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Protein and Vesicle Trafficking, Cytoskeleton
1 isoform activation is required for EGF-induced NF-
B inactivation and IB
stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers
Division of Digestive Diseases, Department of Internal Medicine, Department of Pharmacology,and Department of Molecular Physiology, Rush University Medical Center, Chicago, Illinois 60612
Submitted 31 July 2003 ; accepted in final form 2 November 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-
1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-1 prevents oxidant-induced activation of NF-B and the consequences of NF-B activation, F-actin, and barrier dysfunction. We used wild-type (WT) and transfected cells. The latter were transfected with varying levels of cDNA to overexpress or underexpress PKC-1. Cells were pretreated with EGF or PKC modulators ± oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-1 activity, decreasing IB
phosphorylation/degradation, suppressing NF-B activation (p50/p65 subunit nuclear translocation/activity), enhancing stable actin (increased F-actin-to-G-actin ratio), increasing stability of actin cytoskeleton, and reducing barrier hyperpermeability. Cells stably overexpressing PKC-1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IB stabilization, NF-B inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-1 substantially prevented all measures of EGF's protection against NF-B activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-1 activation and NF-B suppression. The molecular event underlying this unique effect of PKC-1 involves inhibition of phosphorylation and increases in stabilization of IB. The ability to inhibit the dynamics of NF-B/IB and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms. F-actin; cytoskeleton; growth factors; Caco-2 cells; gut barrier; protection; protein kinase C isoforms
Barrier disruption (hyperpermeability) has been implicated in the pathogenesis of a wide range of gastrointestinal and systemic disorders, including inflammatory bowel disease (IBD) (3, 30, 31, 36, 37). For instance, intestinal hyperpermeability ("leaky gut") has often been reported and implicated in the pathogenesis of IBD (see, e.g., Refs. 29-31, 58). Although the pathophysiology of mucosal barrier disruption in IBD is not fully characterized, it is known that chronic gut inflammation in IBD is associated with high levels of oxidants such as H2O2 (3, 11, 18, 36, 38, 39, 42) as well as activation of NF-B (17, 20, 49, 53, 55). Indeed, both oxidants and NF-B have been implicated in mucosal injury and inflammation in IBD (4, 5, 49, 36, 37, 53, 55, 58). NF-B is composed of two subunits (p50 and p65), and its activation is tightly regulated by an endogenous cytoplasmic inhibitor, IB (34, 47). Once activated, NF-B appears to regulate several important cellular events involved in inflammatory response including the upregulation of oxidative processes (17, 20). We recently reported (4, 5) on the importance of NF-B-dependent mechanisms in oxidant-induced barrier disruption. Accordingly, understanding how gut barrier function is protected against oxidative, proinflammatory NF-B conditions is of fundamental clinical and biological value.
We have been investigating endogenous defensive mechanisms against oxidant- and NF-B-induced mucosal damage and barrier disruption not only to better understand endogenous protective mechanisms (e.g., by growth factors such as EGF) but also to devise a rational basis for the development of potentially more effective treatments. Using monolayers of intestinal Caco-2 cells, we reported (2, 3, 6, 9, 10, 12, 13) that cytoskeletal depolymerization and instability is a critical event in oxidant-induced barrier disruption and that growth factor [EGF or transforming growth factor- (TGF-)] prevents damage by stabilizing the cytoskeleton through the activation of protein kinase C (PKC).
The PKC family includes at least 12 known isoforms that can be classified into three subfamilies (1, 7, 9, 10, 13,, 24, 32, 40, 51): the classic isoforms (, 1, 2, 
), the novel isoforms (
, 
, 
, 
, µ), and the atypical isoforms (
, 
, 
). Intestinal cells (e.g., Caco-2) express at least 10 isoforms of PKC, including PKC-, PKC-1, PKC-2, PKC-, PKC-, PKC-, PKC-, PKC-, PKC-, and PKC- (9, 10, 13, 24, 41). Because these isozymes are different in their intracellular distribution, expression, substrate type, and activation, it is thought that each isoform may perform unique tasks in signal transduction (8, 13, 43, 46, 50, 51). We showed (9, 10) with wild-type intestinal Caco-2 cells that EGF induces the membrane association of the native 1- and -isoforms of PKC (i.e., PKC-1 and PKC-) and therefore considered each as a possible contributor to EGF-afforded protection against oxidant-induced disruption. Using transfected cells, we then found (13) that PKC-, an atypical, diacylglycerol (DAG)-independent isoform of PKC, is required for a fraction, but not all, of the protection of the monolayer barrier function via inactivation of NF-B-dependent mechanisms.
