INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A FUNDAMENTAL PROPERTY OF epithelial cells of the gastrointestinal (GI) tract is to function as a highly selective permeability barrier, permitting the absorption from the lumen of nutrients, water, and electrolytes but restricting passage of harmful proinflammatory and toxic molecules (e.g., immunoreactive antigens, endotoxin) into the mucosa or the systemic circulation. 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 (NSAID)-induced chemical injury, and a variety of other GI disorders as well as several systemic disorders (e.g., alcoholic liver disease) (5, 30, 37). Pathogenesis of mucosal barrier dysfunction in these disorders remains poorly understood, but several studies, including our own (5-7, 9, 15, 17, 37), have shown that chronic gut inflammation is associated with high levels of reactive oxygen metabolites and that oxidants appear to be a key underlying cause of injury (31, 39, 42, 59). Oxidative injury is of clinical importance not only because reactive oxygen metabolites are common in inflammation but also because they can cause mucosal barrier hyperpermeability and, in turn, lead to the initiation and/or perpetuation of mucosal inflammation and injury (30, 31, 37, 38). For example, increases in epithelial barrier permeability after the injection of bacterial endotoxin into the mucosa in animal models can initiate an oxidative and inflammatory condition similar to IBD (59). Similarly, genetically engineered mice with a leaky gut develop intestinal inflammation (29).

We have been investigating endogenous protective mechanisms (e.g., growth factor signaling) against oxidant-induced barrier dysfunction in an effort to develop a rational basis for more effective treatment regimens for inflammatory disorders of the GI tract. We recently showed, using monolayers of human intestinal cells (Caco-2) as a model of barrier function, that epidermal growth factor (EGF) or transforming growth factor- protect intestinal barrier integrity by stabilizing the microtubule cytoskeleton (5, 6, 8-11) in large part through the activation of protein kinase C (PKC) (8, 10, 11). Because involvement in protective mechanisms by PKC was a novel finding, we surmised that one or more specific isoforms of PKC might mediate the protective actions of PKC.

PKC consists of a family of serine and threonine-specific kinases. The PKC family, which includes at least 12 known isoenzymes, can be classified into three subfamilies on the basis of differences in sequence homology and cofactor requirement (2, 4, 17, 18, 25, 32, 34, 41, 44, 46, 48, 49, 50, 51, 53, 57, 60, 61). 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 (21). Intestinal epithelial cells, including Caco-2 cells, express at least five of these isoforms: PKC-, PKC-₁, PKC-₂, PKC-, and PKC- (1, 8, 11, 16, 21, 43, 53, 58). 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 (1, 16, 32, 41, 44, 46, 49, 50).

We (8) previously showed in wild-type Caco-2 intestinal cells that EGF induces the membrane translocation of native PKC-₁ and PKC- isoforms, and therefore we considered each as a possible contributor to EGF-afforded protection. Using transfected cells that either stably overexpressed PKC-₁ or could not express PKC-₁, we recently found (6, 8, 11) that PKC-₁, a conventional or classic, DAG-dependent isoform of PKC, was necessary for a substantial fraction, but not all, of EGF protection. We noted that protection mediated by PKC-₁ was DAG dependent because neither PKC-₁ overexpression nor low doses of 1-oleoyl-2-acetyl-sn-glycerol (OAG) alone afforded protection but together they led to protection. In the current report, we have explored the role of the -isoform of PKC because: 1) it is translocated to the membranes in wild-type Caco-2 cells by EGF; 2) unlike PKC-₁, it is an "atypical" PKC isoform; 3) it is of clinical and biological importance to more fully establish the idea that specific isoforms of PKC play fundamental roles in endogenous protective mechanisms of cells; and 4) a better understanding of the pathophysiology of hyperpermeability of the intestinal barrier and its prevention could lead to the development of novel therapeutics for inflammatory diseases of the GI tract related to oxidative injury.

Accordingly, we studied the PKC- isoform utilizing targeted molecular interventions (transfection) that enabled us to develop two novel and stably transfected intestinal cell lines. In one, the atypical isoform PKC- was reliably overexpressed; in the other, PKC- expression was almost completely inhibited. Using these new models, we tested the hypothesis that EGF-induced protection against oxidant injury to both the microtubule cytoskeleton and intestinal barrier depends on activation of the -isoform but without the requirement for DAG (OAG).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell culture. Both Caco-2 and HT-29 cells were chosen because they form monolayers that morphologically resemble small intestinal cells, with defined apical brush borders, junctional complexes, and a highly organized microtubule network. Utility and characterization of these cell lines have been previously reported (4, 13, 24, 45).

