Key role of PLC-{gamma} in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly

A. Banan, L. J. Zhang, M. Shaikh, J. Z. Fields, A. Farhadi, and A. Keshavarzian

Departments of Internal Medicine (Section of Gastroenterology and Nutrition), Pharmacology, and Molecular Physiology, Rush University School of Medicine, Chicago, Illinois 60612

Submitted 1 April 2003 ; accepted in final form 29 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Upregulation of inducible nitric oxide synthase (iNOS) is key to oxidant-induced disruption of intestinal (Caco-2) monolayer barrier, and EGF protects against this disruption by stabilizing the cytoskeleton. PLC-{gamma} appears to be essential for monolayer integrity. We thus hypothesized that PLC-{gamma} activation is essential in EGF protection against iNOS upregulation and the consequent cytoskeletal oxidation and disarray and monolayer disruption. Intestinal cells were transfected to stably overexpress PLC-{gamma} or to inhibit its activation and were then pretreated with EGF ± oxidant (H2O2). Wild-type (WT) intestinal cells were treated similarly. Relative to WT monolayers exposed to oxidant, pretreatment with EGF protected monolayers by: increasing native PLC-{gamma} activity; decreasing six iNOS-related variables (iNOS activity/protein, NO levels, oxidative stress, actin oxidation/nitration); increasing stable F-actin; maintaining actin stability; and enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected monolayers overexpressing PLC-{gamma} (+2.3-fold) were protected, as indicated by decreases in all measures of iNOS-driven pathway and enhanced actin and barrier integrity. Overexpression-induced inhibition of iNOS was potentiated by low doses of EGF. Stable inhibition of PLC-{gamma} prevented all measures of EGF protection against iNOS upregulation. We conclude that 1) EGF protects against oxidative stress disruption of intestinal barrier by stabilizing F-Actin, largely through the activation of PLC-{gamma} and downregulation of iNOS pathway; 2) activation of PLC-{gamma} is by itself essential for cellular protection against oxidative stress of iNOS; and 3) the ability to suppress iNOS-driven reactions and cytoskeletal oxidation and disassembly is a novel mechanism not previously attributed to the PLC family of isoforms.

actin cytoskeleton; gut barrier; growth factors; oxidative stress; nitration and carbonylation; reactive nitrogen metabolites; phospholipase C isoform; inflammatory bowel disease; Caco-2 cells


THE EPITHELIUM of gastrointestinal (GI) mucosa is a highly selective permeability barrier that normally excludes the passage of harmful proinflammatory molecules (e.g., bacterial endotoxin, immunoreactive antigens) but allows the absorption from the lumen of nutrients and water into the mucosa and the systemic circulation. Disruption of the GI epithelial barrier, in contrast, permits the penetration of these normally excluded luminal substances into the mucosa and leads to the initiation or perpetuation of inflammatory processes and mucosal disruption (30, 31, 37, 38). Indeed, disruption of mucosal barrier integrity has been implicated in the pathogenesis of several GI disorders such as inflammatory bowel disease (IBD), necrotizing enterocolitis, multiple organ system dysfunction, and chemical (ethanol, NSAID)-induced injury as well as systemic disorders (e.g., alcohol-induced liver disease) (29-31, 37, 38). The fundamental difficulty in managing these disorders is due in part to a lack of effective preventive strategies, which is due in turn to our limited understanding of their pathophysiology and of the endogenous protective pathways.

An important discovery in the GI inflammation (IBD) field was the realization that a leaky and disrupted gut barrier can cause intestinal inflammation and that maintaining a normal mucosal epithelial barrier is required for intestinal health. For instance, intestinal barrier hyperpermeability that is induced by the injection of bacterial endotoxin into the mucosa of rodents can elicit an oxidative and inflammatory condition similar to IBD (60). Moreover, transgenic animals with a leaky gut barrier exhibit symptoms of intestinal inflammation (29). However, the pathophysiology of mucosal barrier disruption in IBD remains poorly understood. Nonetheless, several studies have shown that chronic gut inflammation in IBD is associated with excessive amounts of oxidants (e.g., H2O2) and that a high level of these oxidants appears to be a key contributor to mucosal injury (2, 10, 17, 18, 37, 39, 40, 43). Oxidant-induced disruption is of substantial clinical and biological value not only because oxidants are common in inflammation (e.g., they are elaborated by neutrophils that infiltrate the mucosa during inflammation) but also because they can lead to mucosal barrier dysfunction and, in turn, to the initiation and/or continuation of mucosal inflammation and injury (29-31, 38, 39, 60). Accordingly, understanding how gut barrier integrity can be protected against oxidative, proinflammatory conditions is of fundamental clinical and biological importance.

We have been investigating the mechanisms underlying oxidant-induced mucosal injury and barrier disruption as well as protection against this injury by growth factor pathways. Using monolayers of intestinal cells as a well-established model of gut barrier integrity, we have shown that cytoskeletal disassembly and disruption is a key event in oxidant injury and that growth factors [EGF or transforming growth factor (TGF)-{alpha}] appear to prevent damage by stabilizing the cytoskeleton in large part through a signaling pathway mediated by phospholipase C-{gamma} (PLC-{gamma}) (1-3, 12, 18). The involvement in protective mechanisms by PLC-{gamma} in the GI epithelium was a novel finding (3, 12). We showed, using wild-type Caco-2 intestinal cells, that EGF induces the membrane translocation of the native PLC-{gamma} isoform and therefore considered it as a possible contributor to EGF-mediated protection of the GI epithelial barrier. We then noted that maintaining an intact cytoskeleton is required for protection of intestinal barrier integrity by EGF apparently via PLC-{gamma} (3, 18). Despite the critical importance of the {gamma}-isoform of PLC to intestinal barrier permeability, the fundamental mechanism for PLC-{gamma}-mediated, EGF-induced protection of monolayer barrier and actin cytoskeletal integrity remains elusive.

Inducible nitric oxide synthase (iNOS)-dependent processes are key in the underlying mechanism of oxidant-induced disruption of intestinal barrier integrity (9, 10). Indeed, overproduction and uncontrolled generation of iNOS-derived reactive nitrogen metabolites (e.g., NO, ONOO-) have been proposed to be an important factor in tissue damage during inflammation, including in IBD (17, 34, 37, 39, 40, 55). For example, we have shown that a number of these oxidative reactions, including cytoskeletal nitration and oxidation, also occur in intestinal mucosa from patients with IBD (17, 37) as well as in intestinal cell monolayers in culture (9, 10).

Accordingly, investigating the role of the {gamma}-isoform of PLC in the prevention of oxidative stress of iNOS-driven reactions in cells, we believe, is both novel and significant because it is of substantial clinical and biological importance to establish the idea that specific isoforms of PLC play fundamental roles in endogenous protective mechanisms of cells against oxidative stress to essential cellular structural proteins required for the maintenance of GI integrity. Moreover, an improved understanding of effectively suppressing (e.g., by PLC-{gamma}) the leakiness and disruption of the intestinal barrier under conditions of oxidative stress should lead to the development of new therapeutic modalities for inflammatory diseases of the GI tract that are related to oxidative injury caused by hyperactivation of iNOS and NO pathway.

In view of the above considerations, we tested the hypothesis that PLC-{gamma} not only prevents oxidant-induced iNOS upregulation and its injurious consequences but also that it is key to EGF-mediated protection of F-actin cytoskeleton and intestinal barrier integrity against the oxidative stress of this upregulation. To this end, we utilized both pharmacological and targeted molecular interventions employing several transfected intestinal cell lines that we developed. In several clones the PLC-{gamma} isoform was reliably overexpressed; in the other clones, PLC-{gamma} activity was severely inhibited. Here, we report new mechanisms—prevention of the oxidative stress of iNOS upregulation and of cytoskeletal protein nitration and oxidation—by the {gamma}-isoform of PLC in cell monolayers. To our knowledge, this is the first report that PLC-{gamma} can inhibit the dynamics of iNOS-induced oxidative stress and cytoskeletal oxidation and disassembly in cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. Caco-2 cells were obtained from ATCC (Rockville, MD) at passage 15. This cell line was chosen for our studies because they form monolayers that morphologically resemble small intestinal cells, with defined apical brush borders and a highly organized actin network upon differentiation. Cells grown for barrier function and related experiments were split at a ratio of 1:2 and seeded at a density of 200,000 cells/cm2 into either 0.4 µM Biocoat collagen I cell culture inserts (0.3-cm2 growth surface; Becton Dickinson, Bedford, MA) or 6- and 96-well plates or T-75 flasks. At this seeding density, Caco-2 cells form confluent and differentiated monolayers in typically 7-10 days (e.g., see Refs. 14, 18, 33, 36, 42, 45, 58). We were therefore able to measure alterations in intestinal barrier integrity and related outcomes in differentiated and nonleaky monolayers. In addition, Caco-2 cells, once differentiated, closely resemble normal intestinal cells in that they express intestinal hydrolases such as sucrase-isomaltase and alkaline phosphatase. Furthermore, these cells, once differentiated, are similar to native intestinal epithelial cells in that they have receptors for prostaglandins, growth factors, VIP, LDL, insulin, and specific substrates such as dipeptides, fructose, glucose, hexoses, and vitamin B12. All experiments were performed at least 7 days postconfluence. The utility and characterization of this cell line has been extensively reported (14, 45).

Plasmids and stable transfection. The sense and dominant negative plasmids of PLC-{gamma} were constructed and then stably transfected by using Lipofectin (Lipofectin reagent; GIBCO BRL) as we previously described (3, 12). Expression was controlled by SV40 early promoter present in pXf vector. The dominant negative PLC-{gamma}1 fragment from the Z region (designated as PLCz) of human PLC-{gamma}1, which covers the src homologous 2 and 3 (i.e., SH2 and SH3) domains (amino acids 517-901), was isolated by RT-PCR and cloned into a eukaryotic expression vector, pXf (23, 57). Control conditions included vector (pXf) alone. Multiple clones stably overexpressing PLC-{gamma} or lacking PLC-{gamma} activity were assessed by immunoblotting as well as tested for PLC-{gamma} activity (see below). These cells were then plated on Biocoat collagen I cell culture inserts (Becton Dickinson) and subsequently used for experiments.

