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Protein and Vesicle Trafficking, Cytoskeleton

-Isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-

Division of Digestive Diseases, Department of Internal Medicine, Department of Pharmacology, and Department of Molecular Physiology, Rush University Medical Center, Chicago, Illinois 60612

Submitted 19 December 2003 ; accepted in final form 17 February 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the -isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC- is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC- (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC- showed monolayer injury as indicated by decreased native PKC- activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC- was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC- was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-/tubulin complex. Second, DN transfection to inhibit the endogenous PKC- led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC- led to a mostly cytosolic distribution of -isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1) PKC- isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2) the molecular event underlying this novel biological effect of PKC- involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-.

microtubules; tubulin; occludin; epithelial barrier permeability; protein kinase C isoform

An important advance in our understanding of GI inflammation, especially IBD, has been the realization that a poorly maintained barrier permeability, the so-called leaky gut, can lead to intestinal inflammation and tissue injury. In animal models, loss of gut barrier permeability caused by the injection of bacterial products into the mucosa elicits IBD-like conditions (48). Similarly, transgenic animals with a hyperpermeable gut barrier exhibit symptoms of intestinal inflammation (23). One of the underlying difficulties in managing IBD disorders is due in large part to our limited understanding of the molecular processes involved in alterations of gut barrier.

The stability of intestinal epithelial barrier depends on a complex array of cytoskeletal protein filaments that includes occludin and microtubules (2–4, 7, 9, 11–13, 29, 47). Using monolayers of intestinal Caco-2 cells, we reported (2–4, 7–9, 11) that microtubule cytoskeletal assembly and stability are required for epithelial barrier integrity. Not surprisingly, microtubules play a central role in maintaining cellular integrity, structure, and shape (3, 15, 33, 34, 44). As such, microtubules govern cell membrane morphology and polarity, functions essential to the maintenance of epithelial monolayer (barrier) integrity (3, 12, 15, 19). Protein kinase C (PKC), in general, is known to affect epithelial barrier, but the roles of specific PKC isoforms in cytoskeletal and barrier changes remain largely unknown.

PKC consists of a family of serine- and threonine-specific kinases. This family, which includes at least 12 known PKC isoenzymes, can be classified into three subfamilies on the basis of differences in sequence homology and cofactor requirement (5–8, 10, 14, 18, 26, 35, 43). The conventional (or classical) PKC isoforms (, 1, 2, ) require calcium and phospholipid for their activation, whereas the novel PKC isoforms (, , , , µ) are calcium independent but require phospholipid. Activation of the third group, atypical PKC isoforms (, , ), is independent of calcium and phorbol compounds (10). Intestinal cells (e.g., Caco-2) express at least 10 isoforms of PKC, including PKC-, PKC-1, PKC-2, PKC-, PKC-, PKC-, PKC-, PKC-, PKC-, and PKC- (5–8, 10, 14, 35, 36). These isoenzymes are different in their intracellular distribution, expression, substrate type, and activation. It is therefore thought that each PKC isoform can perform unique biological functions (5, 6, 10, 37, 39, 41, 43). In our previous studies, we investigated both the damaging and protective pathways for gut barrier permeability. We showed that growth factor [epidermal growth factor (EGF) or transforming growth factor- (TGF-)] prevents oxidant-induced barrier disruption through the activation of the classical PKC-1 and the atypical PKC- isoforms, leading to the protection of the cytoskeleton (e.g., 7, 10, 14). Nonetheless, the role of specific PKC isoforms in alterations of intestinal cytoskeletal assembly and barrier permeability still remains largely unexplored.

In the current investigation, we have explored the role of the -isoform of PKC because this member of the novel subfamily of PKC is both present and abundant in our wild-type (WT; naive) Caco-2 cells and therefore could be a possible contributor to epithelial monolayer barrier integrity and cytoskeletal assembly. To this end, we studied the PKC- using targeted molecular interventions, transfecting cells to create the first multiple clones of intestinal cells for this isoform: in several clones, the endogenous (82 kDa) PKC- isoform was reliably inhibited by either antisense underexpression or dominant negative inactivation; in the other clones, native PKC- was ectopically increased by sense expression. Using these targeted approaches, we sought to determine whether PKC- isoform activity is required for changes in the dynamics of the cytoskeleton (e.g., microtubules and occludin) and barrier integrity in intestinal epithelial monolayers. In this article, we describe novel mechanisms dependent on the -isoform of PKC, namely, alterations of the cytoskeletal assembly and permeability clearance in cell monolayers.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Cell culture. Caco-2 cells were obtained from American Type Culture Collection (Rockville, MD) at passage 15. This colonic cell line was chosen for our studies because the cells form monolayers that morphologically resemble intestinal cells, with defined apical brush borders and a highly organized microtubule network upon differentiation (12, 19, 38). Cells were maintained at 37°C in complete Dulbecco's modified Eagle's medium (DMEM) in an atmosphere of 5% CO₂ and 100% relative humidity. WT cells or stably transfected cells (see Plasmids and transfection) were split at a ratio of 1:6 upon reaching confluency and were set up in either 6- or 24-well plates for experiments or in T-75 flasks for propagation. The utility and characterization of this cell line have been previously reported (2–14, 38).

Plasmids and transfection. The sense, antisense, and dominant negative plasmids of PKC- were constructed as we previously described (5, 7). A unique tetracycline-responsive expression (TRE) system was used to overexpress the native PKC-. cDNA encoding the entire reading frame of PKC- plasmid was subcloned into the TRE vector, creating TRE-PKC-. The antisense or dominant negative PKC- plasmid was also subcloned to create AS-PKC- or DN-PKC-, respectively.

Cultures of Caco-2 cells grown to 50–60% confluence were cotransfected with hygromycin resistance plasmid (p-hygro) and with expression plasmids encoding sense PKC-, antisense PKC-, or dominant negative PKC- by using Lipofectin reagent (GIBCO BRL) as we described (5, 7). Control conditions included vector alone. Briefly, cells were incubated for 16 h at 37°C with the plasmid DNA in serum-free medium in the presence of Lipofectamine (25 µl per 25-cm² flask). Subsequently, the DNA-containing solution was removed and replaced with fresh medium containing 10% FBS to relieve cells from the shock of exposure to serum-free medium. After transfection, cells were subjected to hygromycin selection (1 mg/ml). Resistant cells were maintained in DMEM-FBS and 0.2 mg/ml hygromycin (selection medium). For inducible expression of PKC-, Caco-2 cells were transfected with a plasmid expressing the tetracycline-responsive transactivator (tTA) along with a second plasmid conferring resistance to G-418. After selection in 0.6 mg/ml G-418 (selection medium), one such clone (i.e., parental tTA) was then itself transfected with the TRE-PKC- system. p-Hygro was included to confer resistance to hygromycin (selection marker, 1 mg/ml). Control conditions included vector alone (TRE-z). Multiple clones expressing PKC- or lacking PKC- activity were assessed by immunoblotting and activity assay and then used for experiments.

Experimental design. In the first series of experiments, postconfluent monolayers of WT (naive) cells were incubated with vehicle (isotonic saline) for 30 min and then assessed for baseline conditions. Experiments were then repeated by using multiple clones of stably transfected cells. In all experiments, we assessed microtubule cytoskeletal stability (cytoarchitecture, disassembly), tubulin assembly (polymerized and monomeric tubulin pools), tubulin phosphorylation, occludin stability (cytoarchitecture), PKC- subcellular distribution (membrane, cytoskeletal, and cytosolic fractions), PKC- activity (immunoprecipitation and in vitro kinase assay), and monolayer integrity/permeability (clearance of different-sized probes).