In the current report, we have explored the role of the 1-isoform of PKC in EGF protection of the monolayer barrier and F-actin function against NF-B activation. We therefore tested the hypothesis that EGF-mediated protection against oxidant-induced NF-B activation and IB degradation and the consequent injury to both the F-actin cytoskeleton and barrier integrity of epithelial monolayers depend on activation of the PKC-1 isoform. To this end, we utilized both pharmacological and targeted molecular interventions that enabled us to use several stably transfected intestinal cell lines we have recently developed: in several clones the classic (78 kDa) isoform PKC-1 was reliably overexpressed; in the others, PKC-1 expression was almost completely inhibited. We now report novel mechanisms—EGF-mediated prevention of the stress of NF-B activation and of F-actin cytoskeletal depolymerization and disruption— of the classic 1-isoform of PKC in epithelial monolayers.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmids and stable transfection. The sense and antisense plasmids of PKC-1 were constructed and then stably transfected with Lipofectin (GIBCO BRL) as we previously described (9). Expression was controlled by -actin promoter.
Cultures of Caco-2 cells grown to 50-60% confluence were cotransfected with G-418 resistance plasmid and expression plasmids encoding either sense PKC-1 or antisense PKC-1 by Lipofectin. Control conditions included vector alone. After transfection, cells were subjected to G-418 selection. Resistant cells were maintained in culture medium-FBS and 0.2 mg/ml G-418. Multiple clones stably overexpressing PKC-1 or lacking PKC-1 were assessed by immunoblotting and then used for experiments. We confirmed that overexpression of PKC-1 or antisense inhibition of PKC-1 did not affect the relative expression levels of other PKC isoforms and did not injure the Caco-2 cells.
Experimental design. First, postconfluent monolayers of wild (naive)-type cells were preincubated with EGF (1-10 ng/ml) or isotonic saline for 10 min and then exposed to oxidant (H2O2, 0-0.5 mM) or vehicle (saline) for 30 min. H2O2 at 0.1-0.5 mM disrupts F-actin and barrier integrity and activates NF-B (2-4, 9-11, 15, 18). EGF at 10 ng/ml (but not 1 ng/ml) prevents both actin and barrier disruption. These experiments were then repeated with stably transfected cells. In all experiments we assessed actin stability (cytoarchitecture, assembly), F-/G-actin pools, IB levels (expression, phosphorylation, degradation), NF-B subunit (p65/p50) activity (cytosolic levels, nuclear translocation and activity), and PKC-1 subcellular distribution/activity (immunoblotting, in vitro kinase assay).
Second, monolayers that were stably overexpressing PKC-1 were preincubated (10 min) with nonprotective (low) or protective (high) doses of EGF (1 and 10 ng/ml, respectively) or vehicle before exposure (30 min) to a damaging concentration of oxidant (H2O2, 0.5 mM) or vehicle. Outcomes measured were as described above. In other experiments, the same clones were preincubated (10 min) with low or high doses of the PKC modulator 1-oleoyl-2-acetyl-sn-glycerol (OAG, a synthetic DAG; 0.01 or 50 µM) or vehicle before exposure (30 min) to H2O2 (0.5 mM) or vehicle. The vehicle solution for OAG was 0.02% ethanol.
Third, monolayers of antisense-transfected cells stably lacking PKC-1 activity were treated with high doses of EGF and then oxidant. In a corollary series of experiments, we investigated the effects of PKC-1 under- or overexpression on the state of IB degradation and phosphorylation, NF-B activation, F-/G-actin assembly, and actin cytoarchitecture.
