Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

A fundamental property of the epithelium of the gastrointestinal (GI) mucosa is the ability to maintain a highly selective permeability barrier (Unno et al., 1996; Menconi et al., 1997; Hollander, 1998; Banan et al., 1999; Keshavarzian et al., 1999). We (Banan et al., 1996, 1998a,b, 1999, 2000a,b,c, 2001a,b,c,d,e, 2002) and others (Unno et al., 1996; Menconi et al., 1997) have shown that the integrity of the intestinal barrier depends on the stability of complex intracellular networks of the cytoskeletal elements. Among these elements, the actin cytoskeleton, especially the apical ring of actin cortex, plays a key role in the maintenance of an intact GI epithelial barrier (Banan et al., 1996, 2000c, 2001a,e; Unno et al., 1996; Menconi et al., 1997). Actin is the principal protein in the cell cortex localized immediately inside the plasma membrane at areas of cell-to-cell contact and represents a critical structural component. As such it plays a crucial role in maintaining the structure of the cytoplasmic matrix, cell shape, and barrier integrity (permeability).

Intestinal barrier integrity is of clinical and biological importance because this barrier normally restricts the passage of harmful pro-inflammatory and toxic molecules (e.g., bacterial endotoxin, immunoreactive antigens) into the mucosa and systemic circulation (Hollander, 1998; Keshavarzian et al., 1999). Loss of mucosal barrier integrity, on the other hand, is characteristic of multiple organ system dysfunction, inflammatory bowel disease, necrotizing enterocolitis, 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) (Unno et al., 1996; Menconi et al., 1997; Hollander, 1998; Keshavarzian et al., 1999). Although the pathogenesis of mucosal barrier disruption in these illnesses remains unclear, several studies, including our own, have shown that chronic gut inflammation is associated with high levels of oxidants (e.g., H₂O₂) and that oxidative damage is a key contributor to loss of barrier integrity and injury (Keshavarzian et al., 1992; McKenizie et al., 1996; Kimura et al., 1998; Banan et al., 2000a,b,c, 2001a). Not surprisingly, damage to the actin cytoskeleton (especially to the cortical ring of F-actin) can lead to a leaky, hyperpermeable gut, and this damage has been proposed as a key underlying mechanism for the initiation and perpetuation of these oxidant-induced inflammatory disorders. Thus, characterizing the intracellular signaling mechanisms underlying the protection of F-actin is both clinically and biologically important.

In our concerted efforts to enhance our understanding of endogenous protective mechanisms for the cytoskeleton and to provide new insights that might lead to developing more effective treatment regimens for oxidative and inflammatory disorders associated with loss of intestinal barrier integrity, we have been investigating the underlying protective mechanisms used by growth factors to stabilize the cytoskeletal network. Utilizing monolayers of human intestinal (Caco-2) cells exposed to oxidants as a model of cytoskeletal and barrier disruption, we previously showed that growth factors (e.g., EGF or transforming growth factor-) protect intestinal barrier integrity in part by stabilizing the assembly of the apical ring of the F-actin cytoskeleton (Banan et al., 2000c, 2001a,e). We have also shown that the stability of actin is key in mucosal healing under in vivo (Banan et al., 1996) and in vitro (Banan et al., 2000c, 2001a,e) conditions. Stability is based on the ability of monomeric G-actin to polymerize and on the stability of F-actin polymers to resist disassembly. Despite the critical importance of the F- and G- actin cytoskeleton in the maintenance of intestinal barrier integrity, the intracellular signaling mechanism through which EGF stabilizes the actin remains elusive.

In previous studies using naive Caco-2 cells, we also showed that Ca²⁺ is crucial in the maintenance of normal mucosal barrier integrity (Kokoska et al., 1998; Banan et al., 2001b) and that EGF protects the monolayer barrier via normalization of intracellular Ca²⁺ ([Ca²⁺]_i) levels through enhancement of protein kinase C (PKC) in general (Banan et al., 2001b). The specific isoform of PKC responsible for this protective effect of EGF on Ca²⁺ homeostasis remains unknown. Utilizing the first developed stably transfected intestinal (Caco-2) cell lines over-expressing or under-expressing PKC, we recently demonstrated that EGF maintains monolayer barrier permeability, in large part, by increasing activity and membrane association of the 1 isoform of PKC (Banan et al., 2001c).