Experimental design. In the first series of experiments, postconfluent monolayers of wild-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. As we previously showed, H2O2 at 0.5 mM disrupts actin cytoskeleton and barrier integrity and upregulates iNOS (2, 10, 18). EGF at 10 ng/ml (but not 1 ng/ml) prevents both actin and barrier disruption. These experiments were then repeated using transfected cells. In all experiments, we assessed actin cytoskeletal stability (cytoarchitecture, F-actin and G-actin assembly/disassembly), barrier integrity, PLC-{gamma} subcellular distribution, PLC-{gamma} isoform activity, iNOS activity, NO levels, reactive nitrogen metabolites (RNM) levels (e.g., ONOO-), oxidative stress [dichlorofluorescein (DCF) fluorescence], actin nitration (nitrotyrosination), and actin oxidation (carbonylation).

In the second series of experiments, cell monolayers that were stably overexpressing PLC-{gamma} were preincubated (10 min) with EGF (1 or 10 ng/ml) or vehicle before exposure (30 min) to damaging concentrations of oxidant (H2O2, 0.5 mM) or vehicle. Outcomes measured were as described above.

In the third series of experiments, monolayers of dominant negative, namely PLCz, transfected cells lacking PLC-{gamma} activity were treated with high (protective) doses of EGF and then oxidant. In corollary experiments, we investigated the effects of PLC-{gamma} activation or inactivation on the state of 1) actin nitration and oxidation, 2) actin assembly and disassembly, and 3) stability of cytoarchitecture of the F-actin cytoskeleton. Monomeric (G) and polymerized (F) fractions of actin were isolated and then analyzed for outcomes (e.g., oxidation and nitration by immunoblotting) (10, 18). Actin integrity was assessed by 1) immunofluorescent labeling and fluorescence microscopy to determine the percentage of cells with normal actin, 2) detailed analysis by high-resolution laser scanning confocal microscopy (LSCM), 3) immunoblot analysis of G- and F-actin pools, and 4) immunoblot analysis of oxidation and nitration of actin.

Fractionation and imunoblotting of PLC-{gamma}. Cell monolayers grown in 75-cm2 flasks were processed for the isolation of the cytosolic, membrane, and cytoskeletal fractions (7, 8). Protein content of the various cell fractions was assessed by the Bradford method (20). For immunoblotting, samples (25 µg protein/lane) were added to a standard SDS buffer, boiled, and then separated on 7.5% SDS-PAGE. The immunoblotted proteins were incubated with the primary mouse monoclonal anti-PLC-{gamma} (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:2,000 dilution. A horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR) was used as a secondary antibody at 1:4,000 dilution. Proteins were visualized by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL) and autoradiography and subsequently analyzed. The identity of the PLC-{gamma} bands were confirmed by 1) using a PLC-{gamma} blocking peptide in combination with the anti-PLC-{gamma} antibody that prevents the appearance of the corresponding "major" band in Western blots. 2) Additionally, in the absence of the primary antibody to PLC-{gamma}, no corresponding band for PLC-{gamma} was observed. 3) The PLC-{gamma} band ran at the expected molecular weight of 145 kDa as confirmed by a known positive control for PLC-{gamma} (from rat brain lysates). 4) Prestained molecular weight markers (Mr 34,900 and 205,000) were run in adjacent lanes. We also confirmed that overexpression of PLC-{gamma} or dominant negative inhibition of PLC-{gamma} did not affect the relative expression levels of other PLC isoforms and did not injure the Caco-2 cells.

Immunoprecipitation and PLC-{gamma} activity. Immunoprecipitated PLC-{gamma} was collected and processed for its ability to form [3H]inositol phosphates (12). Briefly, after treatments, confluent cell monolayers were lysed by incubation for 20 min in 500 µl of cold lysis buffer [20 mM Tris·HCl, pH 7.4, 150 mM NaCl, anti-protease cocktail (10 µg/ml), 10% glycerol, 1 mM sodium orthovanadate, 5 mM NaF, and 1% Triton X-100]. The lysates were clarified by centrifugation at 14,000 g for 10 min at 4°C. For immunoprecipitation, the lysates were incubated for 2 h at 4°C with monoclonal anti-PLC-{gamma} (1:1,000 dilution, in excess). The extracts were then incubated with protein G-Sepharose for 1 h at 4°C. The immuno-complexes were collected by centrifugation (2,500 g, 5 min) in microfuge tubes and washed three times with immunoprecipitation buffer containing 5 mM Tris·HCl, pH 7.4, and 0.2% Triton X-100. They were then washed one time with sample buffer (20 mM HEPES, pH 7.5) and resuspended in 20 µl of buffer and 5 µl of reaction buffer (5 µCi/ml [3H]myoinositol) plus LiCl (10 mM, which inhibits inositol phosphate hydrolysis) 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 [3H]inositol phosphates (IP) were recovered in the supernatant after centrifugation (16,000 g, 5 min). The extracts were separated on Dowex formate ion-exchange minicolumns (Bio-Rad, Hercules, CA). Radioactivity present (IP content) in samples was quantified by scintillation counting with aqueous counting scintillant. Counts for blanks were subtracted from the sample activity. Sample activity was also corrected for protein concentration (Bradford method), and PLC-{gamma} activity was reported as picomoles per minute per milligram of protein.

Assay of NOS activity. Wild-type and transfected cells grown to confluence were removed by scraping and were centrifuged and homogenized on ice in a buffer containing 50 mM Tris·HCl, 0.1 mM EDTA, 0.1 mM EGTA, 12 mM 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4. Conversion of L-[3H]arginine (Amersham) to L-[3H]citrulline was measured in the cell homogenates by scintillation counting. Experiments in the presence of NADPH, without Ca+2 and with 5 mM EGTA, determined Ca2+-independent NOS (iNOS) activity (1, 4, 9, 10, 16).

Western blot of the level of iNOS. After treatments, the cells were washed once with cold PBS, scraped into 1 ml of cold PBS, and harvested in a standard anti-protease cocktail. For immunoblotting, samples (25 µg protein/lane) were added to SDS buffer (250 mM Tris·HCl, pH 6.8, 2% glycerol, and 5% mercaptoethanol), boiled for 5 min, and then separated on 7.5% SDS-PAGE. Subsequently, proteins were transferred to nitrocellulose membranes and then blocked in 3% BSA for 1 h, followed by several washes (Tris-buffered saline). The immunoblotted proteins were incubated for 2 h in Tris-buffered saline containing Tween 20 and 1% BSA with the primary antibody (mouse monoclonal anti-human iNOS, 1:3,000 dilution; Santa Cruz Biotechnology). An HRP-conjugated goat anti-mouse antibody (Molecular Probes) was used as a secondary antibody, at 1:3,000 dilution. Membranes were visualized by ECL and then autoradiographed (4, 9, 10, 16).

Chemiluminescence analysis of NO. NO production was assessed by a chemiluminescence procedure (4, 9, 10, 16). Briefly, cells were homogenized, and the endogenous nitrate (NO3 -) and nitrite (NO2 -), the metabolic degradation products of NO, were then reduced to NO by using vanadium (III) (Sigma, St. Louis, MO) and HCl at 90°C before measuring the NO concentration with a model 280 nitrix oxide analyzer (NOA) from Sievers (Boulder, CO). NO was expressed in micromolar concentration and calculated by comparison to the chemiluminescence of a standard solution of NaNO2. The absolute NO values were reported as the number of micromoles per 1 x 106 cells.

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 the fluorescent dye DCF (1, 2, 4, 10, 15). Monolayers grown in 96-well plates were preincubated with the membrane-permeable DCFD (10 µg/ml for 30 min) before the treatments. Subsequently, fluorescent signals (i.e., DCF fluorescence) from samples were quantitated using a fluorescence multiplate reader set at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. DCF fluorescence was then expressed as a percentage of baseline oxidative stress. The dependence of the assay on reactive oxygen species (ROS) production (e.g.,.O2 - generation) was shown as we previously reported (1, 4, 9, 10) by adding either catalase, an active H2O2 oxidant scavenger, or SOD, an active superoxide radical scavenger, or, for control conditions, either an inactive H2O2 or inactive superoxide scavenger [heat-inactivated catalase or SOD (iSOD), respectively]. Similarly, we previously showed (1, 4, 9, 10) the dependence of this assay on RNM production (e.g., NO or ONOO- generation) by adding either an RNM scavenger (e.g., cysteine or urate) or an inhibitor of RNM biosynthesis [e.g., N6-(1-iminoethyl)-L-lysine (L-NIL)].

Immunofluorescent staining and high-resolution LSCM of actin cytoskeleton. Cells from monolayers were fixed in cytoskeletal stabilization buffer and then post-fixed in 95% ethanol at -20°C as we previously described (10, 15, 18, 59). Cells were subsequently processed for incubation with FITC-phalloidin (specific for F-actin staining; Sigma), at 1:40 dilution for 1 h at 37°C. After staining, cells were observed with an argon laser ({lambda} = 488 nm) using 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 (1, 2, 10, 11, 13, 14, 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- and G-actin. Polymerized (F) actin and monomeric (G) actin were isolated by using a especially developed series of extraction and ultracentrifugation steps as we described previously (10, 18). Fractionated F- and G-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 loading controls (5 µg/lane) were also run concurrently with each run. To quantify the relative levels of actin, the optical density of the bands corresponding to immunolabeled actin were measured with a laser densitometer.

