytoskeletal pools, reaching near-total activation of PKC-. In WT cells, in contrast, PKC- is found in a mostly cytosolic distribution (indicating inactivity) with smaller pools in the particulate fractions. In these WT cells, there was rapid translocation (i.e., activation) of native PKC- into particulate fractions only after exposure to high doses of EGF.

View this table: [in this window] [in a new window]   Table 2.   Analysis of the subcellular distribution of over-expressed PKC- in various cell fractions from stably transfected Caco-2 cells or from WT cells

Stable antisense inhibition of PKC- and its prevention of EGF-induced protection against oxidative stress of iNOS upregulation. The above findings indicate that PKC- might, by itself, play an essential role in cellular protection against oxidative stress of iNOS-driven reactions. To specifically investigate a possible role for PKC- in EGF-mediated protection against iNOS-pathway upregulation and consequent RNM-driven oxidative stress, we used Caco-2 cells that were transfected with PKC- antisense plasmid and cDNA-encoding G-418 resistance. We confirmed earlier reports that this manipulation substantially and stably reduces (~95%) the steady-state levels of PKC- protein as well as attenuates EGF protection of intestinal monolayer barrier integrity (8).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Stable antisense underexpression of PKC- isoform inhibits the protective/suppressive effects of EGF against oxidant-induced iNOS activation. A novel antisense transfected cell clone previously developed in our laboratory (see MATERIALS AND METHODS), which almost completely lacks PKC- protein, was grown as monolayers and subsequently pretreated with a high dose of EGF (10 ng/ml) and then exposed to 0.5 mM H2O2. Monolayers of WT Caco-2 cells were also treated in a similar fashion. *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2. &P < 0.05 vs. EGF + H2O2 in WT cells. [AS], antisense inhibition of PKC- protein (n = 6/group).

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Immunoblotting analysis showing the suppressive effects of the antisense underexpression of PKC- isoform on EGF's attenuation of both tubulin nitration and oxidation in Caco-2 cells. Cells either lacking PKC- protein (antisense transfected) or expressing native PKC- protein levels (WT) were incubated with EGF before H2O2. Nitration and carbonylation immunoreactivities were assessed as described in Fig. 1. *P < 0.05 vs. vehicle. +P < 0.05 vs. H2O2. &P < 0.05 vs. EGF + H2O2 in WT cells (n = 6/group).

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Prevention of the protective effects of EGF on the downregulation of both NO levels (chemiluminescence assay) and oxidative stress (DCF fluorescence intensity) in intestinal cells by the stable antisense inhibition of PKC- protein expression. Caco-2 cells almost totally lacking PKC- protein were treated as in Fig. 8. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. corresponding H2O2. &P < 0.05 vs. corresponding EGF + H2O2 in WT cells (n = 6/group).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Exploring the role of the -isoform of PKC in the suppression of oxidative stress of iNOS-driven reactions in cells, as we have done in the current investigation, is critical because 1) it is of significant clinical and biological importance to fully establish the idea that specific isoforms of PKC play fundamental roles in endogenous protective mechanisms of cells against oxidative stress to essential cellular proteins required for the maintenance of GI integrity and 2) a better understanding of effectively preventing (e.g., by PKC-) the hyperpermeability of the intestinal barrier under conditions of oxidative stress could lead to the development of novel therapeutics for inflammatory diseases of the GI tract that are related to oxidative injury due to iNOS and NO upregulation.

In the current study, we demonstrated that the -isoform of PKC is required for EGF-mediated protection against oxidant-induced iNOS upregulation and the consequent oxidative stress injury to the integrity of the microtubule (tubulin based) cytoskeleton and the intestinal epithelial barrier. A second conclusion, and also a novel finding, is that PKC- by itself appears to be key in the protection of cells against this oxidative stress injury. To our knowledge, this is the first time this mechanism has been ascribed to the defense and repair of epithelial cells. These conclusions are based on several independent lines of evidence as discussed below.

First, overexpression of PKC-, which we previously reported to prevent H₂O₂-induced barrier hyperpermeability, induces an EGF-like protection against oxidant-induced iNOS upregulation. PKC- evokes a cascade of alterations that are consistent with the proposed mechanism, including downregulation of iNOS activation, normalization of NO levels, reduction of RNM footprints, and decreases in oxidative stress (DCF fluorescence). This protection appears to require overexpression and constitutive activation of the PKC-. Second, overexpression of PKC- inhibits the footprints of oxidative injury (i.e., RNM formation) to the tubulin (50 kDa) protein of the microtubule cytoskeleton. Overexpression of PKC- decreases the nitration (nitrotyrosination) of tubulin, reduces the oxidation (carbonylation) of the tubulin, and maintains normal-appearing microtubule cytoskeleton. These new findings expand on the preliminary observations we made in which the overexpression of PKC- decreased the unstable monomeric (S1) tubulin and increased the stability of polymerized (S2) tubulin and enhanced monolayer barrier integrity (8). Third, a low, nonprotective concentration of EGF potentiates all measures of PKC--mediated protection against oxidative stress. Fourth, antisense to PKC-, which leads to expression of PKC- at only 5% of WT levels, substantially interfered with EGF-mediated suppression of the iNOS upregulation (by ~92%), of nitration and carbonylation of tubulin, and of the instability of microtubules. Additionally, EGF was unable to normalize NO levels or reduce DCF fluorescence in these antisense transfected cells. Finally, PKC- activation quantitatively correlates with decreases in all outcomes indicating protection against oxidative stress.

