PKC-zeta prevents oxidant-induced iNOS upregulation and protects the microtubules and gut barrier integrity

A. Banan1, L. Zhang1, J. Z. Fields1, A. Farhadi1, D. A. Talmage2, and A. Keshavarzian1

1 Departments of Internal Medicine (Section of Gastroenterology and Nutrition), Pharmacology, and Molecular Physiology, Rush University Medical Center, Chicago, Illinois 60612; and 2 Institute of Human Nutrition, Columbia University, New York, New York 10032


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Using intestinal (Caco-2) monolayers, we reported that inducible nitric oxide synthase (iNOS) activation is key to oxidant-induced barrier disruption and that EGF protects against this injury. PKC-zeta was required for protection. We thus hypothesized that PKC-zeta activation and iNOS inactivation are key in EGF protection. Wild-type (WT) Caco-2 cells were exposed to H2O2 (0.5 mM) ± EGF or PKC modulators. Other cells were transfected to overexpress PKC-zeta or to inhibit it and then pretreated with EGF or a PKC activator (OAG) before oxidant. Relative to WT cells exposed to oxidant, pretreatment with EGF protected monolayers by 1) increasing PKC-zeta activity; 2) decreasing iNOS activity and protein, NO levels, oxidative stress, tubulin oxidation, and nitration); 3) increasing polymerized tubulin; 4) maintaining the cytoarchitecture of microtubules; and 5) enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected cells overexpressing PKC-zeta (+2.9-fold) were protected as indicated by decreases in all measures of iNOS-driven pathways and enhanced stability of microtubules and barrier function. Overexpression-induced inhibition of iNOS was OAG independent, but EGF potentiated this protection. Antisense inhibition of PKC-zeta (-95%) prevented all measures of EGF protection against iNOS upregulation. Thus EGF protects against oxidative disruption of the intestinal barrier by stabilizing the cytoskeleton in large part through the activation of PKC-zeta and downregulation of iNOS. Activation of PKC-zeta is by itself required for cellular protection against oxidative stress of iNOS. We have thus discovered novel biologic functions, suppression of the iNOS-driven reactions and cytoskeletal oxidation, among the atypical PKC isoforms.

tubulin cytoskeleton; growth factors; oxidative stress; nitrogen metabolites; molecular biology; inflammatory bowel disease


    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)-alpha ] 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 (alpha , beta 1, beta 2, gamma ) require calcium, diacylglycerol (DAG), and phospholipid for their activation, whereas the novel PKC isoenzymes (delta , epsilon , theta , eta , µ) are calcium independent but require DAG and phospholipid. Activation of the third group, atypical PKC isoforms (lambda , tau , zeta ), is independent of both calcium and DAG (8, 25). Intestinal epithelial cells, including Caco-2 cells, express at least six isoforms of PKC: PKC-alpha , -beta 1, -beta 2, -delta , -epsilon , and -zeta 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-zeta 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-zeta , the other clones underexpressing PKC-zeta . Banan et al. (8) found that PKC-zeta , an atypical, DAG-independent isoform of PKC, is required for a substantial fraction of EGF-mediated protection of the monolayer barrier function. Also, PKC-zeta -mediated protection was DAG independent, because overexpression by itself afforded substantial protection. Despite the critical importance of the zeta -isoform of PKC, the fundamental mechanism for the protection afforded by PKC-zeta (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-zeta 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-zeta was reliably overexpressed; in the other clones, PKC-zeta 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 zeta -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-zeta were constructed and then stably transfected by Lipofectin reagent (GIBCO-BRL) as previously described (7, 8, 25). Expression was controlled by beta -actin promoter. The antisense PKC-zeta plasmid (pbeta -actin SP-As-PKC-zeta ) was constructed by ligating the 2.3-kb EcoRI fragment of PKC-zeta cDNA from pJ6-PKC-zeta into the unique EcoRI sites of the pbeta -actin SP vector (8, 25). The antisense orientation of the plasmid was confirmed by SamI restriction digestion. Multiple clones stably overexpressing PKC-zeta or lacking PKC-zeta 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-zeta 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.