Plasmids and stable transfection. Sense and antisense plasmids of PKC- were constructed and then stably transfected as we previously described (10, 11, 21). Expression was controlled by -actin promoter. The antisense PKC- plasmid (p-actin SP72-As-PKC-) was constructed by ligating the 2.3-kb EcoRI fragment of PKC- cDNA from pJ6-PKC- (21) into the unique EcoRI sites of the p-actin SP72 vector. The antisense orientation of the plasmid was confirmed by SamI restriction digestion (21).

Experimental design. First, postconfluent monolayers of wild-type cells were preincubated with EGF (1 or 10 ng/ml) or isotonic saline for 10 min and were then exposed to oxidant (0.5 mM H₂O₂) or vehicle (saline) for 30 min. As we have previously shown (5-7, 11, 13, 15), H₂O₂ at 0.5 mM disrupts microtubules and barrier integrity; EGF at 10 ng/ml (but not 1 ng/ml) prevents this disruption. These experiments were then repeated using cell monolayers either stably overexpressing or almost completely lacking PKC-. Reagents were applied on the apical side of monolayers unless otherwise indicated. In all experiments, barrier function, microtubule cytoskeletal stability (cytoarchitecture, tubulin assembly/disassembly), and PKC- subcellular distribution were then assessed.

Fractionation and Western 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 by others and by us (1, 8, 11). Protein content of the various cell fractions was assessed by the Bradford method (19). For total PKC extraction, scraped monolayers were placed directly into 1.5 ml of a standard cold lysis buffer (4°C) and subsequently ultracentrifuged. Supernatant was used for bulk protein determination.

Immunofluorescent staining and high-resolution LSCM of microtubules. Cells from monolayers were fixed in cytoskeletal stabilization buffer and then postfixed in 95% ethanol at 20°C as we previously described (5, 6, 8, 12-14). Cells were subsequently processed for incubation with a primary antibody, monoclonal mouse anti--tubulin (Sigma, St. Louis, MO) at 1:200 dilution for 1 h at 37°C, and were then incubated with a secondary antibody (FITC-conjugated goat anti-mouse; Sigma) at 1:50 dilution for 1 h at room temperature. Slides were washed three times in D-PBS and subsequently mounted in aquamount. After being stained, cells were observed with an argon laser ( = 488 nm) using a ×63 oil immersion plan-apochromat objective, 1.4 numerical aperture (Zeiss). Single cells and/or a clump of two to three cells from desired areas of monolayers were processed using the image processing software on a Zeiss ultra high-resolution LSCM so as to create "neat black" areas surrounding the cells. The cytoskeletal elements were examined in a blinded fashion for their overall morphology, orientation, and disruption as we have described (5, 6, 8, 12, 13). Two hundred cells per slide were examined in four different fields by LSCM, and the percentage of cells displaying normal microtubules was determined. Slides were decoded only after examination was complete.

Microtubule (tubulin) fractionation and quantitative immunoblotting of tubulin assembly and disassembly. Polymerized (S2) and monomeric (S1) fractions of tubulin were isolated after a series of centrifugation and extraction steps as we have described (5, 6, 8, 13). 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 per lane) were placed in a standard SDS sample buffer, boiled for 5 min, and then subjected to PAGE on 7.5% gels. Procedures for Western blotting were performed as previously described (5, 6, 8, 13). To quantify the relative levels of tubulin, the optical density of the bands corresponding to immunoradiolabeled tubulin were measured with a laser densitometer.

Determination of barrier permeability by fluorometry. Barrier integrity was determined 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 (5-8, 9-11, 15) and others (33, 35, 52, 56) have described. In select experiments, higher molecular mass fluorescein dextran (FD) probes such as the 4 kDa FD and 70 kDa FD (1 mg/ml) were also utilized. 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 four to six observations per group. Statistical analysis comparing treatment groups was performed using ANOVA followed by Dunnett's multiple range test (27). 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