In the second series of experiments, transfected cell clones overexpressing PKC- were used. Multiple clones that were stably overexpressing PKC- (i.e., TRE-PKC-) were grown as monolayers and then exposed to vehicle. Outcomes were measured as described above. In these overexpression studies, cells were grown for 48 h in the absence of tetracycline before experiments were performed.

In the third series of experiments, monolayers of antisense-transfected cells lacking PKC- activity were treated with vehicle. In all experiments, PKC- activity was determined in immunoprecipitated samples (see Immunoprecipitation and PKC- activity assay). In a corollary series of experiments, we investigated the effects of PKC- dominant negative mutant on the state of monolayer integrity, tubulin assembly, and microtubule disassembly and cytoarchitecture. To this end, monomeric (S1) and polymerized (S2) fractions of tubulin (the structural protein subunit of microtubules) were isolated and then analyzed by immunoblotting (2, 3, 12). Microtubule integrity was assessed by 1) immunofluorescent labeling and fluorescence microscopy, to determine the percentage of cells with normal microtubules; 2) detailed analysis with the use of high-resolution laser scanning confocal microscopy; and 3) immunoblot analysis of monomeric and polymerized tubulin pools.

Fractionation and immunoblotting of PKC. Cell monolayers grown in large 75-cm² flasks were processed for isolation of the cytosolic, membrane, and cytoskeletal fractions (7, 14, 20). Protein content of these various fractions was assessed according to the Bradford method (16). Samples (5 µg protein/lane) were separated by PAGE. The immunoblotted proteins were visualized by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL) and autoradiography (e.g., 1 h at –20°C). The exposure times were adjusted to ensure linear responses. Under these conditions, the chemiluminescence assay was linear between 1 and 10 µg of total protein. Standard (purified PKC-) loading controls (1 µg/lane) were also run concurrently. Blots were routinely stained with 0.1% india ink in Tris-buffered saline containing Tween 20 to further verify equal loading.

For isolation of the cell fractions, after treatments, postconfluent monolayers were scraped and ultrasonically homogenized (GE130; amplitude 50, 6 pulses/s, duration 20 s; GE Ultrasonic Processor) 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 PMSF). The homogenates were then ultracentrifuged (100,000 g, 40 min, 4°C), and the supernatant was removed and used as the source of the cytosolic fraction. Next, pellets were washed with 0.2 ml of Tris·HCl buffer, 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 (100,000 g, 1 h, 4°C), and the supernatant was used as the source of the membrane fraction. To this remaining pellet, we added 0.3 ml of cold (4°C) lysis buffer (150 mM NaCl, 50 mM Tris·HCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml PMSF). The samples were then placed on ice for 1 h and ultracentrifuged as described above. The remainder of the lysate or Triton-insoluble cytoskeletal fraction was then removed. For total extraction, which provides the fraction used to confirm total PKC-, 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.

For immunoblotting, samples (5 µg protein/lane) were added to a standard SDS buffer, boiled, and then separated on 7.5% SDS-PAGE (7, 14). The immunoblotted proteins were incubated with a primary monoclonal antibody to PKC- [nPKC- (E7), no. sc-1680 (human reactive); Santa Cruz Biotechnology, Santa Cruz, CA] at 1:3,000 dilution. A horseradish peroxidase-conjugated antibody (Molecular Probes, Eugene, OR) was used as a secondary antibody at 1:4,000 dilution. Proteins were visualized by enhanced chemiluminescence and autoradiography and subsequently analyzed by densitometry. The identity of the PKC- band was assessed according to a procedure that we previously described (5, 7): 1) Use of a PKC- blocking peptide (no. sc-1680 P; Santa Cruz Biotechnology) in combination with the anti-PKC- antibody prevented the appearance of the corresponding "major" band in Western blots. 2) In addition, in the absence of the primary antibody to PKC-, no corresponding band for PKC- was observed. 3) The PKC- band ran at the expected molecular mass of 82 kDa, as confirmed by a known positive control for PKC- (from rat brain lysates). 4) Prestained molecular weight (M_r) markers (M_r 67,000 and 93,000) were run in adjacent lanes. In initial studies, using total PKC extracts, we confirmed that expression of PKC- or inhibition of PKC- did not affect the relative expression levels of other PKC isoforms.

Immunoprecipitation and PKC- activity assay. Immunoprecipitated PKC- was collected and processed for its ability to phosphorylate a synthetic peptide (5, 10). 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, 10 µg/ml anti-protease cocktail, 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 90 min at 4°C with anti-PKC- (1:2,000 dilution, in excess). The extracts were then incubated with protein A/G-agarose for 1 h at 4°C. The immunocomplexes were collected by centrifugation (2,500 g, 5 min) in a microfuge tube 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 kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgCl₂, 2 mM MnCl₂, and 20 µM ATP), resuspended in 20 µl of kinase buffer and 5 µl of 5x reaction buffer (1 mg/ml histone H1 and 0.25 mg/ml L--phosphatidyl-L-serine) plus 5 µCi [-³²P]ATP, and subsequently incubated for 5 min at 30°C. Reactions were then stopped by the addition of 8 µl of 5x sample buffer, and the samples were boiled at 95°C for 5 min before separation by 7.5% PAGE. The extent of histone H1 phosphorylation was determined by scintillation counting of excised Coomassie blue-stained histone polypeptide bands. Counts for blanks were subtracted from the sample activity. Sample activity was also corrected for protein concentration (Bradford method, Ref. 16), and PKC- activity was reported as picomoles per minute per milligram of protein.

Immunofluorescent staining and high-resolution laser scanning confocal microscopy of microtubule cytoskeleton and occludin. Cells from monolayers were fixed in cytoskeletal stabilization buffer and then postfixed in 95% ethanol at –20°C as we described previously (2–4, 7–9, 11, 12). Cells were subsequently processed for incubation with a primary antibody (monoclonal mouse anti--tubulin antibody; Sigma, St. Louis, MO) and then with a secondary antibody (FITC-conjugated goat anti-mouse antibody; Sigma). For occludin staining, cells were processed for incubation with a primary monoclonal antibody (mouse anti-occludin antibody; Zymed, San Francisco, CA) and then with a secondary antibody (FITC-conjugated goat anti-mouse antibody; Zymed). After staining, cells were observed with an argon laser ( = 488 nm) with a x63 oil-immersion Plan-Apochromat objective (NA 1.4; Zeiss). The cytoskeletal elements were examined in a blinded fashion for their overall morphology, orientation, and disruption as we described previously (2–4, 7–9, 11, 12). The identity of the treatment groups for all slides was decoded only after examination was complete.