Our previous work in WT intestinal cells showed significant correlations between protection of the integrity of monolayer barrier permeability and microtubule stability and between the integrity of the intestinal barrier and native PKC in general (r = 0.94; P < 0.05) (9, 14). In the present study, with the use both of transfected clones and WT cells, we show new and robust correlations between PKC--isoform activation and protection against microtubule cytoskeletal oxidation (r = 0.93; P < 0.05) as well as several other parameters of oxidative stress and microtubule integrity. These include protection against tubulin nitration (RNM footprint) and PKC- activation (r = 0.92; P < 0.05), protection against tubulin carbonylation (oxidation) and PKC- activation (r = 0.93; P < 0.05), and protection against tubulin disassembly (increase S1 monomer pool) and PKC- activation (r = 0.92; 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 percent normal microtubules and PKC- activation (r = 0.95; P < 0.05) are used. Furthermore, protection against oxidant-induced iNOS upregulation and PKC- activation (r = 0.91; P < 0.05), NO levels and PKC- activation (r = 0.88; P < 0.05), and DCF fluorescence levels and PKC- activation (r = 0.90; P < 0.05) provide other robust correlations. The high strength of these correlations, which explains 85-95% of the variance, indicates that PKC- activation is essential to the protection against iNOS upregulation and consequent oxidative stress to the assembly of the tubulin cytoskeleton and intestinal barrier function. In this view, activation of PKC- leads to the normalization of NO levels and protection of the microtubule cytoskeleton and barrier against oxidative injury induced by iNOS. Overall, our studies on the -isoform are consistent with a model in which enhanced translocation and activation of PKC- results in downregulation of iNOS, reduction of both NO and RNM levels, decreases in both tubulin nitration and oxidation, increased assembly of polymerized tubulin pools, and concomitant reduction in monomeric tubulin pools and subsequently leads to enhanced stability of the microtubule cytoskeleton and monolayer barrier integrity under proinflammatory conditions of oxidative stress. Although other PKC isoforms may also be involved in protection of intestinal barrier integrity and microtubules against oxidative stress of iNOS-driven reactions, our findings indicate that such protection is mediated, in large part, through the -isoform.

The findings of this report using targeted molecular interventions are consistent not only with our own previous studies, but also with the findings of others using pharmacology. Our current findings on the atypical PKC- are also consistent with reports in non-GI models in which -activation was shown to be independent of PKC activators (e.g., OAG or TPA) (7-9, 20, 30, 55). For example, the activation of PKC- is not dependent on treatment with phorbol esters or DAG (30). Similarly, atypical PKC isozymes  and  do not respond to phorbol esters or OAG (25). In contrast, OAG has been shown to induce activation of classic isoforms of PKC, such as ₁, in non-GI cellular models (e.g., fibroblasts) as well as in GI cells (e.g., Caco-2 cells) (7, 30). Our findings using molecular biological interventions are also consistent with other previous pharmacological reports (9, 19, 20, 49, 57, 59, 61, 62, 65) in which general PKC activation (translocation) was shown to be necessary for the observed effects of PKC. For instance, EGF activates "constitutively" expressed PKC in several naive cells types (19, 65). Moreover, with the use of these WT cellular models and pharmacological approaches, PKC in general has been suggested to be a potential mediator of EGF-induced alterations in the actin component of the cytoskeleton in HeLa and corneal endothelial cells and of EGF inhibition of canine parietal cell function (26, 40, 65). Our current findings on the -isoform of PKC using specific and targeted molecular approaches further expand on these previous reports and, we believe, now establish a novel biological function: protection against oxidative stress of RNM upregulation and of cytoskeletal oxidation, among the atypical subfamily of PKC isoforms in cells.

Our findings regarding the subcellular distribution of PKC isoforms are consistent with known biochemical properties of PKC isoforms. All PKCs consist of NH₂-terminal regulatory domains and COOH-terminal catalytic domains (which are separated by a flexible hinge region) (31). In resting cells, PKC is mainly found in an inactive conformation. In this inactive phase, PKC is mainly distributed in the soluble (cytosolic) fraction and is 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 (7, 25, 31, 53). For example, OAG (or DAG)-binding sites are absent from the regulatory ("zinc finger") domain of PKC- . Not surprisingly, PKC- has very low affinity for OAG (8, 31).