Fractionation and immunoblotting of PKC. Cell monolayers grown in 75-cm2 flasks were processed for isolation of the cytosolic, membrane, and cytoskeletal fractions (9). Protein content of the various cell fractions was assessed by the Bradford method (21). For immunoblotting, samples (25 µg protein/lane) were added to a standard SDS buffer, boiled, and then separated on 7.5% SDS-PAGE (9). The immunoblotted proteins were incubated with the appropriate primary monoclonal antibody (e.g., anti-PKC-1) (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1,000 dilution. A horseradish peroxidaseconjugated antibody (Molecular Probes, Eugene, OR) was used as a secondary antibody at 1:3,000 dilution. Proteins were visualized by enhanced chemiluminescence (ECL; Amersham) and autoradiography and subsequently analyzed by densitometry. The identity of the PKC isoform bands (e.g., 78-kDa PKC-1) was confirmed as we described previously (7-10, 13).
Immunoprecipitation and PKC isoform activity assay. Immunoprecipitated PKC-1 or PKC- was collected separately and processed for its ability to phosphorylate a synthetic peptide (7, 13). Briefly, after treatments, confluent cell monolayers were lysed by incubation for 20 min in 500 µl of a standard cold-lysis buffer and then immunoprecipitated by corresponding monoclonal anti-PKC-1 or - (1:2,000 dilution, in excess). The immunocomplexes were collected by centrifugation (2,500 g, 5 min) in a microfuge tube and washed three times with a standard immunoprecipitation buffer. They were then washed once with kinase buffer (in mM: 20 HEPES pH 7.5, 10 MgCl2, 2 MnCl2, and 0.1 CaCl2 with 20 µM ATP) and resuspended in 20 µl of kinase buffer and 5 µl of 5x reaction buffer (0.25 mg/ml L--phosphatidyl-L-serine and 1 mg/ml histone H1) plus 5 µCi of [-32P]ATP and subsequently incubated for 5 min at 30°C. Reactions were then stopped by the addition of 8 µl of 5x sample buffer, and the samples were boiled at 95°C for 5 min before separation by 7.5% PAGE. The extent of histone H1 phosphorylation was determined by scintillation counting of excised Coomassie blue-stained polypeptide bands. Counts for blanks were subtracted from the sample activity. Sample activity was also corrected for protein concentration (Bradford method), and PKC-1 or PKC- activity was reported as picomoles per minute per milligram of protein.
Analysis of NF-B activation. NF-B (both p65 and p50 subunits) activation was assessed by a unique ELISA procedure (4, 52). Cells grown in 25-cm2 flasks were processed for the isolation of the cytosolic and nuclear fractions. Cell fractions were added to a 96-well plate to which oligonucleotides containing a consensus binding site for NF-B had been immobilized (Trans-Am; Active Motif, Carlsbad, CA). The binding of NF-B to its consensus sequence was then detected with a primary anti-NF-B (p65 or p50) antibody (Santa Cruz Biotechnology), followed by a secondary antibody conjugated to horseradish peroxidase. The results were quantitated by a chromogenic reaction (52), which was then read for absorbance at 450 nm by a Seivers NOA 280 microplate analyzer.
Western blot analysis of changes in NF-B subunit levels and nuclear translocation. Cellular nuclear and cytosolic extracts from naive and transfected cells were prepared as described in Fractionation and immunoblotting of PKC. NF-B nuclear translocation was determined by comparing the levels of NF-B protein expression in the cytosolic vs. nuclear extracts by anti-p65 and anti-p50 antibodies with a nondenaturing gel (6%) (4, 34).
Western blot analysis of IB degradation, expression levels, and phosphorylation. IB levels of expression in the cytosolic extracts and its degradation (i.e., disappearance from cytosol) were assessed by anti-IB antibody (Santa Cruz Biotechnology) with a standard Western blot protocol (10% gel) (4, 47). IB phosphorylation was assessed by with anti-phospho-IB (Ser32/36). Proteins were visualized by enhanced chemiluminescence and subsequently autoradiographed.