Microtubule (tubulin) fractionation and immunoblotting analysis of polymerized tubulin and monomeric tubulin (tubulin assembly/disassembly). Polymerized (S2) and monomeric (S1) fractions of tubulin were isolated according to a method we described previously (3, 12). Cells were gently scraped and pelleted with centrifugation at low speed (700 rpm, 7 min, 4°C) and resuspended in microtubule stabilization-extraction buffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% DMSO, 1 mM MgSO₄, 10 µg/ml anti-protease cocktail, 1 mM EGTA, and 1% Triton X-100) at room temperature for 20 min. Tubulin fractions were separated after a series of centrifugation and extraction steps. Specifically, cell lysates were centrifuged at 105,000 g for 45 min at 4°C, and the supernatant containing the soluble monomeric pool of tubulin (S1 fraction) 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, and 10 mM CaCl₂) and incubated on ice for 60 min. Subsequently, samples were centrifuged at 48,000 g for 15 min at 4°C, and the supernatant (S2 fraction or cold/Ca²⁺-soluble fraction) was removed. To ensure complete removal of the S2 fraction, we treated the remaining pellet with the Ca²⁺-containing depolymerization buffer twice more by resuspension and centrifugation. The "microtubules" were recovered by separate incubation (at 37°C for 30 min) of the S1 and S2 fractions with the stabilizing agents taxol (10 µM) and GTP (1 mM) in microtubule stabilization buffer (MSB: 0.1 M PIPES, pH 6.9, 30% glycerol, 5% DMSO, 10 µg/ml anti-protease cocktail, 1 mM EGTA, 1 mM MgCl₂, and 1 mM GTP) to promote polymerization of tubulin. Tubulin was then recovered by centrifugation and resuspended in MSB. Fractionated S1 and S2 samples were then flash frozen in liquid N₂ and stored at –70°C until immunoblotting. For immunoblotting, samples (5 µg protein/lane) were placed in a standard SDS sample buffer, boiled, and then subjected to PAGE on 7.5% gels. Standard (purified) tubulin controls (5 µg/lane) were run concurrently with each run. To quantify the relative levels of tubulin, we used a laser densitometer to measure the optical density of the bands corresponding to immunolabeled tubulin.

Immunoprecipitation and Western blot analysis of tubulin phosphorylation. Immunoprecipitated tubulin was collected and then assessed for phosphorylation by PAGE (7). For immunoprecipitation, cell lysates were incubated for 4 h at 4°C with monoclonal anti--tubulin (1:50 dilution, in excess). The extracts were then incubated with protein G-Sepharose 4B (Zymed) for 2 h at 4°C. The immunocomplexes were collected by centrifugation (2,500 g, 5 min) in a microfuge tube and washed three times with immunoprecipitation buffer containing 5 mM Tris·HCl, pH 7.4, and 0.2% Triton X-100. The resultant pellets were resuspended in a standard SDS sample buffer and boiled at 95°C for 5 min before separation by PAGE. Gels were transferred to nitrocellulose membranes, blocked with 1% bovine serum albumin and 0.01% Tween 20 in phosphate-buffered saline for blotting by anti-phosphothreonine (1:3,000 dilution; Transduction Labs, Lexington, KY) and then, for detection of immune complexes by horseradish peroxidase-conjugated secondary antibody, incubated with chemiluminescent reagents (ECL) and autoradiographed.

Determination of monolayer barrier integrity by fluorometry. Status of the monolayer barrier integrity was assessed by a widely used and validated technique that measures the apical-to-basolateral paracellular flux of fluorescent markers such as fluorescein sulfonic acid (FS; 200 µg/ml, 0.478 kDa) as we (2–5, 7–9) and others (27, 29, 45, 47) described. In select experiments, higher molecular mass fluorescein dextran (FD4; 1 mg/ml, 4 kDa) also was used. Briefly, fresh phenol-free DMEM (800 µl) was placed into the lower (basolateral) chamber, and phenol-free DMEM (300 µl) containing probe (FS or FD4) 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 probes were as follows: excitation = 485 nm, emission = 530 nm. Clearance was calculated as Cl (nl·h^–1·cm^–2) = F_ab/([FS or FD4]_a x S), where F_ab is the apical-to-basolateral flux of FS or FD4 (light units/h), [FS or FD4]_a is the probe 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 with analysis of variance followed by Dunnett's multiple-range test (22). 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 GRANTS REFERENCES

 
Stable underexpression of novel PKC- isoform in multiple clones of intestinal cells. Intestinal cells cotransfected with cDNA encoding both hygromycin resistance (for selection) and PKC- antisense (AS-PKC-) stably underexpressed the novel -isoform of PKC (Fig. 1). Cell lysates of confluent monolayers were prepared from these transfected cell clones and then analyzed by immunoblotting. Multiple clones of intestinal cells transfected with 1, 2, 3, 4, or 5 µg of AS-PKC- demonstrated a dose-dependent underexpression of the PKC- (82 kDa) isoform. The clone transfected with 4 µg of AS-PKC- provided maximum reduction (–99.5%) in the levels of PKC- protein. As might be expected, transfection of only the empty vector (vector alone) did not underexpress PKC- (Fig. 1). In empty vector-transfected cells, PKC- levels were comparable to those of the WT cells, which exhibited native, steady-state levels of -isoform. Underexpression of PKC- caused no cellular toxicity (0% cell death assessed by ethidium homodimer probe).

View larger version (13K): [in this window] [in a new window]   Fig. 1. Stable underexpression of novel PKC- in intestinal cells transfected with a plasmid encoding antisense to native PKC- (AS-PKC-). Caco-2 cells were transfected with varying amounts (1–5 µg) of AS-PKC- cDNA to create multiple, unique cell clones, and then lysates from these clones were processed for immunoblotting and subsequently probed with anti-PKC-. Note the dose-dependent underexpression of native PKC- protein (82 kDa). Transfection with 4 µg of AS-PKC- led to the maximum reduction (>99%) in the level of endogenous PKC- protein. Control empty vector (vector alone), as might be expected, did not underexpress PKC-. Results for wild-type (WT) cells expressing the native, steady-state level of PKC- isoform are also shown. Note that the PKC- level in empty vector-transfected cells is similar to that in WT cells. Prestained molecular weights (Mr 67,000 and 93,000) were run in adjacent lanes. Representative blots are shown; n = 6 observations per treatment group.

Reduction of native PKC- isoform levels and disruption of monolayer integrity.

View larger version (28K): [in this window] [in a new window]   Fig. 2. A: underexpression of native PKC- isoform induces a dose-dependent loss of monolayer integrity in intestinal cells. Assessment of monolayer integrity in multiple PKC- anti-sense clones showed a graded disruption of integrity as demonstrated by increased probe clearance. The 4 µg of AS-PKC- showed the largest loss of monolayer integrity (paralleling findings on reduction of PKC- protein levels in Fig. 1). WT cells (those expressing native PKC- levels), in contrast, exhibited normal monolayer integrity. The empty vector clone (vector alone), as might be expected, did not affect monolayer stability. Monolayer integrity was expressed as flux of the fluorescent probe fluorescein sulfonic acid (FS, 0.478 kDa) from the apical to the basolateral compartment of cell culture Transwell inserts, divided by the concentration of probe in the apical chamber. When normalized for the surface area of the monolayer, this expression has units of clearance. B: deleterious effects of endogenous PKC- underexpression on intestinal cell monolayer integrity as determined by fluorescein dextran (FD4). Note the size (molecular mass)-dependent permeation of probes FD4 (B) and FS (A). There is an inverse relationship between probe size and clearance in order of increasing size (0.478-kDa FS > 4-kDa FD4). Values are means ± SE; n = 6 observations per treatment group. *P < 0.05 vs. WT cells. +P < 0.05 vs. vector alone-transfected cells.

Underexpression of endogenous PKC- isoform causes instability of cytoskeletal assembly and cytoarchitecture. Native PKC- underexpression, also in a dose-dependent fashion, injured the microtubule cytoskeleton as demonstrated by a low percentage of intestinal cells displaying normal microtubules (Fig. 3A). In WT cells, similarly to barrier permeability, microtubules were normal. Furthermore, transfection of vector alone, as might be expected, did not affect the microtubules (%normal microtubules = 99 ± 1% for empty vector-transfected cells exposed to vehicle and 98 ± 2% for WT cells exposed to vehicle).