Despite the fundamental importance of PKC signal transduction, as our studies indicate, the role of PKC isoforms in cell function, especially in epithelial cells, has remained poorly understood. Most cells express more than one type of PKC isoform, and the differences among these isozymes with respect to activation conditions and subcellular locations suggest that individual isoforms of PKC mediate distinct biological functions (1, 7, 8, 17, 18, 37, 47, 50, 52, 55). For example, we recently reported (7, 17) that the ₁ (78 kDa)-isoform of PKC is required for EGF-induced protective effects on the normalization of Ca²⁺ homeostasis, indicating that this isoform performs a unique protective task in cells. We further note that our series of studies on PKC to date were designed to investigate beneficial effects of PKC isoforms in the GI tract. Indeed, novel findings of our laboratory are that 1) PKC activation in general can be protective to cells, 2) specific PKC isoforms mediate this protection, and 3) each protective isoform of PKC works through a specific downstream mechanism (7-9). For example, our previous study linked the protective effects of activation of the classic PKC-₁ isoform to normalization of intracellular Ca²⁺ levels via the enhancement of Ca²⁺ efflux (7, 17), whereas the current study links the protective effects of activation of the atypical PKC- isoform with prevention of iNOS upregulation and normalization of NO levels.

We further note that there do exist reports that PKC may also have other effects that are not beneficial. These include the suspected role of PKC and tumor promoters (e.g., phorbol esters) in carcinogenesis (18, 37, 47, 50, 52, 53). For example, a recent immunofluorescent study suggested that PKC- appears to be involved in TNF--induced injury in intestinal (IEC-18) cells (23). Also, PKC- causes disruption of pig kidney cell monolayers (LLC-PK1) (53). Thus it appears that activating or mimicking just different isoforms of PKC will have distinct beneficial (or damaging) effects on the GI epithelium.

Our conclusions are potentially relevant for developing new treatment strategies for IBD. They suggest a novel antioxidative defensive mechanism that might, if it occurred in vivo, protect against oxidative stress and either prevent initiation or manifestation of the acute IBD attack. This defensive mechanism is seen in the ability of PKC- to prevent oxidant-induced cellular injury and barrier disruption through suppression of oxidation. The potential therapeutic use of this antioxidative mechanism would be consistent with the current characterizations of the pathophysiology of IBD (e.g., 12, 17, 42, 44, 45, 48, 56, 60). This is especially true for the transition 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 leads to sustained oxidative stress, hyperpermeability, inflammation, and tissue damage. The current study extends our previous investigation into the role of damaging oxidants in the pathophysiological mechanisms of this disease (5, 12, 17, 42, 44). In our intestinal model, oxidants induce barrier hyperpermeability (5, 9, 17), and this oxidative stress is prevented by the atypical PKC- as shown herein. We previously traced the in vitro cause of this monolayer hyperpermeability to disruption of the functioning and architectural integrity of the cytoskeletal filaments and iNOS upregulation (5, 9, 10, 12, 17). We also established the original concept that the biochemical cause of injury to the cytoskeletal networks and the GI barrier is the oxidation of essential cytoskeletal protein subunits, especially tubulin. We recently confirmed some of these findings on the cytoskeleton in vivo by showing that mucosa of patients with active IBD exhibits increases in tissue nitrotyrosination and oxidation of key cytoskeletal proteins as well as upregulation of iNOS and NO (16, 42). Consistent with these findings, tissue nitration, which was detected by immunofluorescent staining of nitrotyrosine, has been associated with the inflamed human mucosa in IBD and was linked with the upregulation of iNOS (45, 56, 60). It remains to be seen whether PKC- isoform confers protection against oxidative stress in IBD animal models.

The mechanism through which PKC- isoform suppresses iNOS upregulation remains unclear. On the basis of the known regulatory mechanisms for iNOS (22, 24, 28), we propose two mechanisms by which the iNOS upregulation might be prevented: inactivation of NF-B, a proinflammatory transcription factor, and inactivation of the iNOS enzyme molecules by any of several well-known cellular mechanisms such as phosphorylation or dephosphorylation (22, 24, 28). Studies are underway in our laboratory to determine to what extent PKC- protection is mediated by either of these mechanisms in GI epithelial cells.

Finally, with the use of another intestinal cell line, HT-29, we recently reported (8) that both Caco-2 cells and HT-29 cells behaved in an almost identical manner in terms of their responses to PKC--isoform transfection including activation, translocation, and over- and underexpression. Other similar responses included several barrier function-related outcomes such as FSA clearance, cytoskeletal assembly/integrity, and tubulin pools (8). Not surprisingly, they also responded similarly to EGF and oxidant treatment regimens. We recognize that Caco-2 cells are a transformed cell line and that tumor cells may not perfectly model barrier integrity and NO pathways in nontransformed cells, including enterocytes in native tissue. Nonetheless, our findings, using the first GI cells stably over- or underexpressing "protective PKC isoforms," demonstrate an original concept that PKC- appears to be responsible for a substantial portion of protection of the intestinal mucosal epithelium against oxidative stress induced by iNOS upregulation and perhaps is key in preventing amplification and perpetuation