Second, cell monolayers that were stably overexpressing PKC-zeta were preincubated (10 min) with EGF (1, 10 ng/ml) or vehicle before exposure (30 min) to damaging concentrations of oxidant (H2O2, 0.5 mM) or vehicle (9). Outcomes measured were as described above.

Third, monolayers of antisense-transfected cells stably lacking PKC-zeta protein were treated with high (protective) doses of EGF and then oxidant. In corollary experiments, we investigated the effects of PKC-zeta under- or overexpression on the state of tubulin nitration and oxidation and tubulin assembly and disassembly and on stability of the cytoarchitecture of the microtubules. Monomeric (S1) and polymerized (S2) fractions of tubulin (the structural protein subunit of microtubules) were isolated and then analyzed by immunoblotting to assess the oxidation and nitration of these fractions (4, 5, 9, 10, 12, 14).

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-zeta (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-zeta band was confirmed as previously described (8). We also confirmed that overexpression of PKC-zeta or antisense inhibition of PKC-zeta 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<UP><SUB>3</SUB><SUP>−</SUP></UP>) and nitrite (NO<UP><SUB>2</SUB><SUP>−</SUP></UP>), 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<UP><SUB>2</SUB><SUP>−</SUP></UP> 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-beta -tubulin (Sigma) and then with a secondary antibody (FITC-conjugated goat anti-mouse; Sigma). After being stained, cells were observed with an argon laser (lambda  = 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-zeta sense stably overexpress the zeta -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-zeta 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-zeta protects.

Stable overexpression of PKC-zeta isoform protects against oxidative damage to the cytoskeleton: inhibition of both tubulin nitration and oxidation. Because PKC-zeta 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-zeta ), oxidant H2O2 alone resulted in substantial levels of nitration and oxidation of the tubulin cytoskeleton (Fig. 1A). In contrast, overexpression of PKC-zeta by itself afforded protection against oxidant-induced tubulin nitration and tubulin carbonylation compared with those in WT cells. Indeed, only cells stably overexpressing PKC-zeta 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-zeta . 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-zeta 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-zeta 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-zeta 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-zeta by 2.9-fold was used. These intestinal monolayers stably overexpressing PKC-zeta or wild-type (WT) cells were incubated with growth factor [epidermal growth factor (EGF)] before exposure to oxidant (H2O2). Transfected cells overexpressing PKC-zeta [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-zeta -overexpressing cells transfected with PKC-zeta 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-zeta -overexpressing cells exposed to vehicle (b); WT cells exposed to 0.5 mM H2O2 (c); PKC-zeta -overexpressing cells exposed to 0.5 mM H2O2 (d); WT cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (e); PKC-zeta -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-zeta -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-zeta -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).

Figure 1, B and C, shows representative Western blots of the alterations in tubulin nitration and carbonylation. For example, PKC-zeta overexpression substantially inhibits both tubulin nitration and oxidation. This is shown by reduced band (lane) densities to a level close to control levels in cells overexpressing PKC-zeta , indicating prevention of oxidative damage to the microtubule (tubulin) cytoskeleton. As before, only high doses of EGF (e.g., 10 ng/ml) prevent tubulin oxidation and nitration in WT cells.

PKC-zeta -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-zeta 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-zeta -induced protection.