View larger version (60K): [in this window] [in a new window]   Fig. 3. A: percentage of intestinal (Caco-2) cells with a normal microtubule cytoskeleton from multiple cell clones underexpressing native PKC- isoform. PKC- underexpression in a dose-dependent fashion injures the microtubule cytoskeleton, as demonstrated by a low percentage of cells with normal microtubules. Transfection of 4 µg of AS-PKC- clone led to the maximum number of cells with unstable microtubules. WT cells, in contrast, exhibited a high percentage of cells with stable (normal) microtubules. Empty vector clone, as for monolayer integrity, did not affect the microtubules. Cell monolayers were processed for staining with a primary monoclonal anti--tubulin antibody, and subsequently, the microtubule elements were examined in a blinded fashion for their overall normal morphology. Values are means ± SE; n = 6 observations per treatment group. *P < 0.05 vs. vehicle in WT cells. +P < 0.05 vs. vector alone-transfected cells. B: representative fluorescence images revealing the intracellular organization of the microtubule cytoskeleton in intestinal cells of WT and transfected origin. Cell monolayers grown on specially coated coverslips were processed for immunofluorescent staining, and the microtubule elements were captured by high-resolution laser scanning confocal microscopy (LSCM). In WT cells (a), microtubules appeared as intact filamentous structures that dispersed throughout the cytosol in a stellar fashion. In PKC--underexpressing clone (b), in contrast, the microtubules showed a clear fragmentation, disorganization, and collapse of their architecture. In empty vector clone (c), as expected, microtubule cytoarchitecture was highly preserved, resembling that of WT cells. Bar = 25 µm. Representative images are shown; n = 6 observations per treatment group. C: intracellular organization of occludin (another index of monolayer integrity) stained by fluorescein-conjugated phalloidin in intestinal cells of WT and transfected origin. Cell monolayers were processed for immunofluorescent staining and LSCM. Occludin in WT cells (a) appeared as an intact structure, as shown by a continuous and smooth distribution of occludin ring at areas of cell-cell contact (arrow). Only in PKC--underexpressing clone (b) did the occludin ring appear disrupted, fragmented, and beaded. In empty vector clone (c), as might be expected, occludin architecture was highly preserved and resembled morphology detected in WT cells. Bar = 25 µm. Representative images are shown; n = 6 observations per treatment group.

We then determined the effects of native PKC- underexpression on the dynamic alterations in the polymerization and depolymerization states of the microtubule cytoskeleton by assessing the 50-kDa structural protein of microtubules, namely, tubulin. To this end, we isolated and analyzed the tubulin pools (S1 and S2 fractions) using a unique SDS-PAGE fractionation procedure. Analysis of tubulin fractions (Fig. 4A) corroborated the microtubule studies noted above. Only the PKC--underexpressing clones (1–5 µg of AS-PKC-) showed an abnormal tubulin assembly as demonstrated by graded reductions in the polymerized S2 tubulin and increases in the monomeric S1 tubulin, indicating disruption of microtubules. As before, the 4-µg inhibitory clone was the most effective at inducing injurious alterations, in this case to the assembly of tubulin. In WT cells, in contrast, neither any decreases in polymerized S2 tubulin nor any increases in monomeric S1 tubulin were observed, indicating normal assembly of the microtubule cytoskeleton. In these WT cells, tubulin polymerization was comparable to the levels seen in the empty vector-transfected cells. Indeed, transfection of vector alone, similarly to its lack of effects on microtubules and barrier permeability, did not affect tubulin assembly (%tubulin assembly = 66% ± 0.4% for empty vector-transfected cells exposed to vehicle and 65% ± 0.3% for WT cells exposed to vehicle).

View larger version (46K): [in this window] [in a new window]   Fig. 4. A: immunoblotting analysis of disruptive effects of PKC- underexpression on stable polymerized tubulin from intestinal cell monolayers. Underexpression of native PKC- (induced by 1–5 µg of AS-PKC-) caused a dose-dependent disruption of tubulin polymerization. Similarly to its destabilizing effects on microtubules, 4 µg of AS-PKC- led to the maximum disruption of tubulin polymerization, indicating an abnormal tubulin assembly (and microtubule damage). WT cells, on the other hand, showed normal assembly of tubulin, which is comparable to the vector clone. Tubulin extracts from intestinal cells were processed by SDS-PAGE fractionation and immunoblotting and then were autoradiographed. To quantify the relative levels of tubulin polymerization, the optical density of the autoradiograph bands corresponding to immunolabeled tubulin was measured with a laser densitometer and expressed as a percentage. Percent polymerized tubulin = [(S2)/(S2 + S1)], where S2 + S1 is the total intracellular tubulin pool. Values are means ± SE; n = 6 observations per treatment group. *P < 0.05 vs. vehicle in WT cells. +P < 0.05 vs. vehicle in vector alone-transfected cells. B: representative immunoblots for the state of tubulin assembly (polymerized S2 and monomeric S1) in intestinal cells of AS-PKC--transfected cells compared with WT cells. Intracellular tubulin pools (S2 and S1) were extracted and then subjected to SDS-PAGE fractionation and immunoblotting with the use of monoclonal anti--tubulin antibody followed by horseradish peroxidase-conjugated-secondary antibody and were subsequently autoradiographed. Bands representing the polymerized tubulin (S2, index of microtubule assembly) and the monomeric tubulin (S1, index of microtubule disassembly) are from WT cells exposed to vehicle (lane a), vector alone clone exposed to vehicle (lane b), PKC--underexpressing clone (4 µg) exposed to vehicle (lane c), and tubulin standard (50 kDa; lane d). Note the destabilizing effects of PKC- isoform underexpression on the dynamics of tubulin assembly (lane c), namely, decreases in the stable polymerized tubulin pool (top) and concomitant increases in the unstable monomeric tubulin pool (bottom). WT cells (lane a) showed normal assembly of tubulin pools (S2, S1) as indicated by the steady-state levels of both polymerized tubulin (lane a, top) and monomeric tubulin (lane a, bottom). In the vector alone-transfected clone (lane b), as might be expected, assembly of tubulin pools resembled that of WT cells. Representative blots are shown; n = 6 observations per treatment group.

Antisense inhibition of PKC- induces alterations in phosphorylation state of microtubule (tubulin based) cytoskeleton in intestinal monolayers. We next probed potential molecular mechanisms underlying the observed effects of PKC- on the monolayer cytoskeleton and barrier integrity. To this end, we subjected tubulin, fractionated from both transfected and WT monolayers and then immunoprecipitated, to Western immunoblotting to assess phosphorylation (Fig. 5). Tubulin was markedly phosphorylated in WT cells (Fig. 5, lane a), whereas antisense inhibition of native PKC- substantially reduced tubulin phosphorylation (Fig. 5, lane c). In the empty vector-transfected cells (expressing steady-state levels of PKC-), as might be expected, such suppression of tubulin phosphorylation was not seen (Fig. 5, lane b). Indeed, the level of tubulin phosphorylation in the empty vector clone was similar to that of WT cells (which also exhibited normal tubulin phosphorylation and native, steady-state levels of PKC-).