In view of the aforementioned considerations, we hypothesized that the PKC-1 isoform not only is essential to EGF-induced protection of the dynamic assembly of the F- and G-actin cellular pools and the stabilization of the cortical actin ring, but it is key to the normalization of Ca²⁺ homeostasis. To this end, we utilized pharmacological and targeted molecular interventions employing several novel and stably transfected intestinal cell lines we have recently developed. In several clones the classical isoform PKC-1 was reliably over-expressed; in other clones, PKC-1 expression was inhibited. Herein, we report novel biologic functions, protection of actin and Ca²⁺ balance, by the classical 1 isoform of PKC.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Culture. Caco-2 cells, which were obtained from American Type Culture Collection (Manassas, VA) at passage 15, were chosen because they form monolayers that morphologically resemble small intestinal cells, with defined apical brush borders, junctional complexes, and a highly organized actin network (Gilbert et al., 1991; Meunier et al., 1995; Banan et al., 1998b). Cells were maintained at 37°C in complete Dulbecco's minimum essential medium in an atmosphere of 5% CO₂ and 100% relative humidity. Naive or stably transfected cells (see below) were split at a ratio of 1:6 upon reaching confluence and set up in either 6- or 24-well plates for experiments or T-75 flasks for propagation. Cells grown for barrier function experiments were split at a ratio of 1:2 and seeded at a density of 200,000 cells/cm² into 0.4 µM Biocoat collagen I cell culture inserts (0.3-cm² growth surface; BD Biosciences-Discovery Labware, Bedford, MA), and experiments were performed at least 7 days post-confluence. The media were changed every 2 days. The utility and characterization of this cell line has been previously reported (Gilbert et al., 1991; Meunier et al., 1995; Banan et al., 1998b).

Plasmid. The sense and antisense plasmids of PKC-1 were constructed as we previously described (Cho et al., 1998; Banan et al., 2001c,d). Expression was controlled by -actin promoter. The antisense PKC-1 plasmid (p-actin SP72-As-PKC-1) was constructed by ligating the 2.3-kb EcoRI fragment of PKC-1 cDNA from pJ6-PKC-1 (Cho et al., 1998) into the unique EcoRI sites of the p-actin SP72 vector. The antisense orientation of the plasmid was confirmed by SamI restriction digestion (Cho et al., 1998).

Stable Transfection. Cultures of Caco-2 cells grown to 50 to 60% confluence were cotransfected with G-418 (selection) resistance plasmid and expression plasmids encoding either PKC-1 or antisense-PKC-1 by Lipofectin (Lipofectin reagent; Invitrogen, Carlsbad, CA) as we previously described (Banan et al., 2001c). Control conditions included vector alone. Briefly, cells were incubated for 16 h at 37°C with the plasmid DNA in serum-free media in the presence of LipofectAMINE (25 µl/25-cm² flask). Subsequently, the DNA-containing solution was removed and replaced by fresh media containing 10% fetal bovine serum to relieve cells from the shock of exposure to serum-free media. Following transfection, cells were subjected to G-418 selection (0.6 mg/ml) over 4 weeks. Resistant cells were maintained in Dulbecco's minimum essential medium/fetal bovine serum and 0.2 mg/ml G-418 (selection medium). PKC protein expression or lack of it was verified by Western blot analysis of cell lysates (see below). Multiple clones stably over-expressing PKC-1 or lacking PKC-1 were assessed by immunoblotting, plated on cell culture inserts, allowed to form confluent monolayers, and subsequently used for experiments.

Experimental Design. In the first series of experiments, post-confluent monolayers of naive Caco-2 cells were preincubated with EGF (1 to 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 we have previously shown (Banan et al., 2000c, 2001a,e), H₂O₂ at 0.5 mM disrupts actin and barrier integrity; EGF at 10 ng/ml (but not 1 ng/ml) prevents this disruption. These experiments were then repeated using monolayers composed of cells either stably over-expressing or almost completely lacking PKC-1. Reagents were applied on the apical side of monolayers unless otherwise indicated. Since our previous studies (Banan et al., 2000a,c) showed that regardless of whether apical or basolateral exposure of oxidants was used, the results were qualitatively similar; all current studies utilized apical application. In all experiments, actin cytoskeletal stability (ring cytoarchitecture, assembly, disassembly), PKC-1 subcellular distribution, and Ca²⁺ homeostasis ([Ca²⁺]_i and Ca²⁺ efflux) were assessed.

Immunofluorescent Staining and High-Resolution Laser Scanning Confocal Microscopy of Actin. Cells from monolayers were fixed in cytoskeletal stabilization buffer and then post-fixed in 95% ethanol at 20°C, as we previously described (Banan et al., 2000c, 2001a). Cells were subsequently processed for incubation with fluorescein isothiocyanate-phalloidin (specific for F-actin; Sigma-Aldrich, St. Louis, MO), 1:40 dilution, for 1 h at 37°C. Slides were washed three times in D-phosphate-buffered saline, once with deionized H₂O, and subsequently mounted in Aquamount (Fisher Scientific, Fair Lawn, NJ). Following staining, cells were observed with an argon laser ( = 488 nm) using a 63× oil immersion plan-apochromat objective, NA 1.4 (Carl Zeiss GmbH, Jena, Germany). Desired areas of monolayers were processed using the image processing software on a Zeiss Ultra high-resolution LSCM (Carl Zeiss). The apical (cortical) rings of actin, which are known to regulate paracellular permeation through monolayers of Caco-2 cells, were examined in a blinded fashion for their overall morphology and disruption as we previously described (Banan et al., 2000c, 2001a,e). At least 1200 cells per group (200 × 6 slides) were examined in four different fields by LSCM, and the percentage of cells displaying normal actin ring was determined. The slides were decoded only after examination was complete.