Stable overexpression of PKC- isoform. Intestinal cells were cotransfected with complementary DNA (cDNA) encoding both G-418 resistance (for selection) and PKC-. Western immunoblotting analysis of cell lysates of these transfected cells from confluent monolayers demonstrates (Fig. 1A) the overexpression of the PKC- isoform (3 µg of DNA plasmid shown). The PKC- isolated from transfected cells ran at the expected molecular mass of 72 kDa as confirmed by a known positive control for PKC-. Identity of the PKC- band was further ascertained by using the PKC- blocking peptide in combination with the anti-PKC- antibody that prevented the appearance of the corresponding major band in the Western blots. Additionally, in the absence of the primary antibody to PKC-, no corresponding band for PKC- was observed. Immunoblotting assessment of PKC- protein levels (Fig. 1B) showed that total levels of this overexpressed isoenzyme were increased by ~2.9-fold compared with wild-type cells. Optical densities (means ± SE) for these PKC- overexpression studies were 12,210 ± 118 vs. wild type 4,175 ± 79. Preliminary studies confirmed that overexpression of PKC- did not injure intestinal cells as indicated by a lack of change in viability assessed by ethidium homodimer-1 probe (5, 6).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   A: stable overexpression of atypical PKC- in intestinal cells transfected with a plasmid encoding PKC-. Caco-2 cell lysates from G-418-resistant confluent monolayers were processed for immunoblotting and probed with a monoclonal anti-PKC-. An "overexpressed" PKC- protein band ran at the expected molecular mass of 72 kDa as confirmed by a commercially obtained positive control for PKC- (from rat brain lysates). Exclusion of primary antibody (No 1° Ab) led to the disappearance of the corresponding 72 kDa PKC- band. Addition of the antipeptide to primary antibody also caused the disappearance of the PKC- band. Prestained molecular weights 67,000 and 93,000 were also run in adjacent lanes. Shown is a representative blot;. n = 6 cells per group. B: total levels of atypical PKC- protein overexpression in transfected intestinal cells vs. wild-type (WT; untransfected) cells. Caco-2 cell extracts were subjected to SDS-PAGE fractionation and Western immunoblotting with the PKC--specific antibody. Quantitative analysis of X-ray film scanned with a densitometer showed a 2.9-fold increase in the steady-state levels of the PKC- protein in transfected intestinal cells. Corresponding optical densities from left to right are: WT 4,153 and overexpressed 12,115. Shown is a representative blot from n = 6 cells per group.

Protective effects of the overexpressed PKC- isoform against oxidant-induced injury. Multiple clones of intestinal Caco-2 cells (Table 1) or HT-29 cells (Table 2) transfected with 1, 2, 3, 4, or 5 µg of PKC- sense cDNA showed a dose-dependent protection of barrier integrity in monolayers against oxidant-induced injury as assessed by FSA clearance. In Caco-2 monolayers, the clone transfected with 3 µg of PKC- sense provided the maximum protection observed (Table 1). This was comparable to 3 µg PKC- sense, which also provided the maximum protection in HT-29 cells (Table 2). Accordingly, we used the appropriate clones for overexpressing PKC- in all subsequent experiments.

View this table: [in this window] [in a new window]   Table 1.   Effects of transfection of varying amounts of PKC- sense or antisense DNA on Caco-2 monolayer barrier integrity

View this table: [in this window] [in a new window]   Table 2.   Effects of transfection of varying amounts of PKC- sense or antisense plasmids on monolayer barrier integrity of another intestinal cell line, HT-29

View larger version (26K): [in this window] [in a new window]   Fig. 2.   A: overexpression of PKC- protects the barrier integrity of Caco-2 monolayers against oxidant injury. Intestinal monolayers stably overexpressing PKC- were incubated with epidermal growth factor (EGF) before exposure to oxidant H2O2 (0.5 mM). Transfected cells overexpressing PKC- (Z) maintain their barrier integrity against oxidant-induced injury. WT monolayers, in contrast, were protected only by a high dose of EGF (10 ng/ml), whereas a low dose of EGF (1 ng/ml) did not protect WT cells. Also, note potentiation-induced protection of barrier integrity in PKC--overexpressing cells exposed to EGF. Both PKC--overexpressing cells and WT cells responded in the same fashion to vehicle treatment. Barrier integrity was expressed as flux of the fluorescent probe fluorescein sulfonic acid (FSA) from the apical to basolateral compartment of cell culture transwell inserts divided by the concentration of probe in the apical chamber. When normalized for the surface area of the monolayer, this expression has units of clearance. * P < 0.05 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 cells per group). B: comparison of the protective effects of PKC- overexpression on monolayer barrier integrity of Caco-2 cells exposed to oxidant H2O2 (0.5 mM) as determined by several fluorescein-conjugated probes of different molecular mass: 70 kDa fluorescein dextran (FD), 4 kDa FD, and 0.478 kDa FSA. Note the size (molecular mass)-dependent permeation of these probes in both WT and transfected Caco-2 cells. There is an inverse relationship between the probe size and clearance in order of increasing size (0.478 kDa FSA > 4 kDa FD > 70 kDa FD). * P < 0.05 vs. corresponding vehicle; + P < 0.05 vs. corresponding H2O2 in WT (n = 6 cells per group).