View larger version (9K): [in this window] [in a new window]   Fig. 5. Alterations in the phosphorylation state of tubulin in intestinal cells of transfected origin compared with WT cells. Tubulin was immunoprecipitated, and then analyzed by Western immunoblotting to assess tubulin phosphorylation. The tubulin phosphothreonine bands are from WT cells exposed to vehicle (lane a), empty vector clone exposed to vehicle (lane b), and PKC--underexpressing clone (4 µg) exposed to vehicle (lane c). Underexpression of endogenous PKC- isoform reduced tubulin phosphorylation (lane c), paralleling its disruptive effects on tubulin assembly (see Fig. 4B) and on microtubules stability (see Fig. 3). This destabilizing hypophosphorylation was not found in WT cells (lane a), where tubulin is normally phosphorylated. As before, empty vector clone (lane b) was comparable to the WT cells. A representative blot is shown; n = 6 observations per treatment group.

Endogenous PKC- is complexed with tubulin.

View larger version (38K): [in this window] [in a new window]   Fig. 6. A and B: PKC- underexpression caused a reduction in native coassociation of the PKC- isoform and the tubulin in intestinal cells. WT cells exhibited the endogenous PKC-/tubulin complexes (lane b). Note that the underexpression induced reduction (disappearance) in coimmunoprecipitation of the PKC-/tubulin complexes in transfected cells lacking PKC- isoform (lane c). For blots in A, cleared cell lysates were incubated with excess (1:2,000 dilution) anti-PKC- bound to protein A beads for 1 h, and the immune complexes were subsequently assessed by PAGE with the use of appropriate anti-PKC- or anti-tubulin antibodies. WT (vehicle treated) resting cells exhibited native PKC-/tubulin complexes (see lane b). PKC--underexpressing clones displayed no such endogenous PKC-/tubulin complexes (lane c). For blots in B, an opposite protocol was performed in which an anti-tubulin antibody was used for immunoprecipitation and the complexes formed were subsequently examined. Once again, an identical pattern of coassociation (complex formation) was observed between tubulin/PKC-. Bands are from cell lysates (lane a), WT cells exposed to vehicle (lane b), PKC--underexpressing clone (4 µg) exposed to vehicle (lane c), and empty vector clone exposed to vehicle (lane d). WB, Western blot; IP, immunoprecipitated with the antibody shown. Representative immunoblots are shown; n = 6 observations per treatment group. Asterisks denote PKC-/tubulin complexes.

Subcellular distribution and activity levels of PKC- in intestinal cells: native PKC- isoform is constitutively active and found largely in particulate (membrane and cytoskeletal) pools.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Subcellular distribution of native (endogenous) PKC- isoform in cytosolic, membrane, and cytoskeletal fractions of WT intestinal cells (A) and transfected intestinal cell clones underexpressing PKC- (B). Cell monolayers grown in 75-cm2 flasks were processed for the isolation of various cell fractions and then immunoblotted using anti-PKC-. In WT cells exposed to vehicle (A), the 82-kDa PKC- isoform was constitutively active under native conditions, as shown by a mostly particulate (i.e., membrane and cytoskeletal) distribution of this isoform. Only a small cytosolic pool of PKC- is shown in these WT cells, further indicating constitutive activity of endogenous PKC- in the particulate fractions. On the other hand, in antisense-transfected clone exposed to vehicle (B), there was notably an almost complete absence of the PKC- isoform in both the particulate and cytosolic fractions, indicating its inactivation. Representative blots are shown; n = 6 observations per treatment group.

View larger version (52K): [in this window] [in a new window]   Fig. 8. In vitro kinase assay of PKC- activity levels in intestinal cells of antisense-transfected or WT (native) origin. Substantial activity of the native PKC- isoform was found in WT cells. In these WT cells, most of the endogenous PKC- activity was found in the particulate fractions, with a much smaller activity in the cytosolic fractions, indicating constitutive activity of the PKC- isoform under native conditions. Antisense inhibition of PKC- (4 µg of AS-PKC- clone) almost completely inactivated the endogenous PKC- isoform, because PKC- was no longer constitutively active in the particulate fractions. Cell monolayer extracts were immunoprecipitated by the anti-PKC- antibody and processed for in vitro kinase assay to determine their ability to phosphorylate a synthetic peptide. Values are means ± SE; n = 6 observations per treatment group. *P < 0.05 vs. WT, native PKC-. +P < 0.05 vs. vector alone-transfected cells.

Changes in activity of PKC- in intestinal cells robustly correlate with several different indexes of monolayer barrier stability. Using data from all experimental conditions, we found significant (P < 0.05) correlations (r = 0.97) between PKC- levels (in vitro kinase activity assay or optical density from the particulate fractions) and enhanced monolayer barrier integrity (decreased FS clearance). Similarly, we found other robust correlations when two other markers of stability, either microtubule integrity (i.e., %normal) or tubulin assembly (i.e., S2 pool), were correlated with the PKC- levels (r = 0.95 or 0.92, respectively, P < 0.05 for each). Still, when other markers of stability, such as enhanced tubulin phosphorylation or reduced tubulin disassembly (i.e., decreased S1 pool), were used against PKC-, additional supporting correlations were found (r = 0.93 or 0.96, respectively, P < 0.05).

Dominant negative targeted approach to inactivate endogenous PKC- isoform and its resultant loss of monolayer barrier integrity and cytoskeletal stability. We then used a dominant negative approach for PKC- to stably decrease the activity of endogenous PKC- isoform. Cells were thus transfected with PKC- dominant negative cDNA (DN-PKC-) and with plasmid encoding hygromycin resistance. Using this dominant negative mutant model, we were able to nearly completely abrogate the steady-state activity levels for native PKC- isoform by 99.7% (Fig. 9, 3 µg of clone). By comparison, in WT cells exposed to vehicle, native PKC- activity was high in the particulate pools, exhibiting the endogenous, steady-state constitutive activity levels for this isoform. The empty vector-transfected cells exposed to vehicle also showed the steady-state constitutive activity of PKC-.

View larger version (23K): [in this window] [in a new window]   Fig. 9. Stable dominant negative inhibition of endogenous PKC- activity in intestinal cells transfected with a dominant negative plasmid to the novel PKC- isoform (DN-PKC-). To inactivate PKC-, an independent approach was followed in which a dominant negative fragment for PKC- was used. Particulate and cytosolic cell extracts were subjected to immunoprecipitation and activity assay. Analysis of PKC- activity for its ability to phosphorylate a synthetic peptide in vitro showed an almost complete inactivation of PKC- in these dominant negative mutant cells (3 µg of clone). WT cells exhibited the native, steady-state activity levels for PKC-. Values are means ± SE; n = 6 observations per treatment group. *P < 0.05 vs. WT, native PKC-. +P < 0.05 vs. empty vector-transfected clones.