Multiple clones of intestinal cells transfected with 1, 2, 3, or 5 µg of PKC-zeta sense cDNA showed (Table 1 and Fig. 2A) a dose-dependent inhibition of iNOS upregulation {L-[3H]citrulline formation} against oxidant (H2O2)-induced challenge. The clone transfected with 3 µg of PKC-zeta sense provided the maximum inhibition of iNOS upregulation against oxidative insult (Table 1) without any loss of cell viability (0% cell death assessed by ethidium homodimer probe). Accordingly, we used this clone for overexpressing PKC-zeta in subsequent experiments.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effects of transfection of varying amounts of PKC-zeta 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-zeta 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-zeta 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-zeta overexpression on downregulating iNOS protein levels in Caco-2 cell monolayers. The iNOS bands are from WT cells exposed vehicle (a); PKC-zeta -overexpressing cells exposed to vehicle (b); WT cells exposed to 0.5 mM H2O2 (c); PKC-zeta -overexpressing cells exposed to 0.5 mM H2O2 (d); WT cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (e); PKC-zeta -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-zeta -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-zeta -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.

Figure 2A shows that PKC-zeta overexpression using the 3-µg sense transfected clone, which protects barrier integrity (8), also causes a substantial reduction in calcium-independent iNOS activity (~94% lower iNOS activity). This is comparable with controls (displaying only low iNOS activity). These measurements were done in lysates of both transfected and untransfected Caco-2 monolayers. In WT cells, this same dose of H2O2 causes both hyperpermeability and increases in iNOS activity. PKC-zeta -induced inhibition of iNOS upregulation did not require EGF. However, a low EGF concentration, 1 ng/ml, which did not by itself afford inhibition of iNOS in WT cells, potentiated PKC-zeta -induced iNOS downregulation in transfected cells. WT cells required a higher dose of EGF (10 ng/ml; Fig. 2A). As expected, transfection of only the vector did not confer protection against oxidant-induced iNOS activation (iNOS activity was 0.41 ± 0.05 pmol · min-1 · mg protein-1 for vector-transfected cells exposed to vehicle, 5.86 ± 0.22 pmol · min-1 · mg protein-1 for vector-transfected cells exposed to H2O2 alone, and 0.62 ± 0.10 pmol · min-1 · mg protein-1 for PKC-zeta sense transfected cells incubated in H2O2). This did not appear to be caused by changes in the ability of oxidants to cause iNOS upregulation, because vector-transfected cells and WT cells responded in a similar fashion to H2O2, exhibiting comparable iNOS upregulation.

Figure 2B depicts a representative Western blot showing that H2O2 significantly increases iNOS protein levels in WT cells, whereas transfected cells overexpressing PKC-zeta exhibit only low basal levels of the iNOS protein. For example, the corresponding OD values for control were 987 ± 82, 4,400 ± 112 for 0.5 mM H2O2, and 1,089 ± 123 for PKC-zeta sense transfected cells incubated in H2O2. Transfection of vector alone, similar to its lack of effects on iNOS activity and tubulin oxidation, was ineffective in preventing iNOS protein upregulation (not shown).

NO is the product of the iNOS-catalyzed reaction. Figure 3 and Table 1 show NO levels in both transfected monolayers and in WT monolayers exposed to H2O2 as determined by sensitive chemiluminescence analysis of cell lysates. PKC-zeta overexpression markedly prevented oxidant-induced NO overproduction (Fig. 3). In WT cells, for protection against tubulin oxidation and iNOS upregulation, NO production was inhibited only by high doses (e.g., 10 ng/ml) of EGF. Table 1 also depicts the results on NO analysis from multiple clones of transfected intestinal cells showing a dose-dependent inhibition of NO overproduction, paralleling findings on the suppression of iNOS. As expected, transfection of vector alone did not confer protection against NO overproduction (not shown).


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-zeta 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-zeta -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).

Figure 4 shows the time course for increases in iNOS protein, iNOS activity, and NO levels under oxidative conditions and their prevention in transfected cells. PKC-zeta overexpression prevented the effects of H2O2 on all three parameters. Maximalfold increases under H2O2 alone are ~5.0 for iNOS protein, ~11 for iNOS activity, and ~11 for NO levels; these increases were prevented by PKC-zeta overexpression.


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-zeta -overexpressing cells. Units are pmol · mg-1 · protein-1 for iNOS activity; 10-3 × optical density for iNOS protein levels; µmoles/106 cells for NO levels. Cells were exposed to 0.5 mM H2O2 at time 0.