Determination of cell oxidative stress. Oxidative stress was assessed by measuring the conversion of a nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (DCFD) (Molecular Probes) into a fluorescent dye, dichlorofluorescein (DCF) (2, 6, 11). Briefly, monolayers grown in 96-well plates were preincubated with the membranepermeant DCFD (10 µg/ml for 30 min) before treatments. Fluorescent signals (i.e., DCF fluorescence) from samples were quantitated with a fluorescence multiplate reader set at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
Immunofluorescent staining and high-resolution laser scanning confocal microscopy of actin cytoskeleton. Cells from monolayers were fixed in cytoskeletal stabilization buffer and then postfixed in 95% ethanol at -20°C (8, 11, 16, 18). Cells were subsequently processed for incubation with FITC-phalloidin (specific for F-actin staining; Sigma, St. Louis, MO), at 1:40 dilution for 1 h at 37°C. After staining, cells were observed with an argon laser ( = 488 nm) with a x63 oil immersion plan-apochromat objective (NA 1.4; Zeiss). The cytoskeletal elements were examined in a blinded fashion for their overall morphology, orientation, and disruption (2, 3, 8, 10, 11, 14, 15, 18). The identity of the treatment groups for all slides was decoded only after examination was complete.
Actin fractionation and quantitative Western immunoblotting of F-actin and G-actin. Polymerized (F-) and monomeric (G-) actin were isolated with a method we described previously (8, 11, 18). Isolated actin samples were then flash frozen in liquid N2 and stored at -70°C until immunoblotting. For immunoblotting, samples (5 µg protein/lane) were placed in a standard SDS sample buffer, boiled, and then subjected to PAGE on 7.5% gels. Standard (purified) actin controls (5 µg/lane) were run concurrently with each run. To quantify the relative levels of actin, the optical density (OD) of the bands corresponding to immunolabeled actin was measured with a laser densitometer.
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 with analysis of variance followed by Dunnett's multiple-range test (28). Correlational analyses were done with the Pearson test for parametric analysis or, when applicable, the Spearman test for nonparametric analysis. P values < 0.05 were deemed statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 sense stably overexpress the classic 1 (78 kDa) isoform of PKC (
3.1 fold compared with wild-type/naive cells) (Fig. 1A; see immunoblot) and that this overexpression synergizes with low (nonprotective) doses of PKC modulators (e.g., 0.01 µM OAG) or growth factor (e.g., 1 ng/ml EGF) to protect monolayer barrier integrity [fluorescein sulfonic acid (FSA) clearance] against exposure to oxidant challenge. In contrast, in wild-type cells, higher (protective) doses of OAG (50 µM) or EGF (10 ng/ml) were required for protection of barrier integrity (9).
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Key role of PKC-1 isoform in EGF-mediated protection against oxidant-induced NF-B activation: Suppression of nuclear translocation and activation of both NF-B subunits. We surmised that this protection may be due to the inhibition of oxidant-activated pathways such as the proinflammatory pathway triggered by NF-B. Therefore, using our wild-type and transfected clones, we assessed the nuclear translocation of NF-B subunits (p65 and p50) and their activation under oxidant challenge. In wild-type cells (those not overexpressing PKC-1), oxidant (H2O2) alone caused a substantial activation of both p50 and p65 subunits (Fig. 1). Overexpression of PKC-1 potentiated the ability of a low dose of EGF (1 ng/ml; Fig. 1) to prevent oxidant-induced NF-B subunit activation in transfected clones. In wild-type cells, the low dose of EGF did not suppress NF-B activation where higher doses of EGF (10 ng/ml) were required (Fig. 1). A similar synergy for suppression of NF-B was seen between PKC-1 and a low dose of the PKC modulator OAG (0.01 µM) in transfected clones (Table 1). In fact, OAG caused effects almost identical to those of EGF. Additionally, the extent of suppression of NF-B in PKC-1-overexpressing clones was not significantly different from that in wild-type cells of higher doses of these same modulators. As expected, transfection of the empty vector alone did not prevent oxidant-induced NF-B activation. For instance, the level of p65 subunit that was activated was 0.085 ± 0.02 (OD at 450 nm) for vector-transfected cells exposed to vehicle, 1.34 ± 0.08 for vector-transfected cells exposed to H2O2 alone, and 1.30 ± 0.11 for vector-transfected cells incubated with EGF (1 ng/ml) + H2O2 compared with 0.16 ± 0.03 for PKC-1 sense-transfected cells incubated with EGF (1 ng/ml) + H2O2. Similarly, the level of p50 subunit that was activated was 0.074 ± 0.04 for vector-transfected cells exposed to vehicle, 1.28 ± 0.12 for vector-transfected cells exposed to H2O2 alone, and 1.19 ± 0.15 for vector-transfected cells incubated with EGF (1 ng/ml) + H2O2 compared with 0.20 ± 0.04 for PKC-1 sense-transfected cells incubated with EGF (1 ng/ml) + H2O2. These alterations did not appear to be caused by changes in the ability of oxidants to cause NF-B activation because empty vector-transfected cells and wild-type cells responded in a similar fashion to H2O2, exhibiting comparable NF-B activation.