View larger version (79K): [in this window] [in a new window]   Fig. 3.   A: percent of Caco-2 cells displaying normal microtubule cytoskeleton in PKC--overexpressing cells. Treatments and conditions were as explained in Fig. 2A. Cell monolayers grown on coverslips were processed for immunofluorescent staining with a primary monoclonal anti--tubulin antibody, and subsequently the microtubule elements were examined in a blinded fashion for their overall morphology. Microtubules are protected against oxidant injury in PKC--overexpressing cells. A high dose of EGF (10 ng/ml), which protects microtubules in WT cells, is also shown. PKC- overexpression by itself is protective. Note the potentiation of protection in PKC-- overexpressing cells exposed to EGF. PKC--overexpressing cells and WT cells responded comparably with vehicle treatment. * P < 0.05 vs. vehicle; + P < 0.05 vs. H2O2 in WT; & P < 0.05 vs. PKC- 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 cells per group). B: intracellular distribution of the microtubules as imaged by ultra high-resolution laser confocal microscopy (LSCM) in intestinal cells from monolayers. Monolayers of WT Caco-2 cells were incubated with vehicle (isotonic saline) (a) or 0.5 mM H2O2 (b). PKC--overexpressing monolayers were exposed to 0.5 mM H2O2 (c) or vehicle (d). Microtubules in controls (a and d) appear as normal microfilamentous structures that disperse throughout the cytosol. In cells exposed to H2O2 (b), the microtubules show a clear collapse, disorganization, and disruption of their architecture. In contrast, in cells overexpressing PKC- and incubated with oxidant, intact microtubule cytoarchitecture are highly preserved and protected, resembling the controls (c). Cells from desired areas of monolayers were processed using the image processing software on a Zeiss LSCM so as to create "neat black" areas surrounding the cells. Shown is a representative photomicrograph. Bar = 25 µm; n = 6 cells per group.

View this table: [in this window] [in a new window]   Table 3.   Transfection of PKC- sense plasmid protects the microtubule cytoskeleton and tubulin assembly while antisense inhibits protection in intestinal HT-29 cells

View larger version (56K): [in this window] [in a new window]   Fig. 4.   A: immunoblotting analysis of the polymerized tubulin (S2, index of microtubule assembly) and the monomeric tubulin (S1, index of microtubule disassembly) in Caco-2 cell monolayers overexpressing atypical PKC-. Tubulin fractions were extracted from monolayers and subjected to SDS-PAGE fractionation and immunoblotted using monoclonal anti--tubulin antibody followed by horseradish peroxidase conjugated-secondary antibody and subsequently autoradiographed. To quantify the relative levels of tubulin bands, the optical density of the bands corresponding to immunoradiolabeled tubulin were measured with a laser densitometer. Results for %tubulin assembly for WT cell extracts are also shown. Conditions were as described in Figs. 2A and 3A. Percent polymerization of tubulin = [(S2)/(S2 + S1)], where S2 + S1 is the total cellular tubulin pool. * P < 0.05 vs. vehicle. + P < 0.05 vs. H2O2 in wild type; & P < 0.05 vs. 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 per group). B and C: photomicrographs representing typical Western immunoblots of the polymerized tubulin/S2 fractions (B) and monomeric tubulin/S1 fractions (C) from PKC--transfected and WT intestinal Caco-2 monolayers. Tubulin fractions extracted from intestinal cells were processed for Western immunoblotting as described in Fig. 4A. Immunoblotted tubulin on nitrocellulose membranes was visualized by enhanced chemiluminescence (ECL) and autoradiography. The tubulin bands from left to right correspond to: a, WT cells exposed vehicle; b, PKC--overexpressing cells exposed to vehicle; c, WT cells exposed to 0.5 mM H2O2; d, PKC--overexpressing cells exposed to 0.5 mM H2O2; e, WT cells treated with EGF (1 ng/ml) + 0.5 mM H2O2; f, PKC--overexpressing cells treated with EGF (1 ng/ml) + 0.5 mM H2O2; g, WT cells treated with EGF (10 ng/ml) + 0.5 mM H2O2; h, PKC--overexpressing cells treated with EGF (10 ng/ml) + 0.5 mM H2O2; i, tubulin standard (50 kDa). PKC- overexpression in transfected cells by itself maintains normal dynamics of tubulin polymerization against oxidant-induced disassembly. This is shown by a tubulin band density comparable to that of the control (vehicle) levels. In WT cells, only a high dose of EGF, but not a low dose, enhanced tubulin assembly to levels similar to that of transfected or control cells. Shown is a representative blot from each tubulin fraction (n = 6 per group).