Immunoblotting determination of protein actin oxidation and actin nitration. Oxidation and nitration of the actin cytoskeleton were assessed, respectively, by measuring protein carbonyl and nitrotyrosine formation (10, 18). To avoid unwanted oxidation of actin samples, all buffers contained 0.5 mM dithiothreitol (DTT) and 20 mM 4,5-dihydroxy-1,3-benzene sulfonic acid (Sigma). To determine the carbonyl content, samples were blotted to a polyvinylidene difluoride (PVDF) membrane, followed by successive incubations in 2 N HCl and 2,4-dinitrophenylhydrazine (DNPH; 100 µg/ml in 2 N HCl; Sigma) for 5 min each. Membranes were then washed three times in 2 N HCl and subsequently washed seven times in 100% methanol (5 min each), followed by blocking for 1 h in 5% BSA in 10x PBS-Tween 20 (PBS-T). Immunologic evaluation of carbonyl formation was performed for 1 h in 1% BSA/PBS-T buffer containing anti-DNPH (1:25,000 dilution; Molecular Probes). Membranes were then incubated with an HRP-conjugated secondary antibody (1:4,000 dilution, 1 h; Molecular Probes). To determine nitrotyrosine content, after the blocking step described above (i.e., BSA/PBS-T buffer), membranes were probed for nitrotyrosine by incubation with 2 µg/ml monoclonal anti-nitrotyrosine antibody for 1 h (Up-state Biotech, Lake Placid, NY), followed by the HRP-conjugated secondary antibody (as above). Processing and film exposure were as in a standard Western blot protocol. The relative levels of oxidized or nitrated actin were then quantified by measuring, with a laser densitometer, the optical density (OD) of the bands corresponding to anti-DNPH (carbonylation) or anti-nitrotyrosine (nitration) immunoreactivity. Immunoreactivity was reported as the carbonyl or nitrotyrosine formation (OD) in the treatment group compared with the maximally oxidized or nitrated actin standards, expressed as a percentage. Oxidized actin standards (5 µg/lane) were run concurrently with corresponding treatment groups.

Determination of barrier permeability by fluorometry. The status of the integrity of monolayer barrier function was confirmed by a widely used and validated technique that measures the apical-to-basolateral paracellular flux of fluorescent markers such as fluorescein sulfonic acid (FSA; 200 µg/ml, 0.478 kDa) as we and others have described previously (1, 2, 6-12, 18, 33, 36, 58). Briefly, fresh phenol-free DMEM (800 µl) was placed into the lower (basolateral) chamber, and phenol-free DMEM (300 µl) containing probe (FSA) was placed in the upper (apical) chamber. Aliquots (50 µl) were obtained from the upper and lower chambers at time 0 and at subsequent time points and transferred into clear 96-well plates (clear bottom; Costar, Cambridge, MA). Fluorescent signals from samples were quantitated using a Fluorescence multiplate reader (FL 600; BIO-TEK Instruments). The excitation and emission spectra for FSA were as follows: excitation = 485 nm, emission = 530 nm. Clearance (Cl) was calculated using the following formula: Cl (nl·h-1·cm-2) = Fab/([FSA]a x S), where Fab is the apical-to-basolateral flux of FSA (light units/h), [FSA]a is the concentration at baseline (light units/nl), and S is the surface area (0.3 cm2). Simultaneous controls were performed with each experiment.

Statistical analysis. Data are presented as means ± SE. All experiments were carried out with a sample size of at least six observations per treatment group. Statistical analysis comparing treatment groups was performed using analysis of variance followed by Dunnett's multiple range test (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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We initially confirmed our earlier preliminary findings (3) that intestinal cells transfected with PLC-{gamma} sense stably overexpress the {gamma} (145 kDa)-isoform of phospholipase C (~2.3-fold compared with wild-type cells) and that this overexpression protects monolayer barrier integrity against exposure to oxidant challenge. Because PLC-{gamma} protects against oxidant-induced disruption, we surmised that this protection may be due to the inhibition of oxidant-activated pathways such as the one triggered by reactive metabolites. In the current investigation, utilizing both pharmacological and molecular biological interventions, we studied the underlying mechanism by which PLC-{gamma} protects.

Stable overexpression of PLC-{gamma} isoform protects against oxidative damage to the cytoskeleton: inhibition of both actin nitration and oxidation. Using both our wild-type and transfected cells, we measured the "footprints" of RNM formation, nitrotyrosine moieties, under conditions of oxidant challenge. We also simultaneously measured oxidation footprints by assessing the carbonylation levels. This was done by sequentially fractionating and purifying the 43-kDa actin molecule from cell monolayers and subsequently immunoblotting these fractions. In wild-type cells (those not overexpressing PLC-{gamma}), oxidant H2O2 alone resulted in a substantial levels of nitration and oxidation of the actin cytoskeleton (Fig. 1A). In contrast, overexpression of PLC-{gamma} by itself afforded protection against oxidant-induced actin nitration and actin carbonylation compared with those in wild-type cells. Indeed, only cells stably overexpressing PLC-{gamma} were protected against oxidant-induced nitration and oxidation injuries. Protection did not require the presence of the growth factor EGF in the cell culture media. Although 1 ng/ml EGF did not afford significant protection against actin nitration or oxidation in wild-type cells, this concentration did potentiate the protection observed in cells overexpressing PLC-{gamma}. In wild-type cells, higher doses of EGF (10 ng/ml) were required for protection (Fig. 1A). Transfection of only the empty vector did not confer protection against oxidation and nitration. For instance, the percentage of actin that was nitrated was 0% for vector-transfected cells exposed to vehicle, 0.73 ± 0.28% for vector-transfected cells exposed to H2O2 alone, and 0.11 ± 0.5% for PLC-{gamma} sense-transfected cells incubated in H2O2. Similarly, the percentage of actin that was carbonylated was 0% for vector-transfected cells exposed to vehicle, 0.77 ± 0.25% for vector-transfected cells exposed to H2O2 alone, and 0.09 ± 0.34% for PLC-{gamma} sense-transfected cells incubated in H2O2. These oxidative alterations did not appear to be caused by changes in the ability of oxidants to cause oxidation/nitration because vector-transfected cells and wild-type cells responded in a similar fashion to H2O2, exhibiting comparable actin oxidation.



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Fig. 1. A: overexpression of PLC-{gamma} protects against oxidant-induced nitration (nitrotyrosination) and oxidation (carbonylation) injury to the actin cytoskeleton of Caco-2 cell monolayers. A novel sense-transfected cell line developed in our laboratory that overexpresses PLC-{gamma} 2.3-fold was utilized. These intestinal monolayers stably overexpressing PLC-{gamma} (PLC-{gamma} cells) or not [wild-type (WT) cells] were incubated with growth factor (EGF) before exposure to oxidant (H2O2) and were then processed for immunoblotting. Transfected cells overexpressing PLC-{gamma} show protection of actin-based cytoskeleton against oxidant-induced nitration and carbonylation injuries. Actin in WT monolayers was protected only by a high dose of EGF (10 ng/ml), whereas a low dose of EGF (1 ng/ml) did not protect against actin oxidation in these same cells. Nitration or oxidation was normalized to a nitrated or oxidized purified actin standard, expressed as a percentage. Values are means ± SE; n = 6 observations per group. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. WT cells exposed to H2O2. &P < 0.05 vs. PLC-{gamma}-overexpressing cells exposed to H2O2 or pretreated with EGF before exposure to H2O2 in WT cells. #P < 0.05 vs. WT cells treated with EGF (10 ng/ml) before exposure to H2O2. Representative immunoblots (n = 6 per group) of the actin nitration (B) and oxidation (carbonylation, C) are shown after treatments as described in A. The actin nitration (anti-nitrotyrosine) bands (B) or actin carbonylation [anti-dinitrophenylhydrazone (DNP)] bands (C) from left to right correspond to WT cells exposed to vehicle (a), PLC-{gamma}-overexpressing cells exposed to vehicle (b), WT cells exposed to 0.5 mM H2O2 (c), PLC-{gamma}-overexpressing cells exposed to 0.5 mM H2O2 (d), WT cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (e), PLC-{gamma}-overexpressing cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (f), WT cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (g), PLC-{gamma}-overexpressing cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (h), or the corresponding nitrated or oxidized actin standard (43 kDa) (i). PLC-{gamma} overexpression in transfected cells by itself protects actin cytoskeleton against both nitration and oxidation damage by oxidant insult (lane d in B and C). This is comparable to that of the control (vehicle treated) actin, which exhibits no nitration or oxidation (corresponding lanes a and b). In WT cells, only a high dose of EGF (10 ng/ml, lane g in B and C) prevented actin nitration and oxidation.

 

Figure 1, B and C, shows representative immunoblots of the alterations in actin nitration and carbonylation. For instance, PLC-{gamma} overexpression substantially inhibited both actin nitration (Fig. 1B) and oxidation (Fig. 1C) as shown by reduced band (lane) densities to a level close to that of controls, indicating prevention of oxidative damage to the actin cytoskeleton in cells overexpressing PLC-{gamma}. As above, only high (protective) doses of EGF (e.g., 10 ng/ml) prevented actin oxidation and nitration in wild-type cells. In contrast, oxidant caused the oxidation and nitration of actin in these wild-type cells.

PLC-{gamma}-induced protection involves downregulation of iNOS-driven reactions: inhibition of iNOS, NO, RNMs (ONOO-), and oxidative stress. Because oxidants such as H2O2 upregulate iNOS (1, 15), we hypothesized that inhibition of iNOS-driven pathways might be a key mechanism for PLC-{gamma}-induced protection. To this end, multiple clones of intestinal cells transfected with 1, 2, 3, or 5 µg of PLC-{gamma} sense cDNA showed (Table 1) a dose-dependent inhibition of iNOS upregulation (L-[3H]citrulline formation) against oxidant (H2O2)-induced challenge. The clone transfected with 3 µg of PLC-{gamma} sense provided the maximum inhibition of iNOS upregulation against oxidative insult. Accordingly, we used this stable ({gamma}3) clone in subsequent experiments.