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Multiple clones of intestinal cells transfected with 1, 2, 4, or 5 µg of PKC-1 sense cDNA demonstrated a dose-dependent synergy with OAG (Table 1) or EGF (Table 2) to suppress NF-B activation that was induced by oxidant (H2O2). Because the clone transfected with 4 µg of PKC-1 sense (+4) provided maximum (88%) synergy suppression of NF-B, we utilized this stable sense clone for all subsequent experiments.
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Representative immunoblots for the alterations in NF-B subunit translocation into the nuclear fractions are shown in Fig. 2. These data further corroborate the aforementioned findings. PKC-1 overexpression synergizes with a low dose of EGF (1 ng/ml) to suppress oxidant-induced nuclear translocation of NF-B subunits p50 (Fig. 2A) and p65 (Fig. 2B), as shown by reduced band densities that are comparable to those of the controls. As before, only high doses of EGF (e.g., 10 ng/ml) prevented NF-B nuclear translocation in wild-type cells. On the other hand, exposure to oxidant led to the translocation of NF-B subunits to the nucleus in these wild-type cells, paralleling findings on NF-B activation.
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A time course for alterations in NF-B activation in wild-type cells and its inhibition in PKC-1-overexpressing clones in synergy with added EGF is shown in Fig. 3. Maximal fold increase in NF-B activation induced by H2O2 alone was 17; these increases were almost completely suppressed in PKC-1-overexpressing cells, where PKC-1 potentiated EGF protection.
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PKC-1-induced protection involves stabilization of cytosolic IB: Suppression of IB degradation. We then probed potential mechanisms through which PKC-1 prevents oxidant-induced NF-B activation. Because oxidants not only increase IB degradation but also disrupt monolayer barrier permeability (4), we surmised that suppression of the degradation of IB (a 37-kDa endogenous modulator of NF-B) could be a novel mechanism underlying protection by PKC-1.
To this end, multiple clones of cells stably transfected with PKC-1 sense showed a dose-dependent synergy with OAG (Table 1) or EGF (Table 2) to protect and stabilize IB against H2O2 exposure. The 4-µg clone of PKC-1 (+4) led to the highest level of protection against IB degradation, paralleling findings on NF-B inactivation.
Figure 4A shows the synergy-induced protective stabilization of IB by PKC-1 overexpression with the 4-µg sense clone [a clone that also protects gut barrier permeability, as we showed previously (9)]. Assessments of the cytosolic fractions of both transfected and untransfected Caco-2 monolayers indicated that only PKC-1-overexpressing clones led to a substantial decrease in oxidant-induced IB degradation (75% less degradation). Here, a low EGF concentration, 1 ng/ml, which did not stabilize IB in wild-type cells, did so in the transfected, PKC-1-overexpressing cells. This level of IB stability in the cytosol is nearly identical to that of the controls, which exhibit steady-state levels of IB. In wild-type cells, this dose of H2O2 results in both hyperpermeability (9) and IB instability (Fig. 4A). Without the synergy afforded by PKC-1 overexpression, wild-type cells pretreated with low doses of EGF showed substantial H2O2-induced degradation of IB. These wild-type cells, which have native levels of PKC-1, required a higher dose of EGF (10 ng/ml; Fig. 4A) to stabilize IB. Transfection of only the empty vector did not confer protection against oxidant-induced IB degradation [IB levels were 100 ± 1% (arbitrary units) for vector-transfected cells exposed to vehicle, 11 ± 3% for vector-transfected cells exposed to H2O2 alone, and 17 ± 5% for vector-transfected cells incubated with EGF (1 ng/ml) + H2O2 compared with 92 ± 0.8% for PKC-1 sense-transfected cells incubated with EGF (1 ng/ml) + H2O2].