Intracellular distribution and constitutive activation of the overexpressed PKC- in transfected intestinal monolayers. Western immunoblotting assessment of the cytosolic, membrane, and cytoskeletal-associated fractions from transfected cells overexpressing PKC- showed that the -isoform (72 kDa) is found mostly in the membrane and cytoskeletal fractions of these transfected cells with only a small distribution to the cytosolic fractions (Fig. 5A, Caco-2 shown). In wild-type cells (Fig. 5B), in contrast, we found a mostly cytosolic distribution of PKC- with smaller pools in the membrane and cytoskeletal (particulate) fractions, indicating inactivity. Figure 6 shows a graphic depiction of the intracellular distribution of the overexpressed PKC- in various Caco-2 cell monolayer fractions as a fraction of total distribution (expressed in arbitrary units). Finding PKC- in particulate pools indicates that the overexpressed PKC- isoform is "constitutively active" because achieving this distribution by PKC- did not require EGF or OAG (a PKC activator). Pretreatment of transfected cells with EGF, however, further increased the fraction of PKC- isoform into the membrane and cytoskeletal fractions, reaching near-total activation of PKC-. Wild-type cells exposed to vehicle or oxidant show a mostly cytosolic distribution of PKC-. In these wild-type cells, we noted rapid translocation of native PKC- into particulate (membrane + cytoskeletal) fractions of cells only after exposure to high doses of EGF, which confirms our recent and preliminary findings (8).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Subcellular distribution of overexpressed PKC- in the cytosolic, membrane, and cytoskeletal fractions of intestinal cell monolayers. Transfected Caco-2 cells overexpressing PKC- are shown in A. WT monolayers are shown in B. Cell monolayers grown in 75-cm2 flasks were processed for the isolation of various fractions and then Western immunoblotted using monoclonal anti-PKC-. In transfected cells, note the presence of PKC- in the particulate (i.e., membrane and cytoskeletal) fraction, indicating "constitutive" activation of PKC-. Here, the lanes and corresponding optical densities from left to right are: cytosolic 848, membrane 10,055, and cytoskeletal 1,211. In WT cells (B), PKC- is not constitutively active as shown by a mostly cytosolic pool of PKC-. Corresponding optical densities from left to right are: cytosolic 3,758, membrane 332, and cytoskeletal 63. Shown is a representative blot (n = 6 cells per group).

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Subcellular distribution of overexpressed PKC- in various cell fractions in stably transfected intestinal cells and in WT cells (not overexpressing PKC-). Caco-2 cells were pretreated with EGF with or without exposure to H2O2 (0.5 mM) and then processed for isolation of cytosolic, membrane, and cytoskeletal (Triton X-100-insoluble) associated fractions. Note the constitutive activation of overexpressed PKC- in transfected cells as indicated by its distribution mostly in membrane and cytoskeletal-associated monolayer fractions ("particulate fraction"). WT cells show a mainly cytosolic pool of PKC-, indicating inactivity. In these WT cells, native PKC- was rapidly translocated into the particulate (membrane + cytoskeletal) fractions of cells only after exposure to high doses of EGF. Relative levels of PKC- overexpression in these fractions were quantified by measuring the optical density of the bands corresponding to anti-PKC- immunoreactivity with a laser densitometer. The optical density for the membrane pool in the corresponding vehicle-treated cells was assigned an arbitrary value of 100, and all other densities were normalized to that value and thus reported in arbitrary units. * P < 0.05 vs. corresponding fraction in WT cells treated with vehicle; + P < 0.05 vs. corresponding fraction in WT cells exposed to H2O2; & P < 0.05 vs. corresponding fraction from Z cells exposed to H2O2 or EGF before H2O2 in WT cells (n = 6 per group).

Stable antisense inhibition of PKC- to underexpress the -isoform and its prevention of EGF-induced protective effects. The above findings indicate that PKC- might, by itself, play a key role in cellular protection. To show that PKC- specifically contributes to EGF-mediated protection, we utilized an antisense approach to stably decrease the steady-state levels of PKC- protein. Figure 7A shows an immunoblot of cell lysates of wild-type Caco-2 cells transfected with PKC- antisense cDNA (3 µg) and plasmid encoding G-418 resistance. These data show a substantial reduction (95%) in the levels of PKC- protein in these antisense-transfected cells.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   A: stable antisense (AS) inhibition of PKC- protein in differentiated intestinal cells transfected with antisense plasmid to the atypical -isoform of PKC. Whole cell extracts from Caco-2 monolayers were subjected to immunoblotting by the monoclonal anti-PKC- antibody, and subsequently the immunoblotted proteins were visualized by enhanced chemiluminescence and autoradiography. Analysis by densitometry showed an almost complete absence of PKC- protein (95% reduction) in these antisense-transfected cells. Corresponding optical densities from left to right are: positive control 14,090, WT 4,173, and antisense 212. Shown is a representative immunoblot; n = 6 cells per group. B: prevention of the protective effects of EGF on barrier function of differentiated intestinal cell monolayers by the stable antisense inhibition of PKC- protein expression. Caco-2 cells almost totally lacking PKC- protein were incubated with EGF (10 ng/ml) before exposure to H2O2. FSA clearance was determined as described in Fig. 2A. * P < 0.05 vs. vehicle; + P < 0.05 vs. H2O2; & P < 0.05 vs. EGF + H2O2 in WT cells (n = 6 cells per group).