Actin Fractionation and Quantitative Immunoblotting of F- and G-Actin State of Assembly and/or Disassembly. Polymerized (F-) and monomeric (G-) fractions of actin were isolated as we previously described (Banan et al., 2000c, 2001a,e). Cells were gently scraped and pelleted with centrifugation at low speed (700 rpm, 7 min, 4°C) and resuspended in actin stabilization-extraction buffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% dimethyl sulfoxide, 1 mM MgSO₄, 10 µg/ml anti-protease cocktail, 1 mM EGTA, and 1% Triton X-100) at room temperature for 20 min. Actin fractions were separated following a series of centrifugation and extraction steps. Cell lysates were centrifuged at 105,000g for 45 min at 4°C, and the supernatant containing the soluble monomeric pool of G-actin (or S1) was gently removed. The remaining pellet was then resuspended in 0.3 ml of Ca²⁺-containing depolymerization buffer (0.1 M PIPES, pH 6.9, 1 mM MgSO₄, 10 µg/ml anti-protease cocktail, and 10 mM CaCl₂) and incubated on ice for 60 min. Subsequently, samples were centrifuged at 48,000g for 15 min at 4°C, and the supernatant (S2- or F-fraction, or cold/Ca²⁺-soluble fraction) was removed. To ensure the complete removal of the F-fraction, the remaining pellet was treated with the Ca²⁺-containing depolymerization buffer twice more by resuspension and centrifugation. The "actin" was recovered by separately incubating (at 37°C for 30 min) the S1 and S2 fractions with stabilizing agents, phalloidin (1 µM) and ATP (0.1 mM), in actin stabilization buffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% dimethyl sulfoxide, 10 µg/ml anti-protease cocktail, 1 mM EGTA, 1 mM MgCl₂, and 0.1 mM ATP) to promote polymerization of actin. Actin was then recovered by centrifugation and resuspended in the above stabilization buffer. Fractionated S1 and S2 samples were then flash-frozen in liquid N₂ and stored at 70°C until immunoblotting. For immunoblotting, samples (5 µg of protein/lane) were placed in a standard SDS sample buffer, boiled for 5 min, and then subjected to PAGE on 7.5% gels. Procedures for Western blotting were performed as previously described (Banan et al., 2000c, 2001a,e). To quantify the relative levels of actin, the optical density of the bands corresponding to immunoradiolabeled actin was measured with a laser densitometer.

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 we previously described (Banan et al., 2001b,c). Briefly, following treatments, post-confluent monolayers were scraped and ultrasonically homogenized in Tris-HCl buffer (20 mM Tris-HCl, pH 7.5, 0.25 mM sucrose, 2 mM EDTA, 10 mM EGTA, 2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml phenylmethylsulfonyl fluoride). The homogenates were then ultracentrifuged (100,000g, for 40 min at 4°C), and the supernatant was removed and used as a source of the cytosolic fraction. Next, pellets were washed with 0.2 ml of Tris-HCl buffer and resuspended in 0.8 ml of buffer containing 0.3% Triton X-100 and maintained on ice for 1 h. The samples were then centrifuged (100,000g, for 1 h at 4°C), and the supernatant was used as the source of the membrane fraction. To this remaining pellet, 0.3 ml of cold (4°C) lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml phenylmethylsulfonyl fluoride) was added. The samples were then placed on ice for 1 h and ultracentrifuged as above. The remainder of the lysate or Triton-insoluble cytoskeletal fraction was then removed. Protein content of the various cell fractions was assessed by the Bradford method (Bradford, 1976). For total PKC extraction, which provides the fraction used to confirm total PKC-1 expression, scraped monolayers were placed directly into 1.5 ml of cold lysis buffer and subsequently ultracentrifuged as described above. The supernatant was used for bulk protein determination.

Measurement of [Ca²⁺ ]_i. Alterations in [Ca²⁺]_i were determined using the sensitive fluorescence Ca²⁺-indicator Fluo-3-AM (Molecular Probes) as we described previously (Kokoska et al., 1998; Banan et al., 2001b). Briefly, monolayers were washed two times with Hanks' balanced salt solution prior to loading with Fluo-3 for 60 min (final concentration of 4 µM). Monolayers were then washed three times to remove excess Fluo-3 followed by treatment regimens. At the desired time points [Ca²⁺]_i was quantitated using the equation: [Ca²⁺]_i (nM) = K_d [(F  F_min)/(F_max  F)], where F_min (the minimum Fluo-3 signal) is equal to F_MnCl2 minus 0.25 F_max, and K_d (the dissociation constant) equals 400 nM (Vandenberghe and Ceuppens, 1990; Kokoshka et al., 1998). The maximum Fluo-3 signal (F_max) was obtained by permeabilizing Caco-2 cells with 50 µM digitonin (Sigma-Aldrich). The Fluo-3 signal was quenched to obtain F_MnCl2 using 2 mM MnCl₂ and 50 µM digitonin. The heavy metal scavenger, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (50 µM), was used in all solutions. Fluorescent signals from samples were quantitated by a fluorescence multiplate reader (FL 600; Bio-Tek Instruments, Winooski, VT) at 37°C, employing excitation and emission wavelengths of 485 and 530 nm, respectively.