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Figure 4B is a representative immunoblot demonstrating that H2O2 substantially increased IB degradation levels in wild-type cells, whereas transfected cells overexpressing PKC-1 in synergy exhibited near steady-state levels of IB. The corresponding OD findings for control cells (5,424 ± 122), 0.5 mM H2O2-treated cells (471 ± 52), wild-type cells incubated with EGF (1 ng/ml) + H2O2 (551 ± 93), and PKC-1 sense-transfected cells incubated with EGF (1 ng/ml) + H2O2 (3,967 ± 111) demonstrated stabilization of IB in the PKC-1 clones. As expected, transfection of the vector alone was ineffective (not shown).
PKC-1-induced stabilization of cytosolic IB involves inhibition of Ser 32/36 phosphorylation of IB. We next probed potential mechanisms by which PKC-1 enhances IB stability such as alterations in IB phosphorylation. IB phosphorylation (i.e., phospho-IB) levels in both transfected and wild-type monolayers exposed to H2O2 are shown in Fig. 5. Similar to its protective effects against NF-B activation, PKC-1 overexpression synergized with low doses of EGF (1 ng/ml) to markedly reduce IB phosphorylation (Ser 32/36 phospho-IB). In wild-type cells, on the other hand, such suppression of IB phosphorylation was promoted only by high doses (e.g., 10 ng/ml) of EGF. In these same wild-type cells, oxidant led to substantial IB phosphorylation. Once again, transfection of only the empty vector was ineffective (not shown).
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Using immunoprecipitation analysis (Fig. 6), we further investigated possible mechanisms underlying the stabilizing affect of PKC-1 on IB. To this end, cells were immunoprecipitated with a monoclonal PKC-1 antibody and the immunoprecipitates were then analyzed for the presence of IB, examining whether this PKC isoform physically associates with IB. The resting (wild type) vehicle-treated cells did not show any association between these proteins (Fig. 6A). In EGF-pretreated wild-type cells, a small amount of IB coprecipitated with PKC-1. In contrast, the amount of IB coprecipitation was dramatically enhanced in transfected cells overexpressing PKC-1 in the presence of a low dose of EGF, indicating increased formation of a PKC-1-IB complex. OAG caused similar effects (not shown). Without the synergy-induced protection afforded by PKC-1 overexpression, wild-type (naive) cells pretreated with the same low dose of EGF showed little coprecipitation. In a reverse protocol (Fig. 6B), anti-IB antibody was utilized and immune complexes were then analyzed for the presence of PKC-1. As expected, PKC-1 was not detectable in the complex in wild-type/vehicle-treated cells (i.e., no coprecipitation with IB), whereas stable transfection in synergy resulted in an accumulation of IB-PKC-1 complex. In a third protocol, we further saw specificity of the formation of the PKC-1-IB complex. Here, probing cell lysates from another PKC isoform clone, the classic PKC-2-transfected clone, demonstrated no such physical association (not shown).
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In parallel with the inhibition of oxidant-induced affects, PKC-1 overexpression synergistically suppressed oxidative stress as measured by DCF fluorescence (Fig. 7). In wild-type cells, oxidative stress was suppressed only by high doses (10 ng/ml) of EGF. Without oxidant, substantially lower levels of oxidative stress were observed.
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Inhibition of NF-B in transfected cells protects both actin assembly and cytoarchitecture of F-actin cytoskeleton. Because oxidants are known to disrupt the cytoskeleton in our Caco-2 model (8), and because such disruption is required for barrier hyperpermeability (9), we measured the state of F-actin cytoskeleton in wild-type and transfected cells. PKC-1 overexpression in synergy with OAG (Table 1) or EGF (Table 3 and Figs. 8 and 9) conferred protection to the actin cytoskeleton. For instance, PKC-1 overexpression synergized with low doses of OAG or EGF to protect the actin cytoskeleton, as shown by the high percentage of cells with normal actin (Tables 1 and 3, respectively). Transfection of only the empty vector was not protective [for example, % normal actin = 97 ± 3 for vector-transfected cells exposed to vehicle, 41 ± 5 for vector transfected cells exposed to H2O2, and 43 ± 4 for vector transfected cells incubated with EGF (1 ng/ml) + H2O2 compared with 88 ± 6 for PKC-1 sense-transfected cells incubated with EGF (1 ng/ml) + H2O2].