PKC-zeta overexpression also inhibited oxidative stress assessed by decreases in the fluorescence of DCF (Fig. 5). In WT cells, where H2O2 significantly increases DCF fluorescence, oxidative stress was prevented only by high doses (10 ng/ml) of EGF. Under vehicle conditions, we observed significantly lower, but still substantial, levels of oxidative stress [possibly due to the normal generation of DCF reactive oxygen radicals (e.g., · O<UP><SUB>2</SUB><SUP>−</SUP></UP>) by well-known cellular metabolic processes, such as the mitochondrial respiratory chain reactions (4, 10, 12)]. Furthermore, transfection of vector alone did not prevent oxidative stress (not shown).


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-zeta 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-zeta -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-zeta 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-zeta 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-zeta 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-zeta 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-zeta -overexpressing cells exposed to oxidant that also showed a preserved microtubule cytoarchitecture. These findings on the protection of the microtubule cytoarchitecture by PKC-zeta parallel our findings on the protective effects of PKC-zeta 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-zeta -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-zeta (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-zeta in transfected intestinal cells correlates with five different indexes of oxidative stress in monolayers. We initially confirmed our preliminary findings in transfected, PKC-zeta -overexpressing, intestinal cells (Table 2) that the zeta -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-zeta in particulate pools (particulate = membrane + cytoskeletal fractions) indicates that the overexpressed PKC-zeta isoform is "constitutively active," because achieving this intracellular PKC-zeta distribution did not require EGF or OAG (a PKC activator). Pretreatment of these transfected cells with EGF, nonetheless, further increased the fraction of PKC-zeta isoform into the membrane and cytoskeletal pools, reaching near-total activation of PKC-zeta . In WT cells, in contrast, PKC-zeta 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-zeta 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-zeta in various cell fractions from stably transfected Caco-2 cells or from WT cells

We now show a significant (P < 0.05) inverse correlation (r = -0.91) between PKC-zeta levels (OD from the particulate fraction) and iNOS downregulation, suggesting that constitutive activation of the zeta -isoform is important in protection against oxidant-induced iNOS upregulation. Similarly, we note other robust correlations when either NO overproduction or oxidative stress (DCF fluorescence) were correlated with the PKC-zeta levels (r = 0.88, 0.90, respectively; P < 0.05 for each). When two other markers of oxidative stress, tubulin carbonylation and tubulin nitration (RNM generation), were used against PKC-zeta , other robust correlations were observed (r = -0.93, -0.92, respectively; P < 0.05 for each); further suggesting that activation of the zeta -isoform is key in protection against oxidative stress of iNOS-driven pathways.

Stable antisense inhibition of PKC-zeta and its prevention of EGF-induced protection against oxidative stress of iNOS upregulation. The above findings indicate that PKC-zeta 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-zeta in EGF-mediated protection against iNOS-pathway upregulation and consequent RNM-driven oxidative stress, we used Caco-2 cells that were transfected with PKC-zeta 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-zeta protein as well as attenuates EGF protection of intestinal monolayer barrier integrity (8).

We now demonstrate that antisense inhibition of expression of the PKC-zeta protein substantially prevents the protection afforded by 10 ng/ml EGF against iNOS upregulation (Fig. 7). This is an EGF dose that almost completely prevented oxidant-induced iNOS upregulation in naive (WT) cells. Table 2 also shows the effects of varying amounts (1, 2, 3, or 5 µg) of PKC-zeta antisense plasmid on inhibition of EGF-induced iNOS downregulation in intestinal cells. These data indicate a dose-dependent effect of antisense transfection. The clone transfected with 3 µg of zeta -antisense cDNA led to maximum inhibition of EGF-induced protection against iNOS upregulation. Accordingly, this antisense clone was used in subsequent inhibition experiments.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7.   Stable antisense underexpression of PKC-zeta 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-zeta 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-zeta protein (n = 6/group).