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Table 1. Effects of transfection of varying amounts of PLC-{gamma} sense or dominant negative mutant DNA on both iNOS activity and NO levels in intestinal Caco-2 monolayers

 

Figure 2A shows that PLC-{gamma} overexpression using the 3-µg sense-transfected clone, which protects gut barrier integrity (3), also caused a substantial reduction in calcium-independent iNOS activity (~96% lower iNOS activity). This is comparable to that of the controls, which displayed only low iNOS activity. These measurements were done in lysates of both transfected and nontransfected Caco-2 monolayers. In wild-type cells, this same dose of H2O2 caused both hyperpermeability and increases in iNOS activity. PLC-{gamma}-induced inhibition of iNOS upregulation did not require EGF. However, a low EGF concentration, 1 ng/ml, which did not by itself afford inhibition of iNOS in wild-type cells, potentiated PLC-{gamma}-induced iNOS downregulation in transfected cells. Wild-type cells, which have native levels of PLC-{gamma}, required a higher dose of EGF (10 ng/ml, Fig. 2A). Transfection of the empty vector alone did not confer protection against oxidant-induced iNOS hyperactivation (iNOS activity was 0.48 ± 0.03 pmol·min-1·mg protein-1 for vector-transfected cells exposed to vehicle, 5.95 ± 0.28 pmol·min-1·mg protein-1 for vector-transfected cells exposed to H2O2 alone, and 0.65 ± 0.23 pmol·min-1· mg protein-1 for PLC-{gamma} sense-transfected cells incubated in H2O2).



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Fig. 2. A: protective effects of PLC-{gamma} overexpression against upregulation of inducible nitric oxide synthase (iNOS) activity induced by H2O2 in Caco-2 monolayers. Monolayers stably overexpressing PLC-{gamma} (transfected) or WT cells (nontransfected) were preincubated with growth factor (EGF) and then exposed to H2O2 and subsequently processed for assessment of L-[3H]citrulline formation. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. WT cells exposed to H2O2. &P < 0.05 vs. PLC-{gamma}-overexpressing cells exposed to H2O2 or pretreated with EGF before exposure to H2O2 in WT cells. #P < 0.05 vs. WT cells treated with EGF (10 ng/ml) before exposure to H2O2. Values are means ± SE; n = 6 observations per group. B: representative Western blot showing the protective effects of PLC-{gamma} overexpression on downregulating iNOS protein levels in Caco-2 cell monolayers. The iNOS bands are from WT cells exposed to vehicle (a), PLC-{gamma}-overexpressing cells exposed to vehicle (b), WT cells exposed to 0.5 mM H2O2 (c), PLC-{gamma}-overexpressing cells exposed to 0.5 mM H2O2 (d), WT cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (e), PLC-{gamma}-overexpressing cells treated with EGF (1 ng/ml) + 0.5 mM H2O2- (f), WT cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (g), and PLC-{gamma}-overexpressing cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (h). In WT cells, H2O2 resulted in a large increase in the levels of iNOS protein (~130 kDa). In PLC-{gamma}-overexpressing cells, this upregulation was prevented. The region of gel shown was between the Mr = 126,000 and 218,000 prestained molecular weights that were run in adjacent lanes.

 

Figure 2B depicts a representative Western blot showing that H2O2 significantly increased iNOS protein levels in wild-type cells, whereas transfected cells overexpressing PLC-{gamma} exhibited only low, basal levels of the iNOS protein. For example, the corresponding OD values were 857 ± 78 for control, 4,518 ± 92 for 0.5 mM H2O2, and 963 ± 106 for PLC-{gamma} sense-transfected cells incubated in H2O2. Transfection of empty vector alone, similar to its lack of effects on iNOS activity and actin oxidation, was ineffective in preventing iNOS protein upregulation (not shown).

NO is the product of the iNOS-catalyzed reaction. Figure 3 shows NO levels both in transfected monolayers and in wild-type monolayers exposed to H2O2 as determined by sensitive chemiluminescence analysis of cell lysates. PLC-{gamma} overexpression markedly prevented oxidant-induced NO overproduction (Fig. 3). In wild-type cells, as for actin oxidation and iNOS upregulation, NO overproduction was inhibited only by high, protective doses of EGF (e.g., 10 ng/ml). Transfection of vector alone did not confer protection against NO overproduction (not shown).



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Fig. 3. Concentrations of nitric oxide (NO) in the supernatant of homogenates of intestinal cell monolayers of either transfected or WT origin assessed by a sensitive chemiluminescence assay. As for effects on iNOS downregulation, PLC-{gamma} overexpression by itself prevented NO upregulation induced by oxidant challenge. A high dose of EGF (10 ng/ml), which downregulated iNOS (see Fig. 2), also suppressed NO overproduction in WT cells. Values are means ± SE; n = 6 observations per group. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. WT cells exposed to H2O2.&P < 0.05 vs. PLC-{gamma}-overexpressing cells exposed to H2O2 or pretreated with EGF before exposure to H2O2 in WT cells. #P < 0.05 vs. EGF (10 ng/ml) before H2O2 in WT cells.

 

Table 1 also depicts the results of NO analysis from multiple clones of transfected, PLC-{gamma}-overexpressing intestinal cells showing a dose-dependent inhibition of NO overproduction. As for iNOS suppression, the 3-µg stable clone of PLC-{gamma} sense ({gamma}3) provided the highest protection against NO overproduction.

Figure 4 shows the time course for increases in iNOS protein, iNOS activity, and NO levels under oxidative conditions and their prevention in transfected cells. PLC-{gamma} overexpression prevented the effects of H2O2 on all three outcomes. Maximal fold increases under H2O2 alone were ~5.2 for iNOS protein, ~12 for iNOS activity, and ~12 for NO levels; these increases were prevented by PLC-{gamma} overexpression.



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Fig. 4. Time course for the suppression of the induction of iNOS and increases in NO in PLC-{gamma}-overexpressing cells. Cells were exposed to 0.5 mM H2O2 at time 0. Units are pmol/mg protein for iNOS activity, 10-3 x optical density (OD) for iNOS protein levels, and µmol/106 cells for NO levels. Values are means ± SE.

 

In parallel with the suppression of oxidant-induced affects, PLC-{gamma} overexpression inhibited oxidative stress as determined by a reduction in the fluorescence of DCF (Fig. 5). In wild-type cells, where H2O2 substantially increased DCF fluorescence, oxidative stress was suppressed only by high, protective doses (e.g., 10 ng/ml) of EGF. In the absence of oxidant, we observed significantly lower but still substantial levels of oxidative stress [possibly due to the normal generation of DCF reactive oxygen radicals (e.g., *O2-) by well-known cellular metabolic processes such as the mitochondrial respiratory chain reactions (1, 4, 9, 10)]. Transfection of the empty vector alone did not suppress oxidative stress (not shown).



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Fig. 5. Oxidative stress in intestinal cell monolayers that is induced by oxidant is attenuated by PLC-{gamma} overexpression as determined by changes in dichlorofluorescein (DCF) fluorescence intensity. PLC-{gamma} overexpression prevented oxidative stress, as shown by a reduction in the DCF fluorescence intensity that is comparable to control (vehicle) levels. In WT cells, only a high (protective) dose of EGF prevented oxidative stress, which is also similar to the low (basal) oxidative stress levels in controls. Values are means ± SE; n = 6 observations per group. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. H2O2 in WT cells. &P < 0.05 vs. PLC-{gamma}-overexpressing cells exposed to H2O2 or pretreated with EGF before exposure to H2O2 in WT cells. #P < 0.05 vs. WT cells treated with EGF (10 ng/ml) before exposure to H2O2.

 

Suppression of iNOS upregulation in transfected cells protects the assembly of actin and the cytoarchitecture of F-actin cytoskeleton. Because it is known that oxidants in this intestinal model disrupt the cytoskeleton, we assessed the state of actin polymerization and its intracellular architecture. PLC-{gamma} overexpression confered protection to the assembly of F-actin pool (Fig. 6) as well as the cytoarchitecture of actin cytoskeleton (Fig. 7, a-c). For example, to determine effects of PLC-{gamma} overexpression on the dynamic alterations in the polymerization states of the F-actin, we performed immunoblotting of actin cytoskeleton. To this end, the polymerized actin fraction (F-actin, an index of actin stability) was isolated from monolayers. Figure 6 shows that PLC-{gamma}-overexpressing monolayers, which were exposed to oxidant, exhibited a stable F-actin assembly, as indicated by an enhancement in this polymerized actin fraction (i.e., increased band density). This state of assembly is comparable to that of controls. In wild-type cells, in contrast, oxidant decreased polymerized F-actin, indicating disassembly of actin cytoskeleton. In these wild-type cells, only pretreatment with the higher doses (10 ng/ml) of EGF resulted in a stable actin assembly. Indeed, confocal microscopy corroborates this finding, showing that intestinal cells overexpressing PLC-{gamma} had a smooth and normal architecture of the actin cytoskeleton even after exposure to oxidant (Fig. 7c). This preserved appearance was indistinguishable from that of control (and untreated) cells (Fig. 7a), which also showed an intact organization of the actin cytoskeleton. In contrast, wild-type cells (not overexpressing PLC-{gamma}) that are challenged with H2O2 exhibit instability, fragmentation, and disruption of the actin cytoskeleton (Fig. 7b). This protection of both the assembly and cytoarchitecture of actin-based cytoskeleton by PLC-{gamma} overexpression parallels the protective effects of this overexpression against oxidant-induced iNOS and NO upregulation as well as actin oxidation.