View this table: [in this window] [in a new window]   Table 1. Effects of transfection of varying amounts of a dominant negative plasmid for the -isoform of PKC on intestinal monolayer integrity

View larger version (17K): [in this window] [in a new window]   Fig. 10. A: dominant negative inactivation of PKC- induced microtubule instability in intestinal cells. In dominant negative-transfected cells (3 µg of DN-PKC-), microtubules were damaged as demonstrated by a low percentage of cells with normal microtubules, whereas in WT cells, nearly 100% of cells had normal microtubules. Values are means ± SE; n = 6 observations per treatment group. *P < 0.05 vs. vehicle in WT cells. +P < 0.05 vs. empty vector-transfected cells. B: analysis of disruptive effects of the dominant negative suppression of PKC- activity on polymerized tubulin. In dominant negative mutant clone lacking PKC- activity, normal tubulin assembly was largely attenuated, as shown by decreases in polymerized tubulin, paralleling findings on microtubule instability (A). Tubulin was extracted from monolayers and processed for immunoblotting, followed by densitometric analysis. Values are means ± SE; n = 6 observations per treatment group. *P < 0.05 vs. vehicle in WT cells. +P < 0.05 vs. empty vector-transfected cells. C: a representative immunoblot for polymerized tubulin further demonstrates a reduction in tubulin assembly (lane c) in the same mutant clone used for experiment shown in B. As before, either WT cells or empty vector cells showed normal (intact) tubulin assembly. The tubulin bands are from WT cells exposed to vehicle (lane a), empty vector clone exposed to vehicle (lane b), PKC- dominant negative (3 µg of DN-PKC-) clone exposed to vehicle (lane c), and tubulin standard (50 kDa; lane d). Representative immunoblots are shown; n = 6 observations per treatment group. D: dominant negative inactivation of PKC- reduced tubulin phosphorylation in intestinal cells. Tubulin phosphorylation in immunoprecipitated cell extracts from both mutant and WT cells was assessed. The tubulin phosphorylation (anti-phosphothreonine) bands shown are from the same treatment regimes as described in C. In WT cells (lane a), tubulin exhibited normal, steady-state phosphorylation. This phosphorylation was largely attenuated in the dominant negative mutant clone (lane c), as shown by a reduction in the phosphorylation band density. Representative blots are shown; n = 6 observations per treatment group.

Stable overexpression of PKC- isoform effects intestinal monolayer barrier integrity. The aforementioned findings obtained with the use of two independent, targeted molecular approaches (antisense and dominant negative) suggest that PKC- could play a novel role in cytoskeletal and monolayer alterations. In a third independent approach, we further investigated the role of PKC- in these alterations. To this end, we cotransfected parental Caco-2 cells (tTA parental cells) with cDNA encoding both hygromycin resistance (for selection) and a TRE system for full-length native PKC-, namely, TRE-PKC- (Fig. 11). In this TRE system, overexpression of PKC- is achieved in the absence of tetracycline, whereas its presence reduces expression to the levels seen in the parental cell line. Cell lysates from these stably transfected cells were then analyzed by Western immunoblotting. Figure 11 shows overexpression of the 82-kDa PKC- isozyme in these transfected clones. Total PKC- levels were elevated 2.0-fold in these TRE-PKC--overexpressing cells compared with parental cells. Overexpression of PKC- at these levels caused no cellular toxicity (0% cell death assessed by ethidium homodimer probe).

View larger version (21K): [in this window] [in a new window]   Fig. 11. Overexpression of the PKC- isoform protein in intestinal cells transfected with cDNA for a tetracycline-responsive expression (TRE) system for the novel PKC- (i.e., TRE PKC-). Cell monolayers were lysed, sonicated, and processed for SDS-PAGE with the use of an anti-PKC- antibody followed by a horseradish peroxidase-conjugated secondary antibody. Total overexpressed PKC- protein is shown. In these TRE PKC--transfected cells, overexpression of native PKC- was achieved in the absence of tetracycline (TTC). In contrast, the presence of tetracycline reduced expression of PKC- to the levels shown in the parental cell line. In parental cells, tetracycline by itself had no effect on the steady-state levels of PKC-. Expression in the parental cell line without tetracycline is also shown. Quantitative analysis by densitometry showed a 2.0-fold increase in PKC- protein in the overexpressing clone TRE-PKC-. Prestained molecular weights Mr 67,000 and 93,000 were run in adjacent lanes. A representative blot is shown; n = 6 observations per treatment group.

Overexpression of PKC- isoform destabilizes cell monolayers.

View this table: [in this window] [in a new window]   Table 2. Effects of transfection of varying amounts of an inducible TRE expression system for the -isoform of PKC on intestinal monolayer integrity

View larger version (30K): [in this window] [in a new window]   Fig. 12. Overexpression of the native PKC- isoform induces loss of monolayer integrity. Intestinal cells overexpressing TRE-PKC- were incubated with or without tetracycline or vehicle. Parental type (tTA parental) cells (those expressing native levels of PKC-) were treated similarly. Vehicle-treated cells overexpressing TRE-PKC- exhibited disruption of monolayer integrity as demonstrated by increased FS clearance. As might be expected, this disruptive effect was inhibited in the presence of tetracycline in cell medium (TRE PKC- + TTC). Similarly, parental type monolayers exposed to vehicle (with or without tetracycline) showed normal permeability. Monolayer integrity was expressed as flux of the fluorescent probe FS from the apical to the basolateral compartment of cell culture Transwell inserts divided by the concentration of probe in the apical chamber, expressed as a clearance. Values are means ± SE; n = 6 observations per treatment group. *P < 0.05 vs. vehicle-treated parental cells. +P < 0.05 vs. vehicle-treated TRE-PKC- cells incubated with tetracycline.

View larger version (80K): [in this window] [in a new window]   Fig. 13. A: a low percentage of intestinal cells displayed normal microtubule cytoskeleton in the PKC--overexpressing cells. Treatments and conditions were as described for Fig. 12. Microtubules were damaged in vehicle-treated TRE-PKC-. As for monolayer integrity, tetracycline (which is used to inhibit PKC- overexpression) prevented microtubule instability in these transfected clones. Parental cells responded comparably to vehicle treatment (with or without tetracycline), showing a high percentage of cells with normal microtubules. Values are means ± SE; n = 6 observations per treatment group. *P < 0.05 vs. vehicle-treated parental cells. +P < 0.05 vs. vehicle-treated TRE-PKC- cells incubated with tetracycline. B: intracellular distribution of the microtubule cytoskeleton in intestinal cell clones as captured by high-resolution LSCM. Monolayers of intestinal cells overexpressing PKC- were incubated with vehicle (c) or vehicle plus tetracycline (d). In TRE-PKC- clones, the microtubules appeared beaded, disorganized, and fragmented. In the presence of tetracycline (which prevents PKC- overexpression, d) in this same clone, normal microtubule cytoarchitecture was highly preserved. Similarly, in the parental cells with or without tetracycline (a and b, respectively), microtubules appeared as intact filamentous structures throughout the cytosol. Bar = 25 µm. Representative images are shown; n = 6 observations per treatment group. C: monolayer organization of occludin (an index of monolayer integrity) in intestinal cell clones assessed by high-resolution LSCM. Intestinal cells overexpressing PKC- were incubated with vehicle (c) or vehicle plus tetracycline (d). In TRE-PKC- clones (c), occludin showed extensive disorganization, kinking, condensation, and beading of its ring structure. In the presence of tetracycline (d), as might be expected, occludin structure appeared preserved. In the parental cells with or without tetracycline (a and b, respectively), occludin showed an intact pattern. The appearance of occludin ring in these parental cells was indistinguishable from that of transfected clones with tetracycline (d) (tetracycline was used to inhibit PKC- overexpression). Bar = 25 µm. Representative images are shown; n = 6 observations per treatment group.