Immunoblotting analysis of the oxidative state of tubulin from antisense transfected cells demonstrates that antisense inhibition of PKC-zeta expression attenuated protection against both tubulin nitration and oxidation by a high (protective) dose of EGF (Fig. 8). In these cells, EGF cannot prevent oxidant-induced tubulin nitration or tubulin carbonylation. PKC-zeta -isoform underexpression by itself did not lead to oxidation of tubulin.


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-zeta isoform on EGF's attenuation of both tubulin nitration and oxidation in Caco-2 cells. Cells either lacking PKC-zeta protein (antisense transfected) or expressing native PKC-zeta 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).

In parallel, analysis of the NO levels (Fig. 9 and Table 1) and oxidative stress (Fig. 9) from antisense transfected cells further demonstrates that stable underexpression of PKC-zeta isoform substantially attenuates both EGF-induced NO and DCF fluorescence downregulation.


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-zeta protein expression. Caco-2 cells almost totally lacking PKC-zeta 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 zeta -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-zeta ) 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 zeta -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-zeta 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-zeta , which we previously reported to prevent H2O2-induced barrier hyperpermeability, induces an EGF-like protection against oxidant-induced iNOS upregulation. PKC-zeta 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-zeta . Second, overexpression of PKC-zeta inhibits the footprints of oxidative injury (i.e., RNM formation) to the tubulin (50 kDa) protein of the microtubule cytoskeleton. Overexpression of PKC-zeta 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-zeta 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-zeta -mediated protection against oxidative stress. Fourth, antisense to PKC-zeta , which leads to expression of PKC-zeta 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-zeta 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-zeta -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-zeta activation (r = 0.92; P < 0.05), protection against tubulin carbonylation (oxidation) and PKC-zeta activation (r = 0.93; P < 0.05), and protection against tubulin disassembly (increase S1 monomer pool) and PKC-zeta activation (r = 0.92; P < 0.05). Similar correlations are reached when either tubulin assembly (increase S2 polymer pool) and PKC-zeta activation (r = 0.92; P < 0.05) or percent normal microtubules and PKC-zeta activation (r = 0.95; P < 0.05) are used. Furthermore, protection against oxidant-induced iNOS upregulation and PKC-zeta activation (r = 0.91; P < 0.05), NO levels and PKC-zeta activation (r = 0.88; P < 0.05), and DCF fluorescence levels and PKC-zeta 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-zeta 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-zeta 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 zeta -isoform are consistent with a model in which enhanced translocation and activation of PKC-zeta 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 zeta -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-zeta are also consistent with reports in non-GI models in which zeta -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-zeta is not dependent on treatment with phorbol esters or DAG (30). Similarly, atypical PKC isozymes lambda  and tau  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 beta 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 zeta -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-zeta . Not surprisingly, PKC-zeta 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 beta 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-beta 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-zeta 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-epsilon appears to be involved in TNF-alpha -induced injury in intestinal (IEC-18) cells (23). Also, PKC-delta 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-zeta 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-zeta 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-zeta isoform confers protection against oxidative stress in IBD animal models.

The mechanism through which PKC-zeta 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-kappa 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-zeta 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-zeta -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-zeta 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 of an uncontrolled, oxidant-induced, inflammatory stress cascade in IBD, one that can be ignited by free radicals and other oxidants present in the GI tract.


    ACKNOWLEDGEMENTS

This work was supported, in part, by a grant from Rush Univ. Medical Center, Dept. of Internal Medicine and by a grant from the American College of Gastroenterology.


    FOOTNOTES

Portions of this work were presented as a "Poster of Distinction" at the annual meeting of the American Gastroenterological Association, May 2002.

Address for reprint requests and other correspondence: A. Banan, Rush Univ. Medical Center, Div. of Digestive Diseases, Section of Gastroenterology and Nutrition, 1725 W. Harrison, Ste. 206, Chicago, IL 60612 (E-mail: ali_banan{at}rush.edu).