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Fig. 6. Protective effects of PLC-{gamma} overexpression on the assembly of F-actin cytoskeletal pool in Caco-2 cell monolayers assessed by immunoblotting. F-actin (triton insoluble) cytoskeletal extracts from Caco-2 cells were subjected to SDS-PAGE fractionation and immunoblotted using monoclonal anti-actin antibody followed by horse-radish peroxidase-conjugated secondary antibody and subsequently autoradiographed. The F-actin (43 kDa) polymerization bands are from WT cells exposed to vehicle (a), PLC-{gamma}-overexpressing cells exposed to vehicle (b), WT cells exposed to 0.5 mM H2O2 (c), PLC-{gamma}-overexpressing cells exposed to 0.5 mM H2O2 (d), WT cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (e), PLC-{gamma}-overexpressing cells treated with EGF (1 ng/ml) + 0.5 mM H2O2 (f), WT cells treated with EGF (10 ng/ml) + 0.5 mM H2O2- (g), and PLC-{gamma}-overexpressing cells treated with EGF (10 ng/ml) + 0.5 mM H2O2 (h). H2O2 resulted in a large decrease in the assembly F-actin pool in WT cells (lane c), whereas in PLC-{gamma}-over-expressing cells this disassembly was prevented (lane d).

 


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Fig. 7. The intracellular organization of the F-actin cytoskeleton stained by fluorescein-conjugated phalloidin and subsequently captured by high-resolution laser scanning confocal microscopy (LSCM) of intestinal cell monolayers. WT Caco-2 cells were exposed to 0.5 mM H2O2 (b). PLC-{gamma}-overexpressing monolayers were also exposed to 0.5 mM H2O2 (c). Untreated (control) cells were exposed to vehicle (a). Actin in control cells appears as an intact structure or ring (a). This is demonstrated by a normal, continuous, and smooth distribution of actin ring (or cortex) at areas of cell-to-cell contact. Only in WT cells exposed to H2O2 (b) does the F-actin ring appear disrupted, fragmented, and disorganized. In cells overexpressing PLC-{gamma} (c) and incubated with oxidant, normal actin cytoarchitecture is highly preserved and resembles morphology detected in that of controls. Bar, 25 µm. Representative photomicrographs are shown; n = 6 observations per group.

 

Intracellular distribution and constitutive activation of the overexpressed PLC-{gamma} in transfected intestinal cells correlates with several different indexes of iNOS and oxidative stress in monolayers. Overexpressing the 145-kDa PLC-{gamma} in intestinal cells led to its distribution into mostly the particulate fractions (particulate = membrane + cytoskeletal fractions), with a much smaller distribution in the cytosolic fractions (Fig. 8A), suggesting the constitutive activation of the {gamma}-isoform of PLC. In wild-type cells (Fig. 8B), in contrast, we found a mostly cytosolic distribution of PLC-{gamma}, with smaller pools in the membrane and cytoskeletal (particulate) fractions, suggesting inactivity of this isozyme.



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Fig. 8. A and B: subcellular distribution of PLC-{gamma} in the cytosolic, membrane, and cytoskeletal fractions of intestinal cell monolayers of either transfected or WT origin. In transfected cells overexpressing PLC-{gamma} (A), note the presence of PLC-{gamma} in the particulate (i.e, both membrane and cytoskeletal) fraction, indicating constitutive activation of the {gamma}-isoform. In WT cells (B), PLC-{gamma} is inactive, as shown by a mostly cytosolic pool of {gamma}-isoform. Cell monolayers grown in 75-cm2 flasks were processed for the isolation of various fractions and then Western immunoblotted using monoclonal anti-PLC-{gamma}. Representative blots are shown; n = 6 observations per group.

 

Table 2 is an analysis of the intracellular distribution of the PLC-{gamma} in various fractions of either transfected or wild-type Caco-2 cell monolayers. Overexpressed PLC-{gamma} isoform is "constitutively active" because achieving this intracellular distribution did not require EGF or pharmacological intervention. Pretreatment of these cells with EGF, however, enhanced the fraction of PLC-{gamma} isoform in the membrane and cytoskeletal fractions, reaching near total levels for PLC-{gamma}. On the other hand, in wild-type cells PLC-{gamma} is found in a mostly cytosolic distribution (suggesting inactivity), with smaller pools in the membrane and cytoskeletal (particulate) fractions. Wild-type cells incubated with EGF also showed increased membrane and cytoskeletal distribution of native PLC-{gamma}.


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Table 2. Analysis of subcellular distribution of PLC-{gamma} in various cell fractions from either stably transfected cells overexpressing PLC-{gamma} or from WT cells expressing native levels of this isoform

 

Figure 9 shows the activity levels of PLC-{gamma} isoform (determined by in vitro assay) from immunoprecipitated particulate cell fractions of Caco-2 cells, which were stably transfected with PLC-{gamma} cDNA to overexpress this isoform. There was a substantial increase in the activity levels of PLC-{gamma} isoform in these transfected (vehicle exposed) cells, confirming findings in Fig. 8 and Table 2. EGF further activated PLC-{gamma} in these transfected cells, reaching near maximal activation levels for this isoform. Wild-type cells exposed to vehicle, in contrast, showed basal activity levels for PLC-{gamma} in the particulate cell fractions. In these wild-type cells, EGF further activated native PLC-{gamma}, but at much lower levels compared with that of the transfected cells under similar conditions.



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Fig. 9. Increases in PLC-{gamma} activity in differentiated intestinal cells, which were stably transfected with a plasmid encoding the {gamma}-isoform of PLC, as determined by in vitro assay. The constitutive activation of PLC-{gamma} is shown in these transfected PLC-{gamma}-overexpressing cells (3-µg clone) as indicated by its high activation levels. EGF further activates PLC-{gamma} in these transfected cells, reaching near maximal activation levels for this isoform. In contrast, WT cells show a low (basal) level of activity for the native PLC-{gamma} isoform. Native PLC-{gamma} was activated only after exposure to EGF. Although EGF (e.g., 10 ng/ml) further activated native PLC-{gamma}, the overall levels of this activation were lower compared with that of the transfected PLC-{gamma}-overexpressing cells under similar conditions. Also, note the near complete suppression of PLC-{gamma} activity in Caco-2 cells that were transfected with a dominant negative fragment, namely PLCz, to the native PLC-{gamma} isoform. In these dominant negative transfected cells (PLCz mutant), almost complete suppression of native PLC-{gamma} activity is achieved. In these same cells, even the addition of EGF could not increase the {gamma}-isoform activity. Particulate cell extracts from Caco-2 monolayers were subjected to immunoprecipitation by the monoclonal anti-{gamma} antibody, and then the PLC-{gamma} activity was determined in vitro. Values are means ± SE; n = 6 observations per group. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. WT cells exposed to H2O2. &P < 0.05 vs. PLC-{gamma}-overexpressing cells exposed to H2O2 or pretreated with EGF (10 ng/ml) before exposure to H2O2 cells in WT cells or EGF (10 ng/ml) + H2O2 in dominant negative PLCz mutant cells. #P < 0.05 vs. corresponding EGF (10 ng/ml) before H2O2 in WT cells or dominant negative cells.

 

Using data across all experimental conditions, we found significant inverse correlations (e.g., r = -0.93, P < 0.05) between PLC-{gamma} levels (in vitro assay or optical density from the particulate fraction) and iNOS downregulation, further suggesting that activation of {gamma}-isoform of PLC is key in protection against oxidant-induced iNOS upregulation. Other robust correlations were seen when either NO overproduction or oxidative stress (DCF fluorescence) was correlated with the PLC-{gamma} levels (r = -0.90 or -0.89, respectively, P < 0.05 for each). When two other markers of oxidative stress, actin carbonylation and actin nitration (RNM generation), were correlated with PLC-{gamma}, additional robust correlations were observed (r = -0.95 or -0.94, respectively, P < 0.05 for each), further indicating that activation of {gamma}-isoform is key in iNOS downregulation through normalization of NO levels. Similarly, when markers of stability such as either actin integrity or actin assembly were correlated with the PLC-{gamma}, robust correlations were seen (r = 0.88 or 0.91, respectively, P < 0.05 for each).

Stable dominant negative inhibition of PLC-{gamma} by PLCz fragment to inactivate native {gamma}-isoform and its prevention of EGF-induced protection against oxidative stress of iNOS upregulation. The above findings collectively indicate that PLC-{gamma} may play an essential intracellular role in protection against oxidative stress of iNOS-driven reactions. To independently investigate a possible role for PLC-{gamma} in EGF-mediated protection against iNOS upregulation and consequent RNM driven oxidative stress, we used stable dominant negative transfected PLCz clones of Caco-2 cells, which we developed. To this end, cDNA encoding a PLCz dominant negative fragment from the Z region of human PLC-{gamma}1 was utilized. Using this dominant negative approach for PLC-{gamma}, we are capable of substantially reducing the steady-state activity levels for native isoform by ~99.3% (Fig. 9, 3-µg clone). In these dominant negative PLCz cells, EGF could not increase the native PLC-{gamma} isoform activity.

Table 1 further demonstrates the dose-dependent effects of varying amounts (1, 2, 3, or 5 µg) of PLC-{gamma} dominant negative plasmid (i.e., PLCz) on suppression of both EGF-induced iNOS downregulation and NO normalization in intestinal cells. The cell clone that was stably transfected with 3 µg of PLCz dominant negative plasmid resulted in maximum inability of EGF to prevent oxidant-induced iNOS upregulation or NO overproduction. Thus this clone was utilized for other inhibition experiments.

For example, we have shown (Fig. 10) that stable dominant negative inhibition of native PLC-{gamma} activity substantially prevented the protection afforded by 10 ng/ml EGF against iNOS upregulation. In wild-type (naive) cells, on the other hand, this same concentration of EGF almost completely prevented oxidant-induced iNOS upregulation. A very large percentage (~90%) of EGF-induced iNOS downregulation is PLC-{gamma} dependent.