Scanning confocal microscopy of the intracellular organization of the microtubule cytoskeleton also showed (Fig. 13B) that clones overexpressing PKC- (i.e., TRE-PKC- exposed to vehicle) exhibit an abnormal arrangement of the cytoskeleton (Fig. 13Bc). This abnormality was shown by the appearance of a fragmented and collapsed microtubule network. In the presence of tetracycline (which prevents overexpression of PKC-, Fig. 13Bd), these same cells displayed a preserved microtubule network as shown by an intact (stellate) architecture of the cytoskeleton. This normal cytoarchitecture is indistinguishable from that of the parental cells exposed to vehicle (Fig. 13B, a and b, with or without tetracycline, respectively). We also assessed the effects of PKC- overexpression on occludin (Fig. 13C). Confocal microscopy demonstrated that in clones overexpressing PKC- (Fig. 13Cc, TRE-PKC- exposed to vehicle), the occludin appeared beaded, fragmented, and disorganized. In the presence of tetracycline (to inhibit PKC- overexpression, Fig. 13Cd) in this same clone, normal occludin ring architecture was preserved. Similarly, in the parental cells with or without tetracycline (Fig. 13C, a and b, respectively), occludin appeared as an intact and smooth ring at areas of cell-cell contact.

Analysis of tubulin assembly (Fig. 14A) further demonstrated that PKC--overexpressing clone (TRE PKC-, vehicle treated) showed an unstable tubulin assembly as indicated by a reduction in polymerized tubulin. In tetracycline-incubated TRE PKC- cells, as might be expected, tubulin assembly was preserved, which was comparable to the normal tubulin polymerization seen in parental cells. Transfection of vector alone, similarly to its lack of effects on microtubules and monolayers, did not alter tubulin assembly (not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 14. A: analysis of the tubulin assembly from cell monolayers overexpressing the novel PKC- isoform compared with that from parental cells. Tubulin pools (polymerized S2, monomeric S1) were extracted from cells and processed for SDS-PAGE and autoradiography, and the tubulin bands were subsequently analyzed by laser densitometry. The polymerized tubulin pool was substantially reduced in PKC--overexpressing clones. This injury did not occur in the parental cells. Values are means ± SE; n = 6 observations per treatment group. *P < 0.05 vs. vehicle-treated parental cells. +P < 0.05 vs. vehicle-treated TRE-PKC- cells incubated with tetracycline. B: representative Western immunoblots of the intracellular tubulin pools (S2, S1) from the PKC--overexpressing clones and the parental intestinal cells. Tubulin fractions extracted from intestinal cells were subjected to immunoblotting protocols as described previously. Immunoblotted tubulin on nitrocellulose membranes was visualized by enhanced chemiluminescence and autoradiography. Bands representing the polymerized (S2) and monomeric (S1) tubulin are parental cells exposed to vehicle (lane a), parental cells exposed to vehicle plus tetracycline (lane b), transfected PKC--overexpressing cells exposed to vehicle (lane c), transfected PKC--expressing cells exposed to vehicle plus tetracycline (lane d), and tubulin standard (50 kDa; lane e). PKC- overexpression (TRE-PKC-) disrupted the dynamics of tubulin polymerization (lane c, see corresponding S2 and S1 pools of tubulin). This disruption was indicated by a reduction in polymerized S2 tubulin and an increase in monomeric S1 tubulin bands. In the same PKC--transfected clone, when tetracycline was present (lane d), normal tubulin assembly was preserved and was comparable to that of the parental cells (lanes a and b). Parental cells exhibited normal, steady-state pools of tubulin. Representative blots are shown; n = 6 observations per treatment group. C: changes in tubulin phosphorylation in PKC--overexpressing clones compared with parental intestinal cells. Immunoprecipitated tubulin samples were assessed for phosphorylation. The tubulin phosphothreonine bands are parental cells exposed to vehicle (lane a), parental cells exposed to vehicle plus tetracycline (lane b), transfected PKC--overexpressing cells exposed to vehicle (lane c), and transfected PKC--expressing cells exposed to vehicle plus tetracycline (lane d). PKC- overexpressing clone showed a decrease in tubulin phosphorylation (lane c), which was prevented by tetracycline (lane d). Parental cells (with or without tetracycline) showed normally phosphorylated tubulin. Representative blots are shown; n = 6 observations per treatment group.

PKC- expression also reduced tubulin phosphorylation, as determined by immunoblotting of immunoprecipitated tubulin (Fig. 14C). PKC--overexpressing cells showed an abnormally low level of tubulin phosphorylation (Fig. 14C, lane c), which was prevented when tetracycline was present (Fig. 14C, lane d). In parental cells, tubulin phosphorylation was normal (Fig. 14C, lanes a and b). These findings on both tubulin assembly and phosphorylation parallel the destabilizing effects of PKC- overexpression on intestinal microtubule and monolayer integrity.

Expression of PKC- leads to its increased cytosolic activity and reduced particulate activity. Overexpression of the native PKC- in intestinal cells led to the distribution of its activity (in vitro kinase assay, Fig. 15) into mostly the cytosolic fractions, with a much smaller distribution of activity in the particulate fractions, indicating a reduction in the constitutive activity of PKC- isoform. In contrast, parental cells exposed to vehicle (with or without tetracycline) showed constitutive activation of PKC-. PKC- is constitutively active in parental cells because it is found endogenously most active in the particulate fractions under native conditions. In the presence of tetracycline, as might be expected, PKC--transfected cells also showed normal constitutive activity of PKC-.

View larger version (28K): [in this window] [in a new window]   Fig. 15. In vitro kinase activity assay of the overexpressed PKC- in cytosolic and particulate fractions of transfected intestinal cells. Overexpression of the native PKC- (i.e., TRE-PKC-) caused the distribution of its activity into mostly the cytosolic fractions, indicating reduced constitutive activity of PKC- isoform. Only a small level of PKC- activity was found in the key particulate (membrane + cytoskeletal) fractions, further indicating a reduction in the constitutive activity of PKC- in overexpressing clones. Parental cells, on the other hand, showed constitutive activation of the native PKC- isoform, because most of the endogenous PKC- activity was found in the particulate fractions. When tetracycline was present, as might be expected, PKC- transfected cells showed normal constitutive activity of PKC-. Values are means ± SE; n = 6 observations per treatment group.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Using monolayers of intestinal epithelium, we have shown in the current investigation that 1) the 82-kDa -isoform of PKC is required for alterations in the microtubule cytoskeleton, occludin, and barrier permeability in epithelial cell monolayers, and 2) the molecular mechanism underlying this unique effect of the PKC- isoform appears to involve changes in the phosphorylation and/or assembly of the subunits of the cytoskeleton. To our knowledge, this is the first report that PKC- can effect the dynamics of cytoskeletal assembly and permeability clearance in cells.

These two conclusions are supported by independent lines of evidence. First, antisense underexpression of native PKC- induces disruption of monolayers (increases in FS and FD4 clearance). This barrier disruption appears to require underexpression and inactivation of PKC- activity. In particular, monolayer barrier instability is dependent on inactivation of PKC- because of the reduced distribution of PKC- in the key particulate (cytoskeletal and membrane) fractions. Second, antisense underexpression of PKC- appears to induce instability of the cytoskeleton. For example, underexpression of endogenous PKC- reduces the phosphorylation of tubulin; promotes the instability of the molecular dynamics of the tubulin-based cytoskeleton (increases the unstable monomeric tubulin and reduces the stable polymerized tubulin), and decreases the percentage of Caco-2 cells displaying normal microtubules. In WT cells, on the other hand, endogenous PKC- not only was constitutively active in the particulate (e.g., cytoskeletal) fractions but also was complexed with the cytoskeleton. Third, dominant negative inactivation of endogenous PKC- induces an antisense-like disruption in intestinal cells, causing a destabilizing cascade of alterations:

Fourth, overexpression of PKC-, which also leads to a reduction in the amount of PKC- in the cytoskeletal and membrane fractions, promotes all measures of monolayer barrier disruption. In these sense-transfected clones, tubulin, microtubules, occludin, and monolayer barrier permeability were substantially disrupted. Fifth, the high strength of the correlations (e.g., PKC- vs. cytoskeletal assembly or monolayer barrier integrity, etc.), which explains 85–95% of the variance, indicates that PKC- is key for alterations in the dynamic assembly of the cytoskeleton and barrier permeability in intestinal cell monolayers.