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

June 5, 2002;10.1152/ajpgi.00143.2002

Received 12 April 2002; accepted in final form 31 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abraham, C, Scaglione-Swell B, Skarosi SF, Qin W, Bissonnette M, and Brasitus TA. Protein kinase C-alpha modulates growth and differentiation in Caco-2 cells. Gastroenterology 114: 503-509, 1998[ISI][Medline].

2.   Babich, M, Foti LR, and Mathias KL. Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells. J Cell Biochem 65: 276-285, 1997[ISI][Medline].

3.   Balda, MS, Gonzalez-Mariscai L, Matter K, Ceredijo M, and Anderson JM. Assembly of the tight junction. The role of diacylglycerol. J Cell Biol 123: 293-302, 1993[Abstract].

4.   Banan, A, Choudhary S, Zhang Y, Fields JZ, and Keshavarzian A. Ethanol-induced barrier dysfunction and its prevention by growth factors in human intestinal monolayers: evidence for oxidative and cytoskeletal mechanisms. J Pharmacol Exp Ther 291: 1075-1085, 1999[Abstract/Free Full Text].

5.   Banan, A, Choudhary S, Zhang Y, Fields JZ, and Keshavarzian A. Role of the microtubule cytoskeleton in protection by epidermal growth factor and transforming growth factor-alpha against oxidant-induced barrier disruption in a human colonic cell line. Free Radic Biol Med 28: 727-738, 2000[ISI][Medline].

6.   Banan, A, Fields JZ, Farhadi A, Talmage DA, Zhang L, and Keshavarzian A. The beta 1 isoform of protein kinase C mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells. J Pharmacol Exp Ther 301: 852-866, 2002[Abstract/Free Full Text].

7.   Banan, A, Fields JZ, Talmage DA, Zhang Y, and Keshavarzian A. The beta 1 isoform of protein kinase C mediates epidermal growth factor protection of the microtubules and barrier integrity of intestinal monolayers against oxidant damage. Am J Physiol Gastrointest Liver Physiol 281: G833-G847, 2001[Abstract/Free Full Text].

8.   Banan, A, Fields JZ, Talmage DA, Zhang L, and Keshavarzian A. The atypical PKC-zeta isoform is required in protection of microtubules and intestinal barrier integrity. Am J Physiol Gastrointest Liver Physiol 282: G794-G808, 2002[Abstract/Free Full Text].

9.   Banan, A, Fields JZ, Zhang Y, and Keshavarzian A. Key role of general protein kinase C activation in EGF-induced protection of the microtubule cytoskeleton and intestinal epithelial barrier against oxidant injury. Am J Physiol Gastrointest Liver Physiol 280: G828-G843, 2001[Abstract/Free Full Text].

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

11.   Banan, A, Fields JZ, Zhang Y, and Keshavarzian A. Targeted molecular inhibition of phospholipase C-gamma prevents EGF-mediated protection of the microtubule cytoskeleton and intestinal epithelial barrier function against oxidant injury. Am J Physiol Gastrointest Liver Physiol 281: G412-G423, 2001[Abstract/Free Full Text].

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

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

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

15.   Banan, A, Wang JY, McCormack SA, and Johnson LR. Relationship between polyamines, cytoskeletal distribution, and gastric mucosal ulcer healing in rats. Am J Physiol Gastrointest Liver Physiol 271: G893-G903, 1996[Abstract/Free Full Text].

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

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

18.   Batlle, E, Verdu J, Dominguez D, del Mont Llosas M, Diaz V, Loukili N, Paciucci R, Alameda F, and de Herreros A. G protein kinase C-alpha activity inversely modulates invasion and growth of intestinal cells. J Biol Chem 273: 15091-15098, 1998[Abstract/Free Full Text].

19.   Birkenfeld, PA, McIntyre BS, Biriski KP, and Sylvester PW. Role of protein kinase C in modulating epidermal growth factor- and phorbol ester-induced mammary epithelial cell growth in vitro. Exp Cell Res 233: 183-191, 1996.