Measurement of ⁴⁵Ca²⁺ Efflux. Caco-2 cells were preloaded with ⁴⁵Ca²⁺ (10 µCi/ml) for 1 h at 37°C and then incubated with the test agents. After centrifugation, radioactivity in the supernatant and within the lysed cells was determined by scintillation counting (Kokoska et al., 1998; Banan et al., 2001b). The ⁴⁵Ca²⁺ efflux was expressed as: Ca²⁺ efflux (%) = [CPM_supernatant /(CPM_supernatant + CPM_cells)], where CPM is counts per minute.

Statistical Analysis. Data are presented as mean ± S.E.M. All experiments were carried out with a sample size of at least six observations per treatment group that were run in triplicate (or in some cases in duplicate) on 2 to 3 different days. Statistical analysis comparing treatment groups was performed using analysis of variance followed by Dunnett's multiple range test (Harter, 1960). 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

Protection of the F-Actin by EGF and PKC Activators against Oxidant-Induced Damage. Figure 1 demonstrates that preincubation of Caco-2 monolayers with EGF or PKC activators (OAG or TPA) prior to subsequent exposure to oxidant (H₂O₂) dose dependently and significantly protected the F-actin against oxidant-induced disruption as measured by increases in the percentage of cells with normal actin. Since EGF (10 ng/ml), OAG (50 µM), and TPA (30 nM), which we previously reported to prevent oxidant-induced disruption of the monolayer barrier and increased paracellular permeability (Banan et al., 2001b), provided maximal protection of actin, we utilized these doses in subsequent pharmacological studies. OAG or TPA protection of actin was not significantly different from EGF-induced protection.

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Protective effects of EGF or PKC activators (OAG and TPA) on the percentage of naive Caco-2 cells displaying a normal actin cytoskeleton. Monolayers of naive cells were exposed to the shown doses of EGF or the OAG and TPA for 10 min prior to exposure to oxidant H2O2 (0.5 mM) for 30 min. Cell monolayers were processed for actin staining by cellular fixation followed by incubation with fluorescein-conjugated phalloidin (specific for F-actin), and the percentage of cells displaying a normal actin was then assessed as described under Materials and Methods. Note that pretreatment with the protective agents (EGF, OAG, or TPA) dose dependently maintains a high percentage of cells with normal actin against exposure to oxidant injury. , p < 0.05 versus vehicle (control). +, p < 0.05 versus H2O2. &, p < 0.05 versus EGF (10 ng/ml) + H2O2 or OAG (50 µM) + H2O2 or TPA (30 nM) + H2O2. n = 6 per treatment group in all experiments including those shown in other figures.

View larger version (68K): [in this window] [in a new window]   Fig. 2.   Prevention of the protective effects of EGF or PKC activators (OAG and TPA) on the percentage of naive Caco-2 cells with normal actin. EGF (10 ng/ml) or PKC activators (OAG, 50 µM or TPA, 30 nM) were added to the monolayers 10 min before subsequent exposure to oxidant (H2O2, 0.5 mM) for 30 min. In other experiments, monolayers were preincubated with either PKC inhibitors (chelerythrine, 1 µM, or GF 109203 X, 10 nM, or the inactive analog iGF 109203 X) or a biologically inactive phorbol ester (4-PDD, 20 nM). , p < 0.05 versus vehicle (control). +, p < 0.05 versus H2O2. &, p < 0.05 versus EGF (or OAG or TPA) + H2O2. , p < 0.05 versus corresponding GF 109203 X + EGF (or OAG or TPA) + H2O2.

View larger version (65K): [in this window] [in a new window]   Fig. 3.   Fluorescent staining of the apical cortical ring of F-actin by fluorescein-conjugated (fluorescein isothiocyanate) phalloidin revealing its intracellular organization in confluent intestinal cell monolayers. Naive monolayers were treated with (panel a) isotonic saline/control or (panel b) 0.5 mM H2O2. In panels c and e, monolayers were pretreated, prior to exposure to H2O2, with protective agents EGF (10 ng/ml; panel c) or PKC activator OAG (50 µM; panel e). In panels d and f, monolayers were pretreated with PKC inhibitor GF 109203 X and then either EGF (panel d) or OAG and subsequently H2O2 (panel f). Ultra high-resolution LSCM reveals that control cells (panel a) show a normal, continuous, and smooth distribution of actin ring (apical cortex) at areas of cell-to-cell contact. In cells exposed to 0.5 mM H2O2 alone (panel b), the actin ring appears disrupted, condensed, beaded, and fragmented. In contrast, in cells pretreated with EGF (panel c) normal actin cytoarchitecture appears intact and preserved. Similarly, actin ring architecture in cells pretreated with OAG (panel e) is highly preserved and resembles the morphology observed in the control group. Preincubation of monolayers with PKC inhibitor GF 109203 X abolished protection of actin in EGF + H2O2 (panel d) or OAG + H2O2 (panel f) groups as shown by a disrupted appearance of the actin cytoskeleton.