View larger version (87K): [in this window] [in a new window]   Fig. 8.   A: stable antisense inhibition of PKC- protein expression inhibits the protective effects of EGF on the microtubule cytoskeleton. The percentage of Caco-2 cells in monolayers displaying normal microtubule cytoskeletons was assessed in antisense-transfected cells treated with EGF (10 ng/ml) before oxidant (conditions as described in Fig. 7B). * P < 0.05 vs. vehicle; + P < 0.05 vs. H2O2; & P < 0.05 vs. EGF + H2O2 in WT cells (n = 6 cells per group). B: ultra high-resolution laser confocal microscopy of the microtubule cytoarchitecture in PKC- antisense-transfected and WT intestinal cells from monolayers. Monolayers of WT Caco-2 cells were preincubated with EGF (10 ng/ml) and then 0.5 mM H2O2 (a). PKC--underexpressing monolayers were pretreated with EGF (10 ng/ml) plus H2O2 (b) or vehicle (c). Microtubules in EGF-pretreated WT cells (a) appear protected as shown by their intact architecture. In contrast, in antisense-transfected cells the same dose of EGF is not protective (b). Here, the microtubules show collapse and fragmentation. Underexpression of PKC- by itself does not deleteriously effect the microtubule cytoskeleton (c). Shown is a representative photomicrograph. Bar = 25 µm; n = 6 cells per group.

View larger version (48K): [in this window] [in a new window]   Fig. 9.   A: antisense inhibition of PKC- protein inhibits the enhancement of tubulin assembly by EGF as determined by quantitative immunoblotting analysis of tubulin pools from Caco-2 cells. Both the polymerized tubulin (S2, index of assembly) and monomeric tubulin (S1, index of disruption) were assessed. Conditions were similar to those in Fig. 8A. Percent polymerization of tubulin = [(S2)/(S2 + S1)]. * P < 0.05 vs. vehicle; + P < 0.05 vs. H2O2; & P < 0.05 vs. EGF + H2O2 in WT cells (n = 6 cells per group). Representative Western blot photomicrographs of the polymerized S2 tubulin (triton-insoluble) extracts (B) and monomeric S1 tubulin extracts (C) following similar treatment regimens as in Fig. 9A are shown. Tubulin fractions from Caco-2 cells were analyzed by SDS-PAGE and Western immunoblots and subsequently processed for X-ray film exposure. The lanes from right to left are: a, WT cells exposed to vehicle; b, PKC--underexpressing cells exposed to vehicle; c, WT cells exposed to 0.5 mM H2O2; d, PKC--underexpressing cells exposed to 0.5 mM H2O2; e, WT cells treated with EGF (10 ng/ml) + 0.5 mM H2O2; f, PKC--underexpressing cells treated with EGF (10 ng/ml) + 0.5 mM H2O2; and g, tubulin standard (50 kDa). Shown is a representative immunoblot from each tubulin extract (n = 6 cells per group).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have demonstrated that the -isoform of PKC plays an important role in EGF-mediated protection against oxidant damage to the microtubule cytoskeleton and to cell monolayer integrity. This isoform of PKC also appears to be a critical endogenous stabilizer of both cytoskeletal and barrier function. Several lines of evidence in the current study support the aforementioned findings.

First, overexpression of PKC- induces an EGF-like protection against oxidant-induced disruption of barrier integrity. This protection appears to require overexpression and constitutive activation of PKC-. In particular, protection is dependent on constitutive activation through the distribution of PKC- into the particulate (cytoskeletal + membrane) fractions. Second, overexpression of PKC- induces stabilization of the microtubule cytoskeleton, a protective phenomenon we have shown to be key in the maintenance of cell monolayer integrity. Overexpression of PKC- decreases the unstable monomeric (S1) tubulin, increases the stability of polymerized (S2) tubulin, and increases the percentage of Caco-2 cells displaying normal microtubules. Third, a low, nonprotective concentration of EGF potentiates all measures of PKC--induced protection. Fourth, antisense inhibition of the expression of PKC- reduces EGF protection of barrier integrity by ~48 ± 8%, the remaining 52% of EGF protection apparently being PKC- independent. In these antisense-transfected clones, which expressed -isoform at ~5% of wild-type levels, EGF protection of S2 tubulin assembly and microtubules was also significantly prevented. Fifth, increases in expression of PKC- quantitatively correlate with increases in outcomes indicating protection (barrier integrity, tubulin polymerization, microtubule assembly, and integrity of microtubule cytoarchitecture).