20.   Boner, C, Guadagno SN, Fabbro D, and Weinstein IB. Expression of four protein kinase C isoforms in rat fibroblasts. J Biol Chem 267: 12892-12899, 1992[Abstract/Free Full Text].

21.   Bradford, MA. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye binding. Anal Biochem 72: 224-254, 1976.

22.   Bredt, DS, Ferris CD, and Synder SH. Nitric oxide synthase regulatory sites. J Biol Chem 267: 10976-10981, 1992[Abstract/Free Full Text].

23.   Chang, Q, and Tepperman BL. The role of protein kinase C isozymes in TNF-alpha -induced cytotoxicity to a rat intestinal epithelial cell line. Am J Physiol Gastrointest Liver Physiol 280: G572-G583, 2001[Abstract/Free Full Text].

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

25.   Cho, Y, Klein MG, and Talmage DA. Distinct functions of protein kinase C-alpha and protein kinase C-beta during retinoic acid-induced differentiation of F9 cells. Cell Growth Differ 9: 147-154, 1998[Abstract].

26.   Chun, J, Auer KA, and Jacobson BS. Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of beta 1 integrin. J Cell Physiol 173: 361-370, 1997[ISI][Medline].

27.   Ellis, B, Schneeberger EE, and Rabito CA. Cellular variability in the development of tight junction after activation of protein kinase C. Am J Physiol Renal Fluid Electrolyte Physiol 263: F293-F300, 1992[Abstract/Free Full Text].

28.   Garcia-Cardena, G, Fan R, Stern DF, Liu J, and Sessa WC. Endothelial nitric oxide synthase is regulated by tyrosine phosphorylation and interacts with caveolin-1. J Biol Chem 271: 27237-27240, 1996[Abstract/Free Full Text].

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

30.   Goodnight, J, Mischak H, Kolch W, and Mushinski JF. Immunocytochemical localization of eight PKC isoenzymes over-expressed in NIH 3T3 fibroblasts. J Biol Chem 270: 9991-10001, 1995[Abstract/Free Full Text].

31.   Gopalakrishna, R, and Jaken S. Protein kinase C signaling and oxidative stress. Free Radic Biol Med 28: 1349-1361, 2000[ISI][Medline].

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

33.   Hartwig, JH, Thelen M, Rosen A, Janmey PA, Nairn AC, and Aderem A. MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 356: 618-622, 1992[ISI][Medline].

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

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

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

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

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

39.   Ischiropoulos, H, Al-Mehdi HA, and Fisher AB. Reactive species in ischemic rat lung injury: contribution of peroxynitrite. Am J Physiol Lung Cell Mol Physiol 269: L158-L164, 1995[Abstract/Free Full Text].

40.   Joyce, NC, and Meklir B. Protein kinase C activation during corneal endothelial wound repair. Invest Ophthalmol 33: 1958-1973, 1992[ISI].

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

42.  Keshavarzian A, Banan A, Kommandori S, Zhang Y, and Fields JZ. Increased colonic free radicals and oxidation and nitration injury to key cytoskeletal proteins in inflammatory bowel disease. Gut, 2002. In press.

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

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

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

46.   MacRae, TH. Towards an understanding of microtubule function and cell organization: an overview. Biochem Cell Biol 70: 835-841, 1992[ISI][Medline].

47.   Maruvada, P, and Levine AE. Increased transforming growth factor-alpha levels in human colon carcinoma cell lines over-expressing protein kinase C. Int J Cancer 80: 72-77, 1999[ISI][Medline].

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

49.   McKenna, JP, Williams JM, and Hanson PJ. The alpha isoform of protein kinase C inhibits histamine-stimulated adenylate cyclase activity in a particulate fraction of the human gastric cancer cell line HGT-1. Inflamm Res 44: 66-69, 1995[ISI][Medline].