View larger version (71K): [in this window] [in a new window]   Fig. 4.   Quantitative immunoblotting analysis of the polymerized F-actin pool (S2, index of actin stability) and monomeric G-actin pool (S1, index of actin disassembly) in Caco-2 monolayers. Conditions were described in Figs. 2 and 3. Percentage of polymerized actin = [(S2)/(S2 + S1)], where S2 + S1 is the total cellular actin pool. , p < 0.05 versus vehicle (control). +, p < 0.05 versus H2O2. &, p < 0.05 versus EGF (or OAG or TPA) + H2O2. , p < 0.05 versus corresponding GF 109203 X + EGF (or OAG or TPA) + H2O2.

View larger version (8K): [in this window] [in a new window]   Fig. 5.   Representative Western immunoblot photomicrograph of the polymerized actin (S2, Triton-insoluble) extracts from naive cell monolayers following treatments. F-actin fractions were analyzed by SDS-PAGE and Western immunoblots using a monoclonal anti-actin primary antibody followed by a horseradish peroxidase-conjugated secondary antibody and then autoradiographed. The lanes from left to right correspond to: a, vehicle; b, 0.5 mM H2O2 challenge; c, EGF (10 ng/ml) + 0.5 mM H2O2; d, PKC inhibitor (GF 109203 X) + EGF (10 ng/ml) + 0.5 mM H2O2; e, inactive analog of PKC inhibitor (iGF 109203 X) + EGF (10 ng/ml) + 0.5 mM H2O2; f, OAG (50 µM) + 0.5 mM H2O2; g, PKC inhibitor (GF 109203 X) + OAG (50 µM) + 0.5 mM H2O2; h, inactive PKC inhibitor (iGF 109203 X) + OAG (50 µM) + 0.5 mM H2O2; i, actin standard (43 kDa).

Key Role of PKC-1 Isoform in Protection of the F-Actin Cytoskeleton. To this end, we used novel and stably transfected intestinal cell lines we developed (Banan et al., 2001c) that either over- or under-express PKC-1 compared with naive Caco-2 cells. Figure 6 and Table 1 show that over-expression (3.1-fold) of PKC-1-potentiated protection by EGF or PKC activators (e.g., OAG) of the F-actin against oxidant-induced injury. In cells stably over-expressing 1 isoform, actin integrity (as assessed by the percentage of cells displaying normal F-actin ring) was protected against oxidant damage by a low dose of EGF (1 ng/ml). This same dose of EGF did not protect the actin in naive cells. A similar synergy was seen for protection of actin by a low dose of a PKC activator (OAG, 0.01 µM) (Fig. 6). TPA caused identical effects (not shown). In all instances, the extent of protection of PKC-1-over-expressing cells was not significantly different from protection of naive cells by higher doses of these same agents (10 ng/ml EGF; 50 µM OAG). This did not appear to be due to changes in the ability of oxidants to cause damage to actin as PKC-1-over-expressing cells (without EGF or OAG) and naive cells responded comparably to H₂O₂, both with similar and significant damage to actin (Fig. 6). EGF or OAG alone did not affect actin compared with vehicle (percentage of normal F-actin was 99 ± 1 for vehicle versus 98 ± 2 for EGF or 96 ± 4 for OAG). Furthermore, PKC-1 over-expression by itself did not protect or injure actin. As expected, transfection of only the vector (SP-72) did not protect actin against oxidant injury (e.g., percentage of normal F-actin was 98 ± 2 for vector-transfected cells exposed to vehicle; 48 ± 4 for vector-transfected cells exposed to H₂O₂ alone; and 49 ± 5 for vector-transfected cells incubated with 1 ng/ml EGF + H₂O₂ versus 90 ± 5 for PKC-1 sense-transfected cells incubated with 1 ng/ml EGF + H₂O₂).

View larger version (48K): [in this window] [in a new window]   Fig. 6.   Protective actions of PKC-1 over-expression on the percentage of Caco-2 cells from transfected monolayers displaying a normal F-actin in the presence of a low concentration of EGF or PKC activator (OAG). A novel sense-transfected cell line previously developed in our laboratory (see Materials and Methods) that over-expresses PKC-1 by 3.1-fold was utilized. Transfected "(T)" cells stably over-expressing PKC-1 were incubated in low doses of EGF (1 ng/ml) or the PKC activator OAG (0.01 µM) prior to the subsequent exposure to oxidant H2O2 (0.5 mM). These low doses do not protect F-actin against oxidant injury in naive "(N)" cells, but they do protect F-actin in transfected cells over-expressing PKC-1. Only high doses of EGF (10 ng/ml) or OAG (50 µM) protected actin in naive monolayers (not over-expressing 1). Also note synergy-induced protection of the actin in PKC-1-over-expressing (T) cells that were exposed to low doses of EGF or OAG. In the absence of these low doses of EGF or OAG, both PKC-1-over-expressing cells and naive cells responded comparably to oxidant insult to actin, both displaying significant reduction in the percentage of cells with normal actin. , p < 0.05 versus vehicle. +, p < 0.05 versus H2O2. &, p < 0.05 versus corresponding low doses of EGF or OAG + H2O2 in naive cells.