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Fig. 10. Stable dominant negative inhibition of the native PLC-{gamma} isoform by the PLCz fragment prevents the protective (suppressive) effects of growth factor (EGF) against oxidant-induced iNOS hyper-activation. A novel PLCz dominant negative-transfected intestinal cell clone developed in our laboratory (see MATERIALS AND METHODS), which almost completely lacks native PLC-{gamma} activity, was grown as monolayers and subsequently pretreated with a high dose of EGF (10 ng/ml) and then exposed to 0.5 mM H2O2. Monolayers of WT Caco-2 cells were also treated in a similar fashion. iNOS activity was subsequently assessed. Values are means ± SE; n = 6 observations per group. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. H2O2. &P < 0.05 vs. EGF + H2O2 in WT cells.

 

Analysis of both the NO levels and oxidative stress from these dominant negative transfected cells additionally demonstrates that inactivation of native PLC-{gamma} isoform substantially attenuated both EGF's normalization of NO levels (Fig. 11A) and downregulation of oxidative stress (Fig. 11B, DCF fluorescence). As for iNOS downregulation, a large percentage (~90%) of EGF-induced NO normalization and DCF fluorescence downregulation appears to be PLC-{gamma} dependent in intestinal monolayers.



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Fig. 11. A: prevention of the protective effects of growth factor (EGF) on the downregulation of NO levels in intestinal cells by the stable dominant negative inhibition of native PLC-{gamma}. Caco-2 cells almost totally lacking PLC-{gamma} activity were incubated with EGF (10 ng/ml) before exposure to H2O2. NO concentrations were assessed by a sensitive chemiluminescence assay. Values are means ± SE; n = 6 observations per group. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. H2O2. &P < 0.05 vs. EGF + H2O2 in WT cells. B: prevention of the protective effects of growth factor (EGF) on the downregulation of oxidative stress (DCF fluorescence intensity) in intestinal cells by dominant negative inhibition of native PLC-{gamma} activity. Conditions are as in A. Values are means ± SE; n = 6 observations per group. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. H2O2. &P < 0.05 vs. EGF + H2O2 in WT cells.

 

Furthermore, immunoblotting analysis of the oxidative state of actin (Fig. 12A) from these same dominant negative clones further shows that stable inactivation of the {gamma}-isoform prevented EGF-induced protection against both actin nitration and oxidation. PLC-{gamma} isoform inactivation by itself did not cause actin oxidation. Finally, analysis of the state of actin assembly from these dominant negative cells demonstrates (Fig. 12B) that inhibition of native PLC-{gamma} attenuated protection against actin depolymerization by a high (protective) dose of EGF. Here, EGF could no longer prevent oxidant-induced actin disassembly.



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Fig. 12. A: immunoblotting analysis of the suppressive effects of dominant negative inactivation of native PLC-{gamma} isoform on EGF's attenuation of both actin nitration and oxidation in Caco-2 cells. Cells either lacking (i.e., PLCz mutant transfected) or expressing native PLC-{gamma} isoform activity (i.e., WT) were incubated with EGF before exposure to H2O2. Nitration and carbonylation immunoreactivities were assessed as in Fig. 1. Values are means ± SE; n = 6 observations per group. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. H2O2. &P < 0.05 vs. EGF + H2O2 in WT cells. B: immunoblotting analysis of the F-actin assembly in Caco-2 cell monolayers of either the dominant negative or WT origin. Dominant negative inactivation of native PLC-{gamma} isoform prevents EGF's protection (enhancement) of F-actin assembly under oxidant challenge. Actin fractions were extracted from intestinal monolayers and then subjected to SDS-PAGE and subsequently autoradiographed. To quantify the relative levels of F-actin assembly, the optical density of the bands corresponding to immunolabeled actin was measured with a laser densitometer. The percentage of polymerized F-actin is calculated as F/(F + G), where F + G is the total cellular actin pool. Polymerized F-actin is an index of actin assembly, and the monomeric G-actin is an index of actin disassembly. Values are means ± SE; n = 6 observations per group. *P < 0.05 vs. corresponding vehicle. +P < 0.05 vs. H2O2. &P < 0.05 vs. EGF + H2O2 in WT cells.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the current report, we have described mechanisms that play a key role in the defense and repair of epithelial cell monolayers in response to oxyradical stress. We have demonstrated that the 145-kDa {gamma}-isoform of PLC is required for EGF-mediated protection against oxidant-induced iNOS upregulation and the consequent oxidative stress injury to the integrity of F-actin cytoskeleton and the intestinal epithelial barrier. A second conclusion is that PLC-{gamma} by itself is key in cellular protection against stress of iNOS-driven reactions. The underlying mechanism for this protective effect of PLC-{gamma} isoform appears to be the suppression of both nitration and oxidation stress injury to the 43-kDa subunit components of the F-actin network and consequent stabilization of actin assembly and cytoarchitecture.

These conclusions are based on several independent lines of findings. Expression of PLC-{gamma} mimics an EGF-like protection against oxidant-induced iNOS upregulation, including downregulation of iNOS activation, normalization of NO levels, reduction of RNM footprints, and decreases in oxidative stress (DCF fluorescence). Moreover, activation of the PLC-{gamma} suppresses the footprints of oxidative injury (i.e., RNM formation) to the 43-kDa actin protein. These other protective effects include decreases in the nitration (nitrotyrosination) of the actin molecule and reduction of oxidation (carbonylation) of actin. In concert, PLC-{gamma} activation decreased the monomeric (G) actin and enhanced the stability of polymerized (F) actin as well as preserved appearance of normal actin cytoarchitecture. Additionally, a low, nonprotective concentration of EGF potentiated all measures of PLC-{gamma}-mediated protection against oxidative stress of iNOS upregulation. Furthermore, dominant negative PLC-{gamma} (i.e., PLCz mutant), which causes almost complete inactivation of native PLC-{gamma}, substantially prevented EGF's protective ability to suppress iNOS upregulation, actin instability, and F-actin disruption. EGF was also unable to inhibit nitration and carbonylation of actin, normalize NO levels, or even reduce DCF fluorescence in these PLCz mutant cells. Finally, PLC-{gamma} activation quantitatively correlated with decreases in all outcomes indicating protection against oxidative stress.

Using both transfected and wild-type cells, we found correlations 1) between PLC-{gamma} isoform activation and protection against oxidant-induced iNOS upregulation (r = -0.93, P < 0.05) as well as several other key outcomes. These others included 2) protection against oxidant-induced NO overproduction and PLC-{gamma} activation (r = -0.90, P < 0.05), 3) actin nitration (RNM footprint) and PLC-{gamma} activation (r = -0.94, P < 0.05), and 4) oxidative stress (DCF fluorescence) levels and PLC-{gamma} activation (r = -0.89, P < 0.05). Similar correlation was also reached when 5) actin carbonylation (oxidation) and PLC-{gamma} activation (r = -0.95, P < 0.05) are utilized. Furthermore, 6) protection against oxidant-induced actin disassembly (decreased F-actin polymer pool) and PLC-{gamma} activation (r = 0.91, P < 0.05) and 7) the percentage of normal F-actin cytoarchitecture and PLC-{gamma} activation (r = 0.88, P < 0.05) provide other supporting correlations. The high strength as well as consistency of these correlations further indicates that PLC-{gamma} isoform activation is essential to protection against iNOS upregulation and consequent oxidative stress to the assembly of F-actin cytoskeleton and integrity of intestinal barrier function. In this view, activation of PLC-{gamma} leads to the normalization of NO levels and subsequently protects actin cytoskeleton and barrier integrity against oxidative injury induced by iNOS.

Other proteins can also be involved in maintaining the integrity of permeability barrier in the GI epithelium. These include a large heterogeneous family of proteins such as microtubules ({alpha}- and {beta}-tubulin), occludin, ZO proteins (e.g., ZO-1, ZO-2, ZO-3), claudins (e.g., isoforms I and V), and myosin (e.g., type II) as well as others such as E-cadherin, connexin43, {beta}-catenin, and adherin (5, 30, 33, 36, 42). Among these proteins, we choose to study actin because previous studies showed the critical role of actin cytoskeleton, especially the so-called "apical ring of actin," in modulation of barrier paracellular permeability in epithelial cells such as Caco-2 monolayers (e.g., Refs. 6, 10, 18, 36, 58). Moreover, we have consistently shown that actin stability is key to EGF-mediated protection of intestinal barrier permeability (6, 10, 18).

The new findings of this report, using targeted molecular interventions, are consistent not only with our own previous studies but also with the findings of other investigators. It is known that PLC-{gamma} profoundly affects cellular functions in nonepithelial cells as well as epithelial cells (23, 26, 32, 53, 62). For example, migration of intestinal cells that is stimulated by growth factor requires PLC-{gamma} activity (22, 41, 46, 49). Other studies have implicated PLC-{gamma} in the pathway for remodeling of the components of the cytoskeleton such as microtubules, profilin, and gelsolin (3, 12, 19, 26, 35). Furthermore, PLC-{gamma} is the only epithelial PLC isoform with SH2 and SH3 domains that can be activated by growth factor ligands (12, 23, 49, 52). The Z region of human PLC-{gamma}1, namely PLCz, which covers both the SH2 and SH3 domains (amino acids 517-901), is known to specifically inhibit PLC-{gamma}1 activation and not other PLC isoforms in epithelial cells (12, 22, 23, 32, 49). Indeed, the PLCz mutant utilized is specific for inhibition of PLC-{gamma}1 because previous studies have shown that PLC-{gamma}1 is the only epithelial PLC isoform that contains SH2 and SH3 domains and that is activated by EGF (12, 23, 32, 44, 49, 52, 57). In our studies PLCz-dominant mutant expression prevented the activation of PLC-{gamma} while at the same time abrogating EGF protection against oxidative stress. Our new findings on the 145-kDa {gamma}-isoform of PLC, we believe, suggest a unique pathophysiological role among PLC isoforms in intestinal cell monolayers, namely protection against oxidative stress of RNM upregulation and of cytoskeletal oxidation.