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Fluorescent images obtained by high-resolution laser scanning confocal microscopy (Fig. 8) show that overexpression of PKC-1 potentiates protection by low doses of EGF (Fig. 8D). This protection is shown by the appearance of normal, intact, and smooth architecture of the F-actin ring at the areas associated with the plasma membrane (i.e., areas of cell-cell contact). The appearance of the F-actin ring in these transfected clones was almost identical to that of the untreated normal cells, which also showed an intact pattern of the F-actin ring (Fig. 8A). Without the synergy afforded by PKC-1 overexpression (and modulators), wild-type cells pretreated with the low dose of EGF and exposed to H2O2 showed extensive disorganization, kinking, condensation, and beading of the F-actin ring (Fig. 8C), as did wild-type cells exposed to H2O2 alone (Fig. 8B) or transfected cells exposed to H2O2 only (Fig. 8E).
Immunoblotting analysis of F-actin (S2, an index of actin integrity) and G-actin (S1, an index of actin disruption) (Fig. 9A) further demonstrates that only the transfected cells overexpressing PKC-1 exhibited a protective synergy between EGF (1 ng/ml) or OAG (0.01 µM; not shown) and PKC. This protection is indicated by normal actin assembly, which is comparable to the controls. H2O2 alone, in contrast, reduced polymerized F-actin and increased monomeric G-actin in both PKC-1-overexpressing cells without added EGF and wild-type cells, indicating disruption of actin assembly. Pretreatment of wild-type cells with only the higher doses of EGF (10 ng/ml) resulted in normal levels of actin assembly.
A representative Western blot of the polymerized F-actin (Fig. 9B) extracted from monolayers further confirmed the above findings. The protection of both the assembly and the structure of the F-actin cytoskeleton by PKC-1 overexpression paralleled the protective effects of PKC-1 expression against oxidant-induced NF-B activation and IB degradation.
Activation of overexpressed PKC-1 in transfected intestinal cells correlates with several different indexes of NF-B in cell monolayers. After pretreatment with low doses of EGF or OAG, there was a redistribution of the 78-kDa PKC-1 isoform into mostly particulate cell fractions (particulate = membrane + cytoskeletal fractions) with a much smaller distribution in the cytosolic fractions (9), indicating the induced activation of the 1-isoform (see Table 4). Overexpressed PKC-1 isoform is "actively induced" because achieving this intracellular distribution did require EGF or OAG. There was redistribution of native PKC-1 into particulate fractions of wild-type cells only after higher doses of EGF or OAG. In contrast, control untreated cells or cells exposed to oxidant alone showed a mostly cytosolic distribution of PKC-1 (suggesting inactivity), with smaller pools in the membrane and cytoskeletal (particulate) fractions.
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Figure 10 shows the activity measurements for PKC-1 isoform (assessed by a sensitive in vitro kinase assay) from immunoprecipitated particulate cell fractions of intestinal cells of either transfected or wild-type origin with or without EGF. There was a substantial increase in the activity levels of the PKC-1 isoform in these transfected cells (in synergy with added EGF), paralleling findings for other outcomes. Wild-type cells exposed to vehicle showed basal activity levels for PKC-1 in the particulate cell fractions. In these wild-type cells, as might be predicted, EGF further activated native PKC-1, but at lower levels than those of transfected cells under similar conditions. Our findings (Table 4 and Fig. 10) demonstrate that PKC modulators (OAG, EGF) activate the PKC-1 isoform by causing its redistribution from the soluble (cytosolic) pool to the particulate pools (membrane and cytoskeletal).