View this table: [in this window] [in a new window]   TABLE 1 Effects of transfection of varying amounts of PKC-1 sense or antisense DNA on F-actin cytoskeleton in Caco-2 monolayers Values are means ± S.E.M. following treatments. Cells stably transfected with varying amounts of PKC-1 sense DNA (1, 2, 4, or 5 µg) were preincubated (10 min) with a low dose of EGF (1 ng/ml) or OAG (a synthetic diacylglycerol and a PKC activator, 0.01 µM) before subsequent exposure to oxidant (H2O2, 0.5 mM) for 30 min. In separate studies, cells transfected with varying amounts of PKC-1 anti-sense (1, 4, or 5 µg) were treated with a high dose of EGF (10 ng/ml) or OAG (50 µM) prior to oxidant. Select treatments from naive (untransfected) cell monolayers are also shown. One hundred fifty to 200 cells per slide (well) were examined by high-resolution laser confocal and the percentage of cells displaying normal actin cytoskeleton was determined. N = 6 per group.

View larger version (103K): [in this window] [in a new window]   Fig. 7.   The intracellular architecture of the apical cortical ring of F-actin cytoskeleton from confluent monolayers as captured by ultra high-resolution LSCM. Monolayers of naive cells were incubated with vehicle (isotonic saline; panel a), 0.5 mM H2O2 (panel b), EGF (1 ng/ml) plus 0.5 mM H2O2 (panel c), or OAG (0.01 µM) plus 0.5 mM H2O2 (panel d). PKC-1-over-expressing cell monolayers (panels e and f) were also exposed to the same low doses of EGF (panel e) or OAG (panel f) and then incubated with the same H2O2 concentration. Normal cells (panel a) reveal an intact and smooth architecture of apical actin cortex (i.e., ring) on the inner side of the plasma membrane (areas of cell-to-cell contact), whereas cells exposed to H2O2 (panel b) show fragmentation, beading, and disruption of the actin ring. Cells over-expressing PKC-1, which were exposed to low doses of EGF (panel e) or OAG (panel f) before oxidant exposure, exhibit a highly preserved appearance of the apical actin ring, which is indistinguishable from the normal cells (panel a). In contrast, this protection did not occur in naive cells (not over-expressing PKC-1; panels c and d) that were pre-exposed to these same low doses of EGF (panel c) or OAG (panel d), as shown by the abnormal cytoarchitecture of the actin ring.

View larger version (49K): [in this window] [in a new window]   Fig. 8.   A, immunoblotting analysis of the stable polymerized F-actin fraction (S2, index of actin integrity) and the monomeric G-actin fraction (S1, index of actin instability) in Caco-2 monolayers. Conditions as in Fig. 6. Percentage of polymerized actin = [(S2)/(S2 + S1)], where S2 + S1 is the total intracellular actin pool. , p < 0.05 versus vehicle. +, p < 0.05 versus H2O2. &, p < 0.05 versus low doses of EGF or OAG + H2O2 in naive cells. (N), naive. (T), transfected. B, representative blot of the polymerized actin fractions from Caco-2 cell monolayers following treatments. F-actin fractions were isolated and immunoblotted and then processed for X-ray film exposure by enhanced chemiluminescence reagents. The lanes from left to right correspond to: a, vehicle; b, 0.5 mM H2O2 challenge in naive cells; c, 0.5 mM H2O2 challenge in PKC-1-over-expressing cells; d, EGF (1 ng/ml) + 0.5 mM H2O2 in PKC-1-over-expressing cells; e, EGF (1 ng/ml) + 0.5 mM H2O2 in naive cells; f, OAG (0.01 µM) + 0.5 mM H2O2 in PKC-1-over-expressing cells; g, OAG (0.01 µM) + 0.5 mM H2O2 in naive cells; h, EGF (10 ng/ml) + 0.5 mM H2O2 in naive cells; i, OAG (50 µM) + 0.5 mM H2O2 in naive cells; and j, actin standard (43 kDa). In transfected cells the over-expressed PKC-1 in the presence of a low dose of EGF (1 ng/ml) or OAG (0.01 µM) increases the polymerized F-actin band density to almost normal levels. In naive cells, on the other hand, preincubation with these same low doses of EGF or OAG does not increase actin polymerization, which is similar to that of the oxidant-exposed groups. Also shown are high concentrations of EGF (10 ng/ml) or OAG (50 µM), which are the only doses that increase actin assembly in naive cells.