PLC-{gamma} hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce not only inositol 1,4,5-trisphosphate (IP3) but also diacyl glycerol (DAG) in epithelial cells (6, 8, 23, 49, 51, 57). DAG is one of the best-characterized products of PLC-{gamma}-mediated reactions and that is known to lead to downstream activation of serine/thereonine protein kinase C (PKC) (8, 11, 12, 28, 47). This is consistent with the fact that we showed, using the same intestinal model, that PKC signaling is also required for EGF-mediated protection against oxidant-induced GI barrier and cytoskeletal disruption (6, 8, 11, 12, 16). That PKC is downstream from PLC-{gamma} in the cell's protective cascade is also indicated by our previous finding that PKC activators (OAG or TPA) can maintain both cytoskeletal integrity and intestinal barrier function even in the presence of PLC inhibition. Indeed, PKC has also been shown to be downstream of PLC in other systems as well (47, 49, 51, 61). Also, other studies have shown that a naturally occurring intracellular activator of PKC, namely DAG (OAG used in our previous studies is a synthetic version of this compound), modulates intestinal monolayer permeability in Caco-2 cells (11, 42). Overall, it appears that growth factor-induced protection is mediated by PLC-{gamma} and then PKC.

It is noteworthy that we previously established (1, 4, 9, 10, 16) that 1) oxidant (e.g., H2O2, HOCl, ONOO-,or NO compounds)- or oxidative stressor (e.g., ethanol)-induced disruption of the cytoskeleton of intestinal cells and the consequent disruption of the permeability barrier of intestinal monolayers require rapid upregulation of an iNOS-driven reactive pathways—increased levels of RNMs such as NO and ONOO- (the latter from reaction of ·O2- + NO)—which causes increased oxidative stress (DCF fluorescence) and damage through nitration and oxidation of cytoskeletal network. For example, H2O2 concentrations that caused actin damage and nitration and intestinal hyperpermeability also rapidly upregulated iNOS and increased RNMs and oxidative stress, including DCF fluorescence; several different exogenously added NO/RNM compounds [e.g., 3-morpholinosydnonimine (SIN-1), soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) + xanthine + xanthine oxidase, ONOO-] mimicked the effects of H2O2 (and potentiated the effects of low, nondamaging doses of H2O2); and either iNOS inhibitors (e.g., L-NIL) or several selective anti-oxidants (urate, L-cysteine, SOD) that scavenge NO/RNM or ·O2- anion prevented or substantially attenuated the injurious changes (e.g., DCF fluorescence) induced by H2O2 or RNM compounds (4, 9, 10).

2) Additionally, exposure of intestinal cells to oxidants, which models the oxidative stress that occurs during the active phase of IBD, can, surprisingly, further increase endogenous cellular synthesis of RNM compounds (such as NO and ONOO-) as well as ROS compounds (·O2-) (·O2- can, in turn, combine with the NO to generate other damaging reactive species such as ONOO-) (1, 4, 9, 10). Thus we demonstrated that oxidants (e.g., H2O2) per se appear to cause epithelial damage through the generation of reactive species (e.g., NO and RNMs), which then cause increases in oxidative stress (DCF) and nitration. 3) We also showed that growth factors (e.g., EGF) protect against oxidant-induced disruption through the underlying suppression of iNOS pathway upregulation and decreased levels of RNMs such as NO and ONOO-, which then reduce oxidative stress (16). 4) Finally, oxidants H2O2, HOCl, and ONOO- can all rapidly upregulate iNOS and NO in intestinal cells, because these cells appear to have a standing pool of iNOS mRNA and protein that is ready for immediate upregulation (4, 5, 10, 16). We found a similar mechanism of action for other oxidative stressors such as ethanol (1, 9).

Studies in endothelial cells as well as one in vivo study in rat gastric mucosal cells are consistent with our published findings of rapid iNOS changes (54, 56). In the study using isolated rat gastric mucosal cells (56), low basal levels of iNOS were noted in control (untreated) mucosa, whereas, after challenge with endotoxin, significant and rapid increases in iNOS activity were detected in the mucosal cells within 1 h, followed by peak levels at 2-4 h (almost identical to the rapid start and peak times for iNOS changes in our Caco-2 cells). Similarly, other studies showed detectable levels of iNOS activity as early as 60 min after H2O2 (0.1-1 mM) challenge (54). In yet another study in endothelial cells, a slight basal expression of iNOS protein (and iNOS mRNA as detected by RT-PCR) was shown in unstimulated cells (63).

Our findings are potentially relevant for developing new treatment strategies for IBD. They suggest a novel antioxidative defensive mechanism that might protect against oxidative stress of iNOS and NO upregulation and prevent initiation, continuation, or manifestation of the IBD attack. The potential therapeutic use of this antioxidative mechanism is consistent with the current characterizations of the pathophysiology of the inflammation, in general, and of IBD, in particular. iNOS is a key factor in the inflammatory response triggered by an array of conditions promoting oxidative stress. Specifically, upregulation of iNOS and the formation of RNMs (e.g., NO, ONOO-) under conditions of oxidative stress appears to be essential in the promotion of an inflammatory response in non-GI as well as GI models (e.g., 34, 37, 40, 50, 55). Furthermore, we and others have shown that upregulation of iNOS and RNMs is found in the intestinal mucosa of patients with ulcerative colitis and Crohn's disease (17, 37, 50, 55), in which high levels of oxidants (e.g., H2O2) as well as loss of mucosal barrier integrity have been reported (17, 30, 31, 37, 39, 43). In these studies, tissue nitration was associated with the inflamed human mucosa of IBD (37, 50, 55) and was linked with the upregulation of iNOS (37, 55). We further showed that the amount of NO and RNM formation (i.e., nitration) correlated with the degree of mucosal inflammation and disease severity score in IBD (17, 37). Interestingly, some nitrated tissue proteins have also been detected in vivo in non-GI models such as the inflamed lung (34, 48). Thus iNOS and NO upregulation appear to be critical to the pathogenesis of inflammatory conditions such as IBD (17, 37). This upregulation is though to be especially important in the transitions from the inactive to active (flare up) phases of inflammation in IBD in which intestinal oxidants and proinflammatory molecules periodically create a vicious cycle that can lead to sustained iNOS upregulation, oxidative stress, hyperpermeability, inflammation, and consequently to mucosal tissue damage. The protective antioxidative effects of PLC-{gamma}, such as those we have found in intestinal cells, could play a pivotal role in preventing the establishment of such a vicious inflammatory cycle.

A question that remains unanswered is how iNOS might be up- or downregulated in intestinal cells. There are several mechanisms by which the rapid iNOS modulation might occur. Regarding iNOS upregulation, we recently showed that oxidant induces rapid iNOS upregulation (and its injurious consequences such as nitration and DCF increases) through the underlying activation of the {delta} (75-kDa)-isoform of PKC (PKC-{delta}, a member of the "novel" subfamily of PKC isoforms) (4, 5, 10). Specifically, oxidant H2O2 induces loss of epithelial barrier integrity by nitrating and disassembling the cytoskeleton through the activation of PKC-{delta} isoform and consequent upregulation of iNOS-driven reactions (including NO, ONOO-, DCF fluorescence, and cytoskeletal nitration). Moreover, expression and activation of PKC-{delta} is by itself key for cellular injury induced by oxidative stress of iNOS-driven reactions, indicating, once again, the critical role of this "injurious PKC isoform" signal in iNOS upregulation under conditions of oxidative stress.

Regarding iNOS downregulation, we recently showed that EGF can rapidly suppress iNOS and NO upregulation through the downstream activation of the {zeta} (72-kDa)-isoform of PKC (PKC-{zeta}, a member of the "atypical" subfamily of PKC isoforms) (7, 16). Specifically, EGF protects against oxidative disruption of the intestinal barrier integrity by stabilizing the cytoskeleton, in large part, through the downstream activation of PKC-{zeta} and consequent downregulation of iNOS. Consistent with these findings, activation of PKC-{zeta} is by itself required for cellular protection against oxidative stress of iNOS upregulation, further supporting a key role for this "protective PKC isoform" in inhibition of iNOS upregulation. These mechanisms appear to account for how iNOS is rapidly modulated by oxidant and EGF in our intestinal model.

Nonetheless, there are other possible mechanisms for iNOS modulation as reported in non-GI models. For example, NOS contains consensus sequences for sites for protein phosphorylation (21, 25), especially tyrosine phosphorylation of calcium-independent NOS (i.e., iNOS), which has been shown in vitro and in endothelial cells following a variety of stimuli, and it was proposed that this mechanism could rapidly regulate the activity of NOS (21, 25). An alternative mechanism is the rapid assembly of the two known monomeric domains of iNOS into an active dimer, which is known to be required for NOS catalytic activity. Specifically, pools of inactive, monomeric iNOS would be available from a standing intracellular protein pool in unstimulated cells (like in Caco-2 cells). These monomers can be rapidly assembled by an appropriate stimulus into an active dimer (21). It remains to be seen whether there are additional molecular mechanisms underlying the iNOS modulation in GI epithelial cells.