Our findings with the use of molecular biology are consistent with the known biochemical properties of PKC isoforms in cells. All PKCs consist of the NH₂-terminal regulatory domains that are separated by a flexible hinge region from the COOH-terminal catalytic domains (21). In resting cells, some isoforms of PKC can be found in an inactive form, because they are mostly distributed in the cytosolic (soluble) fractions and only loosely bound to membrane and cytoskeletal (particulate) components. In contrast, in the same resting cells, other PKC isoforms are found in an active form, because they are mainly distributed in the membrane and cytoskeletal fractions. Regulatory domains of PKC isoforms vary from one subfamily to the next as well as among individual isozymes within the same subfamily (5, 10, 18, 21, 40). Indeed, the effects of PKC in cells are complex and appear to vary widely with different experimental settings as well as tissue types. This is not surprising because not only do most cells express multiple, different isozymes of PKC from various subfamilies but also the differences among these PKC isotypes with respect to conditions of activation and intracellular distribution allow individual isoforms to perform distinct biological functions (1, 5, 6, 10, 14, 26, 37, 39). For example, an immunofluorescence study proposed that PKC- may be involved in TNF--induced damage in the intestinal (IEC-18) cells (17), whereas a pharmacological study suggested that bryostatin-1 attenuates TNF--induced barrier disruption via PKC- in colonic (T-84) cells (49). A recent study from our laboratory (5) that was based on both pharmacological and molecular biological approaches showed that PKC- is involved in oxidant-induced damage in the intestinal (Caco-2) cells. We also showed that the 78-kDa classical 1-isoform of PKC (PKC-1) is required for both normalization of Ca²⁺ homeostasis and suppression of NF-B activation and consequent protection of colonocytes against oxidant injury (6, 14). In other studies, we showed (10) that the 72-kDa atypical -isoform of PKC (PKC-) is required for the suppression of NF-B and inducible nitric oxide synthase-driven oxidative stress and protection of colonocytes. We thus showed that both PKC-1 and PKC- isoforms are required for growth factor (EGF, TGF-)-induced protection of the intestinal epithelium (6, 10, 14), indicating that these PKC isoforms perform unique tasks in protection of intestinal cells. Thus mimicking or activating different isoforms of PKC appears to mediate distinct biological effects (e.g., EGF protection, oxidant injury) on the epithelium. Nonetheless, our current findings on the 82-kDa -isoform of PKC, we believe, indicate a unique role among the novel subfamily of PKC isoforms, namely, effects on epithelial barrier permeability via alterations in the phosphorylation and/or assembly of the subunit components of cytoskeleton. We have thus discovered a novel biological mechanism among this subfamily of PKC isoforms.

In the current investigation, we chose to utilize well-established and widely used fluorescent probes such as FS and FD4 for assessing intestinal monolayer barrier stability for several reasons. Fluorescently labeled FD4 and FS compounds offer appropriate choices for determination of epithelial monolayer barrier integrity. First, these probes are convenient because they come in a range of sizes, are nontoxic to cells, are membrane impermeable, and are relatively inexpensive (e.g., 2–10, 14, 27, 29, 45, 47). Second, both in vivo and in vitro studies, including our own, have revealed that these probes move through the monolayer paracellular space and thus are appropriate for assessing monolayer stability (7, 27, 29, 45). Third, the probes are nontoxic lipophobic moieties and are considered to be cell membrane impermeable (7, 27, 45), so their permeability characteristics make them ideal for use in studying monolayer barrier integrity. Accordingly, these probes can be and have been used as tracers to examine many aspects of endothelial and epithelial monolayer barrier stability. For example, the FS and FD probes have been widely used by numerous groups studying GI inflammation (2–4, 7–9, 27, 29, 45, 47). In several studies, these fluorescent probes were used to assess intestinal Caco-2 monolayer barrier integrity (2–10, 14, 27, 29, 38, 47).

The ability of PKC- to affect cytoskeleton and monolayer barrier integrity could potentially lead to development of new therapeutics for inflammation, in general, and IBD, in particular, because disruption of mucosal barrier integrity has been documented under these conditions. Specifically, this concept is consistent with characterizations of the pathophysiology of IBD and of the disrupted nature of barrier in IBD (23–25, 30–32, 28, 42, 46). The manifestations of IBD, including ulcerative colitis and Crohn's disease, wax and wane between active (symptomatic) phases of disease, when barrier permeability (i.e., barrier disruption) is thought to be high, and inactive (asymptomatic) phases, when barrier permeability is low. Not surprisingly, we previously showed that the degree of mucosal cytoskeletal instability (an index of barrier stability) correlated with the degree of inflammation and disease severity score in patients with IBD flare-up (30). The presence of unstable cytoskeletal components, which indicates an abnormally leaky barrier, has also been shown in intestinal epithelial monolayers (e.g., Caco-2 cells) under IBD-like conditions (3, 5–7, 10, 14). Consistent with these studies, in other studies loss of gut barrier permeability elicited symptoms of intestinal inflammation and IBD-like conditions in animals (23, 48). Thus induction of barrier hyperpermeability (leaky gut) appears to be crucial to the perpetuation of the active, symptomatic phase of IBD, the so-called IBD flare-up, when intestinal inflammation creates a vicious cycle dependent on oxidative stress, cytoskeletal instability, and, ultimately, mucosal tissue damage. The cytoskeletal and monolayer effects by PKC-, as we have found in intestinal cells, could be pivotal in suppressing and/or attenuating the continuation of barrier leakiness and inflammatory processes. Accordingly, developing a means of maintaining mucosal barrier integrity during IBD by using agents that attenuate endogenous signaling pathways (e.g., PKC-) might be a simple and effective strategy for the treatment of IBD. PKC- mimetics might even synergize with the effects of agents triggering protective pathways (e.g., EGF, PKC-) and/or the currently used antioxidants so that inflammatory processes would be more effectively attenuated via the alterations of key intracellular mechanisms.

In summary, our findings with the use of targeted molecular approaches to stably underexpress, inactivate, or overexpress PKC- demonstrate an original concept: this PKC isoform appears to be crucial for a substantial portion of the endogenous stability of the cytoskeleton and barrier permeability in intestinal epithelial monolayers. PKC- perhaps is also key in attenuating establishment of an uncontrolled, leaky gut-initiated inflammatory process that can be found under pathophysiological conditions in the GI tract.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported in part by a grant from Rush University Medical Center, Department of Internal Medicine, and by two National Institutes of Health (NIH) R01 grants, DK-60511 (to A. Banan) and AA-13745 (to A. Keshavarzian).

	ACKNOWLEDGMENTS

 
Portions of this work were presented in abstract form during the annual meeting of the American Gastroenterological Association (Digestive Disease Week), May 2004.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. Banan, Director of Research, Section of Gastroenterology and Nutrition, Rush Univ. of Chicago School of Medicine, 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.

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