50.   Melloni, E, Pontremoli S, Sparatore B, Patrone M, Grossi F, Marks PA, and Rifkind RA. Introduction of the beta  isozyme of protein kinase C accelerates induced differentiation of murine erythroleukemia cells. Proc Natl Acad Sci USA 87: 4417-4420, 1990[Abstract].

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

52.   Mischak, H, Goodnight J, Kolch W, Martiny-Baron G, Schaechtle C, Kazanietz MG, Blumberg PM, Pierce JH, and Mushinski JF. Protein kinase C-alpha and -beta in NIH 3T3 cells induce opposite effects on growth morphology, anchorage dependence, and tumorigenicity. J Biol Chem 268: 6090-6096, 1993[Abstract/Free Full Text].

53.   Mullin, JM, Kampherstein JA, Laughlin KV, Clarkin CE, Miller RD, Szallasi Z, Kachar B, Soler AP, and Rosson D. Protein kinase C-delta increases tight junction permeability in LLC-PK1 cells. Am J Physiol Cell Physiol 275: C544-C554, 1998[Abstract/Free Full Text].

54.   Persons, DA, Wilkison WO, Bell RM, and Finn O. Altered growth regulation and enhanced tumorigenicity of NIH 3T3 fibroblasts by protein kinase C. Cell 52: 447-458, 1988[ISI][Medline].

55.   Ponzoni, M, Lacarelli E, Corrias MV, and Cornaglia-Ferraris P. Protein kinase C isoenzymes in human neuroblasts: involvement of PKC-alpha in cell differentiation. FEBS Lett 322: 120-124, 1993[ISI][Medline].

56.   Ramchilewitz, D, Stamler JS, Bachwich D, Karmeli F, Ackerman Z, and Podolsky DK. Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease. Gut 36: 718-723, 1995[Abstract].

57.   Reynolds, NJ, Talwar HS, Baldassare JJ, Henderson PA, Elder JT, Voorhees JJ, and Fisher GJ. Differential induction of phosphotidylcholine hydrolysis, diacylglycerol formation and protein kinase C activation by EGF and TGF-alpha in normal human skin fibroblasts and keratinocytes. Biochem J 294: 535-544, 1993[ISI][Medline].

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

59.   Saxon, ML, Zho X, and Black JD. Activation of protein kinase C is associated with post-mitotic events in intestinal epithelial cells in situ. J Cell Biol 126: 747-763, 1994[Abstract].

60.   Singer, II, Kawka DW, Scott S, Weidner JR, Mumford RA, Riehl TE, and Stenson WF. Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease. Gastroenterology 111: 871-885, 1996[ISI][Medline].

61.   Terres, AM, Pajares JM, Hopkins AM, Murphy A, Moran A, Baird AW, and Kelleher D. Helicobacter pylori disrupts epithelial barrier function in a process inhibited by protein kinase C activator. Infection Immun 74: 2943-2950, 1998.

62.   Turner, JR, Angle JM, Black ED, Joyal JL, Sacks DB, and Madara JL. PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase. Am J Physiol Cell Physiol 277: C554-C562, 1999[Abstract/Free Full Text].

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

64.   Wall, RL, Albrecht T, Thompson WC, James O, and Carney DH. Thrombin and phorbol myristate acetate stimulate cytoskeletal polymerization in quiescent cells: a potential link to mitogenesis. Cell Motil Cytoskeleton 23: 265-278, 1992[ISI][Medline].

65.   Wang, L, Wilson EJ, Osburn J, and DelValle J. Epidermal growth factor inhibits carbachol stimulated canine parietal cell function via protein kinase C. Gastroenterology 110: 469-477, 1996[ISI][Medline].

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

67.   Yile, SC, and Parker PJ. Defective microtubule reorganization in phorbol ester-resistant U937 variant: reconstitution of the normal cell phenotype with nocodazole treatment. Cell Growth Differ 8: 231-42, 1997[Abstract].


Am J Physiol Gastrointest Liver Physiol 283(4):G909-G922
0193-1857/02 $5.00 Copyright © 2002 the American Physiological Society