Our findings are consistent with our previous reports that activation of PKC in general is required for EGF protection (8) and that specific isoforms mediate that protection (11). For example, PKC-₁ isoform mediates a substantial portion (60 ± 8%) of EGF protection (11). On the basis of percent mediation of protection, it is reasonable to speculate that activation of both 1 and -isoforms of PKC can account for 100% of EGF-induced protection.

Although PKC-₁ and PKC- share in common the ability to protect, there appear to be differences in their mechanisms of action. Protection by PKC-, as shown herein, does not require the presence of pharmacological activators of PKC (e.g., OAG or EGF), whereas protection by PKC-₁ does require them (8, 11). This difference is fully consistent with the fact that PKC- is an "atypical" isoform of PKC, whereas PKC-₁ is a "conventional" isoform. Indeed, our findings on the atypical PKC- are consistent with reports in non-GI models in which the -isoform activation was shown to be independent of PKC activators (e.g., OAG or 12-O-tetradecanoylphorbol 13-acetate) (18, 25, 50). For example, the activation of PKC- is not dependent on treatment with phorbol esters or DAG (OAG) (25). Similarly, atypical PKC isozymes  and  do not respond to phorbol esters or OAG (21). 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) (25) as well as in GI cells (e.g., Caco-2 cells) as we recently reported (11).

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 (separated by a flexible hinge region) (26). 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 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 (21, 26, 47). For example, OAG (or DAG) binding sites are present in the regulatory "zinc finger" domain of PKC-₁ but are absent from the regulatory domain of PKC-. Not surprisingly, PKC- has severalfold lower affinity for OAG than has PKC-₁ at the zinc finger domains (26). Additionally, in many wild-type cells, PKC-₁ and PKC- appear to be found in different subcellular fractions (11, 21). Consistent with these known facts, our previous and present studies collectively demonstrate a novel concept that increased levels of these PKC isoforms in the particulate fractions (i.e., activation) either by pharmacological manipulation (OAG or EGF for PKC-₁) or by transfection (constitutive activation for PKC-) lead to enhanced cellular protection.

Although most cells express more than one type of PKC isoform, differences among isozymes with respect to activation conditions and subcellular locations suggest that individual PKC isoforms have distinct activation mechanisms as well as mediate distinct biological processes (1, 4, 16, 17, 32, 41, 44, 46, 49-51, 60). In resting cells (in the absence of lipid cofactors), most PKCs assume an inactive structural conformation. This is maintained by an intramolecular interaction between an autoinhibitory sequence (i.e., the pseudosubstrate on the NH₂ terminus) in the regulatory domain and the substrate-binding region of the catalytic domain (26). Moreover, accumulated evidence suggests that modifications of the regulatory domain of a PKC isoform can lead to the activation of that isoform (21, 26). For example, binding of PKC- isoform to phospholipids, especially anionic phosphotidylserine, in membranes (i.e., translocation to membranes) is thought to be necessary to cause conformational changes to its regulatory domain, before activation. Specifically, structural studies (26) demonstrate that in the presence of inducers (e.g., overexpression by transfection), this regulatory domain (especially the zinc finger portion) forms an automatic hairpin-like hydrophobic structure that mediates PKC interaction with the membrane lipids and subsequent conformational changes within the regulatory domain, leading to autoactivation. Thus overexpression of PKC- may promote a conformational change that releases the inherent autoinhibition present and triggers kinase catalytic activity. For DAG-dependent PKC isoforms such as PKC-₁ (11, 21), on the other hand, the inhibitory function of the regulatory domain can be overcome by pharmacological agents that mimic DAG (e.g., OAG or 12-O-tetradecanoylphorbol 13-acetate), thus producing conformational changes within this key domain and, in turn, resulting in an activated form of the PKC isoform.

Our findings that activation of PKC in general (8) and PKC-₁ (11) and PKC- in particular are involved in protection of intestinal cells are supported by two recent pharmacological studies (54, 55). For example, Terres et al. (54) using intestinal T-84 cells showed that Helicobacter pylori-associated decreases in monolayer barrier resistance was inhibited in the presence of a PKC activator 12-O-tetradecanoylphorbol 13-acetate, thus suggesting a possible role for PKC in protection against bacterial-induced damage. Additionally, our findings on the ₁-isoform of PKC (11) and -isoform (current report) utilizing more specific and targeted molecular approaches further expand on these previous pharmacological reports and, we believe, now establish a novel biological function (protection) among the isoforms of PKC subfamilies. Furthermore, we have more recently identified activation of EGF receptor tyrosine kinase and then phospholipase C-1 as the upstream signal for EGF-induced, PKC-mediated protection of intestinal barrier and cytoskeletal integrity (10).