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Using data across all experimental conditions, we found significant inverse correlations (e.g., r = -0.94; P < 0.05) between PKC-1 activity (in vitro kinase assay or OD from the particulate fraction) and NF-B inactivation, further suggesting that activation of the 1-isoform is critical to protection against oxidant-induced NF-B activation. Similarly, when either NF-B nuclear translocation or IB degradation was correlated with PKC-1 levels other robust correlations were observed (r = -0.93 and -0.88, respectively; P < 0.05 for each). Additional robust correlations were seen when other markers of stability such as actin integrity or F-actin assembly were correlated with PKC-1 levels (r = 0.94 and 0.93, respectively; P < 0.05 for each). We found still other supporting correlations such as those between 1) IB phosphorylation or IB stabilization and 2) PKC-1 activation (r = -0.90 and 0.89, respectively; P < 0.05 for each), further suggesting that activation of 1-isoform is key to NF-B inactivation via IB stabilization.
Stable antisense inhibition of PKC-1 to inactivate native 1-isoform and its substantial attenuation of EGF-induced NF-B inactivation and IB stabilization. As indicated by the above findings, PKC-1 can have a key intracellular function in protection against NF-B activation. To further investigate the potential role of this isoform in EGF-mediated protection against NF-B activation, we used an independent approach involving stable antisense transfection of PKC-1 to create clones of Caco-2 cells. Using these recently developed antisense PKC-1 clones from our laboratory (9), we can substantially (-99.5%) reduce the steady-state activity of the native PKC-1 isoform (Fig. 10). Not surprisingly, in this antisense clone, EGF can no longer enhance native PKC-1 isoform activity.
The dose-dependent inhibitory effects of varying amounts (1, 2, 4, or 5 µg) of PKC-1 antisense plasmid on attenuation of OAG- or EGF-induced NF-B inactivation and IB stabilization are shown in Tables 1 and 2, respectively. The 4-µg antisense clone of PKC-1 caused maximum inability of EGF (or OAG) to suppress both oxidant-induced NF-B activation and IB degradation and was thus subsequently utilized.
Because the atypical -isoform of PKC is also key to EGF-induced protection against oxidant-induced NF-B activation (13), we first confirmed that antisense inhibition of PKC-1 does not affect the relative expression and activity levels of PKC-. PKC- activity was 39 ± 8 pmol·min-1·mg protein-1 in clones transfected with PKC-1 antisense, which was comparable to 40 ± 5 pmol·min-1·mg protein-1 in untransfected (wild type) cells. Densitometric analysis of protein expression levels for PKC- further confirms these findings. PKC- protein expression was 2,199 ± 96 (OD) in PKC-1 antisense clones compared with 2,257 ± 73 in wild-type cells. Our findings on other PKC isoforms (Table 5) further confirm that antisense to PKC-1 does not affect the relative expression levels of other PKC isoforms in our intestinal model.
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Figure 11 shows that stable antisense inhibition of native PKC-1 substantially attenuated the protection mediated by high (protective) doses of EGF (e.g., 10 ng/ml) against NF-B activation induced by oxidant (p65 subunit activity shown). This same dose of EGF almost completely suppressed oxidant-induced NF-B activation in wild-type (naive) cells. In fact, a large percentage (60%) of EGF-induced NF-B inactivation appears to be PKC-1 dependent. PKC-1 inactivation did not alter basal NF-B activity.
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Assessment of both IB levels (Fig. 12A) and IB phosphorylation (Fig. 12B) in the same antisense clones additionally demonstrated that the absence of native PKC-1 isoform activity substantially attenuates both EGF's enhancement of IB stabilization (Fig. 12A) and reduction of IB phosphorylation (Fig. 12B). As for effects on NF-B, a substantial percentage (60%) of EGF's stabilization of IB is PKC-1 dependent in these intestinal cells.
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Measurements of oxidative stress (Fig. 13) from these antisense clones further demonstrated that inactivation of the PKC-1 isoform largely attenuates EGF-induced prevention of oxidative stress (failure to decrease DCF fluorescence). Finally, immunoblotting analysis of F-actin assembly shows (Fig. 14) that stable antisense suppression of PKC-1 prevents protection against F-actin disassembly by a high dose of EGF, attenuating EGF's protection against oxidant-induced actin depolymerization. PKC-1 isoform inactivation by itself did not affect th