Activation of Over-Expressed PKC-1 Correlates with Three Different Indices of Actin Integrity. We initially confirmed our recent findings (Banan et al., 2001b,c) in both naive and transfected, PKC-1-over-expressing intestinal cells that EGF or PKC activators translocate the 1 (~78 kDa) isoform of PKC from the cytosol to both membrane- and cytoskeletal-bound fractions. In particular, following pretreatment with low doses of EGF or OAG, there is a rapid redistribution of over-expressed PKC-1 isoform from a mostly cytosolic distribution into particulate fractions (i.e., particulate = membrane + cytoskeletal fractions), indicating the induced activation of the 1 isoform (Fig. 9A). This figure also shows a temporal relationship between PKC-1 (optical density from the particulate fraction) and actin ring integrity. When these two variables were plotted against each other, we found a robust correlation (r = 0.93, p < 0.05). When two other markers of actin stability, F-actin polymerization and G-actin disassembly, were plotted against PKC-1 (Fig. 9, B and C), additional robust correlations were observed (r = 0.92 and 0.91, respectively, p < 0.05 for each), further suggesting that increased activation of 1 isoform is key in protection of actin integrity.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   A, actin integrity and PKC-1 particulate-associated fraction versus time. PKC-1-over-expressing cells were pretreated with EGF (1 ng/ml) or OAG (0.01 µM) prior to incubation with H2O2 (0.5 mM). Variables depicted are optical density (O.D.) of particulate-associated PKC-1 band and percentage of cells with normal actin. B and C, polymerized F-actin assembly (B) or monomeric G-actin disassembly (C) versus the O.D. of particulate-associated PKC-1 band, 1 isoform activation. PKC-1 levels (O.D.) in the particulate bands from PKC-1-over-expressing cells were correlated with two other markers of the condition of actin integrity, namely the percentage of the pool of polymerized F-actin, an index of actin assembly, and the percentage of the pool of monomeric G-actin, an index of actin disassembly.

Stable Antisense Inhibition of PKC-1 and Its Inhibition of EGF-Induced Protection of Actin. To further investigate a possible role for PKC-1 in EGF-mediated protection of actin, we utilized Caco-2 cells that were transfected with PKC-1 antisense plasmid and cDNA encoding G-418 resistance. We confirmed our earlier reports (Banan et al., 2001c) that this manipulation substantially (~90%) and stably reduces the steady-state levels of PKC-1 protein.

View larger version (47K): [in this window] [in a new window]   Fig. 10.   Stable antisense under-expression of PKC-1 isoform inhibits the protective effects of high doses of EGF (10 ng/ml) or PKC activator (OAG, 50 µM) on the actin cytoskeleton as determined by the percentage of cells displaying normal F-actin. A novel antisense-transfected cell line previously developed in our laboratory (see Materials and Methods), which almost completely lacks PKC-1 protein, was grown as monolayers and then exposed to a high dose of EGF (10 ng/ml) or OAG (50 µM) and then to H2O2. , p < 0.05 versus vehicle. +, p < 0.05 versus H2O2. &, p < 0.05 versus high doses of EGF or OAG + H2O2 in naive cells. (N), naive. (AS), antisense inhibition of PKC-1.

View larger version (55K): [in this window] [in a new window]   Fig. 11.   Stable antisense (AS) inhibition of PKC-1 prevents the protective effects of EGF or OAG on the enhancement of actin polymerization. Immunoblotting analysis of the polymerized F-actin (S2) and monomeric G-actin (S1) from cellular extracts was performed following treatment regimens similar to those shown in Fig. 10. Percentage of polymerized actin = [(S2)/(S2 + S1)]. , p < 0.05 versus vehicle. +, p < 0.05 versus H2O2. &, p < 0.05 versus high dose of EGF (10 ng/ml) + H2O2 or high dose of OAG (50 µM) + H2O2 in naive (N) cells.

Normalization of [Ca²⁺]_i Concentration: Prevention of Oxidant-Induced Rise in [Ca²⁺]_i in PKC-1 Transfected Cells. Because our previous studies showed that EGF normalizes [Ca²⁺]_i in the face of oxidant challenge, we surmised that normalization of [Ca²⁺]_i might be a key mechanism for PKC-1-induced, EGF-mediated protection. Indeed, measurement of [Ca²⁺]_i in monolayers prelabeled with the Ca²⁺-sensitive dye Fluo-3 (Fig. 12A) showed that PKC-1 over-expression synergized with low doses of EGF (1 ng/ml) or OAG (0.01 µM) to maintain [Ca²⁺]_i at almost control levels. These same low doses of EGF or OAG did not normalize [Ca²⁺]_i in naive cells. Additionally, the extent of normalization of [Ca²⁺]_i in transfected cells exposed to these low doses was not significantly different from the extent of normalization of [Ca²⁺]_i in naive cells by higher doses of EGF or OAG (Fig. 12A). This did not appear to be due to changes in the ability of oxidants to markedly increase [Ca²⁺]_i since PKC-1-over-expressing cells (without EGF or OAG) and naive cells responded similarly to H₂O₂, both with comparable and significant increases in [Ca²⁺]_i. For example, naive monolayers exposed to H₂O₂ exhibited [Ca²⁺]_i levels at 327 ± 10 nM compared with 122 ± 7 nM for vehicle. Furthermore, transfection of vector alone was ineffective ([Ca²⁺]_i = 125 ± 10 nM for vector-transfected cells exposed to vehicle, 321 ± 9 nM for vector-transfected cells exposed to H₂O₂, and 324 ± 15 nM for vector-transfected cells incubated with 1 ng/ml EGF + H₂O₂ versus 138 ± 15 nM for PKC-1 sense-transfected cells incubated with 1 ng/ml EGF + H₂O₂).