Our series of studies on PKC, to date, were designed to investigate possible beneficial effects of PKC isoform activation in the GI tract. Although our findings are consistent with other published pharmacological studies, several reports (3, 23, 47) have shown that activation of PKC in cellular models may lead to nonprotective effects and these may vary with different experimental conditions and cell types. For example, overexpression of PKC- leads to the disruption of pig kidney epithelial (LLC-PK1) cell monolayers (47). A recent pharmacological study also suggested that PKC- and PKC- appear to be involved in tumor necrosis factor--induced injury in intestinal (IEC-18) cells (59).

Our findings show that stable antisense inhibition of PKC- prevents all measures of EGF-induced protection in our intestinal cell model. Whereas the mechanism for this inhibitory effect on EGF protection needs to be fully established, this attenuation is consistent with our data showing substantial downregulation of PKC- expression by its antisense inhibition as well as by parallel inhibition of three separate EGF-related protective variables: barrier integrity, tubulin assembly, and microtubule stability. A question that remains to be answered is how antisense to PKC- prevents EGF protection. We now suggest a mechanism by which the PKC- downregulation prevents EGF-induced protection: protein phosphorylation by PKC- by any of several well-known cellular mechanisms, such as cytoskeletal phosphorylation/dephosphorylation. This mechanism is consistent with our previous reports that EGF protection is mediated through stabilization of the assembly of the tubulin-based cytoskeleton (6, 8) and that EGF-induction causes PKC to phosphorylate tubulin and enhance tubulin assembly, which correlates significantly (r = 0.90 and 0.88, respectively; P < 0.05 for each) with EGF protection of microtubule stability and of barrier integrity (11). This proposed mechanism is further consistent with several reports in non-GI models that PKC activation phosphorylates and stabilizes cytoskeletal proteins (25, 28). Because cytoskeletal assembly and stability is critical in cellular protection, it follows that downregulation of PKC- (by antisense) can prevent essential protein phosphorylation, thereby preventing EGF protection of cytoskeletal integrity. For example, we recently reported (11) that EGF or the PKC activator OAG led to an enhancement of serine phosphorylation of the tubulin (50 kDa) subunit protein of the microtubules. This increase was substantially attenuated by antisense inhibition of PKC-₁, suggesting that PKC may be acting, directly or indirectly, on the tubulin-based cytoskeleton. It is possible, therefore, that activated PKC-₁ or activated PKC- phosphorylates the same or similar cytoskeletal or membrane targets. This proposed mechanism in our GI model is consistent with several previous studies in non-GI models. For instance, PKC has been shown to be involved in remodeling of the cytoskeletal filaments (2, 22, 25, 28, 40), although it has not been clearly established which PKC isoforms are essential in these processes. PKC can phosphorylate the cytoskeletal proteins talin and vinculin (25). Also, a specific substrate for PKC, myristoylated, alanine-rich PKC substrate protein (MARCKS), has been suggested to be an actin cytoskeletal reorganizer (28). In particular, MARCKS activity is abolished by PKC-induced phosphorylation. It is also possible that PKC isozymes can phosphorylate tubulin-associated capping proteins (e.g., microtubule associated proteins).

Evidence exists for other possible mechanisms for protection by PKC. Our previous reports showed that certain antioxidants (5, 7, 9) or agents that normalize intracellular calcium homeostasis (8) prevent oxidative damage in our model. Therefore, enhancement of either of these mechanisms could conceivably underlie PKC protective effects. Studies are underway in our laboratory to determine to what extent PKC protection is mediated by either of these mechanisms.

In summary, it appears that PKC- is responsible for a substantial portion of normal protection of the GI mucosal epithelium and perhaps is key to preventing amplification and perpetuation of an uncontrolled, oxidant-induced, inflammatory cascade that can be ignited by free radicals and other oxidants present in the GI tract. By creating the first GI cells stably overexpressing "protective PKC isoforms," our laboratory has discovered that these PKC isoforms possess critical functions in protecting cells against oxidative stress. This new knowledge may prove useful because increasing the activity of protective PKC isoforms through activation of endogenous PKC or using PKC mimetics may lead to novel therapeutic strategies for the treatment of a wide variety of oxidant-induced inflammatory disorders of the GI tract, including IBD.

Finally, our proposed mechanism of protection against oxidative stress has laid the groundwork for future "translational research" in humans and animals. We envision that these in vitro experiments will lead to highly focused studies that will test the clinical relevance of these potentially key biochemical pathways in IBD. For example, we (A. Keshavarzian, A. Banan, S. Kommandori, Y. Zhang, and J. Z. Fields, unpublished observations) have shown that a number of these oxidative reactions also occur in intestinal mucosa from patients with IBD. An important question that remains to be answered is whether modulation of PKC activity in vivo might also prevent oxidative damage.