In summary, our findings suggest that PLC-{gamma} is responsible for a substantial portion of the protection of the intestinal epithelium against oxidant stress that is induced by iNOS upregulation and perhaps is key to preventing amplification and perpetuation of an uncontrolled, oxidant-induced, inflammatory cascade in IBD, one that can be ignited by free radicals and other oxidants present in the GI tract. Our studies also suggest that PLC-{gamma} is a potential therapeutic target for intervention against inflammatory conditions in which oxidants are prevalent (e.g., IBD). For instance, one therapeutic approach might be the exogenous delivery of a sense vector for PLC-{gamma} isoform (targeted gene therapy) to the inflamed GI mucosa in vivo. If this gene therapy approach is successful, one should be able to protect and maintain epithelial integrity against oxidative stress such as that occuring during incipient or rampant inflammation and subsequently limit the initiation and progression of GI mucosal inflammation and damage. PLC-{gamma} therapies might even synergize with the use of currently employed antioxidants so that inflammatory processes are more effectively attenuated through the manipulation of both the damaging and protective intracellular pathways.


    DISCLOSURES
 
This work was supported in part by a grant from the Department of Internal Medicine at Rush University Medical Center and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-60511 (to A. Banan). Portions of this work were presented in the abstract form during the annual meeting of the American Gastroenterological Association (Digestive Disease Week), May 2003.


    ACKNOWLEDGMENTS
 
We thank Dr. Allen Wells at the Pittsburgh University Medical Center for generous help in providing the PLCz vector.


    FOOTNOTES
 

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

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


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

2. Banan A, Choudhary S, Zhang Y, Fields JZ, and Keshavarzian A. Oxidant-induced intestinal barrier disruption and its prevention by growth factors in a human colonic cell line: role of the microtubule cytoskeleton. Free Radic Biol Med 28: 727-738, 2000.[ISI][Medline]

3. Banan A, Farhadi A, Fields JZ, Zhang L, and Keshavarzian A. The {gamma} isoform of phospholipase C (PLC-{gamma}) in EGF protection of intestinal F-actin cytoskeletal assembly and barrier integrity (Abstract). Gastroenterology 122, Suppl 1: T863, 2002.

4. Banan A, Farhadi A, Zhang L, Fields JZ, and Keshavarzian A. The {delta}-isoform of protein kinase C causes inducible nitric oxide synthase and nitric oxide up-regulation: key mechanism for oxidant-induced carbonylation, nitration, and disassembly of cytoskeleton and disruption of the microtubule cytoskeleton and hyperpermeability of barrier intestinal epithelia. J Pharmacol Exp Ther 305: 482-494, 2003.[Abstract/Free Full Text]

5. Banan A, Fields JZ, Farhadi A, Talmadge DA, Zhang L, and Keshavarzian A. Activation of {delta}-isoform of protein kinase C is required for oxidant-induced disruption of both the microbule cytoskeleton and permeability barrier of intestinal epithelia. J Pharmacol Exp Ther 303: 17-28, 2002.[Abstract/Free Full Text]

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

7. Banan A, Fields JZ, Talmage DA, Zhang L, and Keshavarzian A. PKC-{zeta} is required in EGF protection of cytoskeleton and intestinal barrier integrity against oxidant injury. Am J Physiol Gastrointest Liver Physiol 282: G794-G808, 2002.[Abstract/Free Full Text]

8. Banan A, Fields JZ, Talmage DA, Zhang Y, and Keshavarzian A. PKC-{beta}1 mediates EGF protection of microtubules and barrier integrity of intestinal monolayers against oxidants. Am J Physiol Gastrointest Liver Physiol 281: G833-G847, 2001.[Abstract/Free Full Text]

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

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

11. Banan A, Fields JZ, Zhang Y, and Keshavarzian A. Key role of PKC and Ca2+ in EGF protection of microtubules and intestinal barrier against oxidants. Am J Physiol Gastrointest Liver Physiol 280: G828-G843, 2001.[Abstract/Free Full Text]

12. Banan A, Fields JZ, Zhang Y, and Keshavarzian A. Phospholipase C-{gamma} inhibition prevents EGF protection of intestinal cytoskeleton and barrier against oxidants. Am J Physiol Gastrointest Liver Physiol 281: G412-G423, 2001.[Abstract/Free Full Text]

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

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

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

16. Banan A, Zhang L, Fields JZ, Talmage DA, and Keshavarzian A. PKC-{zeta} prevents oxidant-induced iNOS upregulation and protects the microtubules and intestinal barrier integrity. Am J Physiol Gastrointest Liver Physiol 283: G909-G922, 2002.[Abstract/Free Full Text]

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

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

19. Bar-Sagii D, Rotin D, Batzer A, Mandiyan V, and Schlessinger J. SH3 domains direct cellular localization of signaling molecules. Cell 74: 83-91, 1993.[ISI][Medline]

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

21. Bredt DS, Ferris CD, and Synder SH. Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites. J Biol Chem 267: 10976-10981, 1992.[Abstract/Free Full Text]

22. Chen P, Gupta K, and Wells A. Cell movement elicited by epidermal growth factor receptor requires kinase and autophosphorylation but is separable from mitogenesis. J Cell Biol 124: 547-555, 1994.[Abstract]

23. Chen P, Xie H, and Wells A. Mitogenic signaling from the egf receptor is attenuated by a phospholipase C-gamma/protein kinase C feedback mechanism. Mol Biol Cell 7: 871-881, 1996.[Abstract]

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

26. Goldschmidt-Clermont PH, Kim JW, Mcheskky LM, Rhee SG, and Pollart TD. Regulation of phospholipase C-gamma1 by profilin and tyrosine phosphorylation. Science 251: 1231-1233, 1996.

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

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

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

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

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

32. Homma Y. and Takenawa T. Inhibitory effect are src homology (SH) 2/SH3 fragments of phospholipase C-gamma on the catalytic activity of phospholipase C isoforms. Identification of a novel phospholipase C inhibitor region. J Biol Chem 267: 21844-21849, 1992.[Abstract/Free Full Text]

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

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

35. Janmey PA, Lamb J, Allen PG, and Matsudaira PT. Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin. J Biol Chem 267: 11818-11823, 1992.[Abstract/Free Full Text]

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

37. Keshavarzian A, Banan A, Farhadi A, Kommandori S, Zhang L, and Fields JZ. Increases in free radicals and cytoskeletal protein oxidation in the colon of patients with inflammatory bowel disease. Gut 52: 720-728, 2003.[Abstract/Free Full Text]

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

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

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

41. Kinashi T, Escobeda JA, Williams LT, Takatsu K, and Springer TA. Receptor tyrosine kinase stimulates cell-matrix adhesion by phosphatidylinositol 3 kinase and phospholipase C-{gamma}1 pathways. Blood 86: 2086-2090, 1995.[Abstract/Free Full Text]

42. Lindmark T, Kimura Y, and Artursson P. Absorption enhancement through intracellular regulation of tight junction permeability by medium-chain fatty acids in Caco-2 cells. J Pharmacol Exp Ther 284: 362-369, 1998.[Abstract/Free Full Text]

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

44. Meisenhelder J, Suh PG, Rhee G, and Hunter T. Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein tyrosine kinase in vivo and in vitro. Cell 57: 1109-1122, 1989.[ISI][Medline]

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

46. Nishimura R, Li W, and Kashishian A, Mondino A, Zhou M, Cooper J, and Schlessinger J. Two signaling molecules share a phosphotyrosine-containing binding site in PDGF receptor. Mol Cell Biol 13: 6889-6896, 1993.[Abstract]

47. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. The role of protein kinase C in cell surface signal transduction and tumor promotion. Science 258: 607-614, 1992.[ISI][Medline]

48. Phelps DT, Ferro TJ, Higgins PJ, Shankar R, Parker DM, and Johnson A. TNF-{alpha} induces peroxynitrite-mediated depletion of lung endothelial glutathione via protein kinase C. Am J Physiol Lung Cell Mol Physiol 269: L551-L559, 1995.[Abstract/Free Full Text]

49. Polk DB. Epidermal growth factor receptor-stimulated intestinal epithelial cell migration requires phospholipase C activity. Gastroenterology 114: 493-502, 1998.[ISI][Medline]

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

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

52. Rhee SG and Chio KD. Regulation of inositol phospholipidspecific phospholipase C isozymes. J Biol Chem 267: 12393-12396, 1992.[Free Full Text]

53. Rotin D, Margolis B, Mohammadi M, Daly RJ, Daum G, Li N, Fischer EH, Burgess WH, Ulrich A, and Schlessinger J. SH2 domains prevent tyrosine dephosphorylation of EGF-R: identification of Tyr992 as the high affinity binding site for SH2 domains of PLC-gamma. EMBO J 11: 559-567, 1992.[Abstract]

54. Shimizu S, Nomoto M, Naito S, Yamamoto T, and Momose K. Stimulation of nitric oxide synthase during oxidative endothelial cell injury. Biochem Pharmacol 55: 77-83, 1998.[ISI][Medline]

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

56. Tripp MA and Tepperman BL. Role of calcium in nitric oxide-mediated injury to gastric mucosal cells. Gastroenterology 111: 65-72, 1996.[ISI][Medline]

57. Turner T, Epps-Fung MV, Kassis J, and Wells A. Molecular inhibitor of phospholipase C-gamma signaling abrogates DU-145 prostate tumor cell invasion. Clin Cancer Res 3: 2275-2282, 1997.[Abstract]

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

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

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

61. Yang LJ, Rhees SG, and Williamson JR. Growth factor-induced activation and translocation of phospholipase C-{gamma}1 to the cytoskeleton in rat hepatocytes. J Biol Chem 269: 7156-7162, 1994.[Abstract/Free Full Text]

62. Yeo E-J, Kalzlasukas A, and Exton JH. Activation of PLC-gamma is necessary for stimulation of Phosholipase by PDGF. J Biol Chem 269: 27823-27826, 1994.[Abstract/Free Full Text]

63. Zadeh MS, Kolb JP, Geromin D, D'Anna R, Boulmerka A, Marconi A, Dugas B, Marsac C, and D'Alessio P. Regulation of ICAM-1/CD54 expression on human endothelial cells by hydrogen peroxide involves inducible NO synthase. J Leukoc Biol 67: 327-334, 2000.[Abstract]





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