View larger version (61K): [in this window] [in a new window]   Fig. 12.   Intracellular Ca2+ alterations assessed by the Ca2+-sensitive probe Fluo-3-AM in PKC-1-over-expressing cells following treatments with EGF or OAG (A). Cells preloaded with Fluo-3-AM were treated under similar conditions to those in Fig. 6. In other experiments (B), cells were pretreated with known membrane "pump" Ca2+-ATPase inhibitors (vanadate or quercetine) and subsequently incubated with H2O2 in the presence or absence of pretreatment with EGF or OAG. A, in transfected (T) cells that were exposed to oxidants, PKC-1 over-expression synergizes with the low dose of EGF (1 ng/ml) or OAG (0.01 µM) to markedly and significantly normalize intracellular Ca2+; this did not occur in naive (N) cells (not over-expressing 1). Only high doses of EGF (10 ng/ml) or OAG (50 µM) normalize intracellular Ca2+ in naive monolayers. B, inhibitors of membrane Ca2+-ATPase prevent the normalization of intracellular calcium. , p < 0.05 versus vehicle. +, p < 0.05 versus H2O2. &, p < 0.05 versus the low dose of EGF or OAG + H2O2 in naive cells. #, p < 0.05 versus the corresponding EGF or OAG + H2O2 in naive or transfected cells.

View larger version (46K): [in this window] [in a new window]   Fig. 13.   Antisense (AS) inhibition of PKC-1 isoform substantially attenuates the normalization of intracellular Ca2+ by high doses of EGF (10 ng/ml) or by PKC activator (OAG, 50 µM). Treatment conditions were as in Fig. 10. , p < 0.05 versus vehicle. +, p < 0.05 versus H2O2. &, p < 0.05 versus high doses of EGF or OAG + H2O2 in naive (N) cells.

Induction of Ca²⁺ Efflux under Protective Conditions in Stably Transfected Intestinal Cells. Since two different Ca²⁺-ATPase inhibitors abolished synergy-induced normalization of [Ca²⁺]_i, thereby maintaining [Ca²⁺]_i at high levels, we hypothesized that Ca²⁺ efflux is an essential mechanism for PKC-1-induced (EGF) normalization of [Ca²⁺]_i. Indeed, assessment of Ca²⁺ efflux from monolayers prelabeled with ⁴⁵Ca²⁺ (Fig. 14A) showed that PKC-1 over-expression synergized with the low doses of EGF (1 ng/ml) or OAG (0.01 µM) to markedly and significantly enhance Ca²⁺ efflux. These same doses were ineffective in naive cells; only high doses were effective. Furthermore, two different inhibitors of the membrane Ca²⁺-ATPase pump (Fig. 14B), quercetine and vanadate, abolished this enhancement in Ca²⁺ efflux. We did not observe any significant changes in Ca²⁺ efflux in vector (SP-72)-alone transfected cell monolayers (Ca²⁺ efflux = 51 ± 4% for vector-transfected cells exposed to vehicle, 43 ± 2% for vector-transfected cells exposed to H₂O₂, and 49 ± 5% for vector-transfected cells incubated with 1 ng/ml EGF + H₂O₂ versus 84 ± 3% for PKC-1 sense-transfected incubated with 1 ng/ml EGF + H₂O₂). These data on efflux parallel our data on [Ca²⁺]_i.

View larger version (56K): [in this window] [in a new window]   Fig. 14.   A, 45Ca2+ efflux from Caco-2 monolayers over-expressing PKC-1 in the presence of a low concentration of EGF or PKC activator (OAG). PKC-1 over-expression synergizes with a low dose of EGF or OAG to result in an enhancement of Ca2+ efflux from transfected monolayers. In naive (N) cells, in contrast, these same low doses of EGF or OAG did not cause Ca2+ efflux. In these naive cells higher doses of EGF or OAG were required to increase Ca2+ efflux. B, prevention of calcium efflux by known inhibitors of Ca2+-ATPase pump (vanadate or quercetine). , p < 0.05 versus vehicle. +, p < 0.05 versus H2O2. &, p < 0.05 versus the low dose of EGF or OAG + H2O2 in naive cells. #, p < 0.05 versus the corresponding EGF or OAG + H2O2 in naive or transfected (T) cells.

View larger version (39K): [in this window] [in a new window]   Fig. 15.   Under-expressing PKC-1 prevents 45Ca2+ efflux from monolayers incubated with high doses of EGF or PKC activator (OAG). Conditions were similar to those in Fig. 13. , p < 0.05 versus vehicle. +, p < 0.05 versus H2O2. &, p < 0.05 versus high doses of EGF or OAG + H2O2 in naive (N) cells. (AS), antisense inhibition of PKC-1.

	Discussion