Differential subcellular targeting of PKC-{epsilon} in response to pharmacological or ischemic stimuli in intestinal epithelia

Joshua M. V. Mammen, J. Cecilia Song, James Yoo, Peter S. Kim, Harold W. Davis, M. Isabel Calvo, Roger T. Worrell, Karl S. Matlin, and Jeffrey B. Matthews

Epithelial Pathobiology Research Group, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio

Submitted 31 March 2004 ; accepted in final form 25 August 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ischemia is the central pathogenic factor underlying a spectrum of intestinal disorders. The study of the cellular signaling responses to ischemic stress in nonepithelial cells has progressed substantially in the previous several years, but little is known about the response in epithelial cells. Unique features of the epithelial response to ischemic stress suggest differential regulation with regards to signaling. The PKC family of proteins has been implicated in ischemic stress in nonepithelial systems. The role of PKC isoforms in chemical ischemia in intestinal epithelial cells is evaluated in this study. Additionally, the phosphorylation of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS) is also studied. Chemical ischemia resulted in the transient activation of only the isoform PKC-{epsilon} as detected by translocation employing the subcellular fractionation technique. The pharmacological agonists phorbol 12-myristate 13-acetate and carbachol also led to the translocation of PKC-{epsilon}. By immunofluoresence, MARCKS is noted to be located at the lateral membrane under control conditions. In response to carbachol, MARCKS translocates to the cytosol, indicating its phosphorylation, which is additionally confirmed biochemically. Consistent with this observation, carbachol induces the translocation of PKC-{epsilon} to proximity with MARCKS at the lateral membrane. In response to chemical ischemia, MARCKS fails to translocate and phosphorylation does not increase. Additionally, the translocation of PKC-{epsilon} is not to the lateral membrane but rather basally. The data suggest that the differential translocation of PKC-{epsilon} in response to pharmacological agonists versus ischemic stress may lead to different effects on downstream targets.

F-actin; hypoxia; ischemia; myristolated alanine-rich C kinase substrate; phorbol ester


ISCHEMIA (oxygen and substrate depletion) is the central pathogenic factor underlying a spectrum of intestinal disorders ranging from acute mesenteric vascular catastrophes to chronic ischemic colitis. Additionally, ischemia and subsequent reperfusion injury contribute to tissue destruction and inflammatory responses in diverse conditions including inflammatory bowel disease, neoplasia, and intestinal obstruction, as well as in the context of organ preservation in the setting of intestinal transplantation. The intestinal epithelium is particularly sensitive to episodes of ischemia-reperfusion (I/R) injury (16, 33, 47). In recent years, substantial advances have been made in understanding the general cellular response to ischemic stress, notably in organs such as the heart and brain. Comparatively little information exists with respect to epithelial cells, particularly in the intestine, yet such information is critical to the development of new intervention strategies (19). It is likely that many aspects of the general response to ischemic stress are conserved in epithelial cells, yet there is scattered evidence to suggest that these cells, particularly epithelial cells from the intestinal tract, may exhibit unique features. A recent report (7), for example, suggests that the tight junctional complex of intestinal epithelial cells may be unusually stable during ischemic stress in contrast to renal epithelia. Moreover, intestinal crypt epithelial cells appear to be more resistant to postischemic apoptosis than villus enterocytes (13).

PKC is a family of 11 serine-threonine kinases that have been implicated in the cellular response to ischemia. PKCs are subdivided into three major categories based on structural features and activation requirements. Conventional PKC (cPKC) isozymes ({alpha}, {beta}1, {beta}2, and {gamma}) are Ca2+ dependent and are activated by diacylglycerol (DAG) or phorbol ester. Novel PKC (nPKC) isozymes ({delta}, {epsilon}, {eta}, and {theta}) are also activated by DAG or phorbol ester but are Ca2+ independent. The atypical PKC isozymes ({zeta}, {iota}, and {lambda}) are insensitive to both Ca2+ and DAG or phorbol ester (14). Certain PKC isozymes have been demonstrated to undergo subcellular translocation (an event associated with PKC activation) both during and following ischemic stress (1, 2, 21, 32, 36, 43). For example, in the myocardium, mild ischemic stress has been shown to induce the activation of nPKC-{epsilon}, which has proven to be a critical mediator of ischemic preconditioning (3, 28, 45). Despite the apparent importance of PKC-{epsilon} in the response to ischemic stress in various cell culture and animal studies, its downstream targets have not been clearly elucidated (9, 24, 31, 34). The activation and downstream effects of PKC isoforms in epithelial cells during ischemia are even more poorly understood than in myocardial and neural cells. In in vivo renal experiments, various PKC isoforms ({alpha}, {beta}, {delta}, {epsilon}, and {zeta}) were noted to be activated following I/R injury (17, 26). Additionally, ischemic preconditioning in the kidney has been shown to be PKC dependent (18). In the lung, hypoxia leads to PKC-dependent vasoconstriction as well as PKC-dependent events in lung epithelial cells such as the modulation of fibrin deposition and ion transport (25, 27, 44, 46). Accumulating evidence indicates that PKC may be activated by hypoxia with or without substrate deprivation as well as by subsequent restoration of cellular oxygen and substrate supply (I/R) (4, 29, 37).

We have previously shown that activation of PKC-{epsilon} by phorbol ester, bryostatin-1, and carbachol leads to cytoskeletal remodeling and alterations in basolateral membrane dynamics, effects postulated to be mediated through the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS). In the present study, we examine whether ischemic stress in polarized intestinal epithelial cells leads to activation of PKC-{epsilon} and seek to define subcellular targeting and potential substrates compared with known pharmacological PKC activators.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. T84 cells obtained from American Type Culture Collection (Rockville, MD) were grown in a 5% CO2 humidified incubator at 37°C on 162-cm2 flasks (Corning Costar, Acton, MA) with medium containing a 1:1 mixture of F-12 nutrient medium (Ham's) and DMEM supplemented with 6% heat-inactivated fetal bovine serum, 15 mM HEPES, 14.3 mM NaHCO3, and antibiotics and antimycotics (100 U/ml aqueous penicillin G, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B) at a pH of 7.4. Cells were passaged weekly on reaching 90% confluence. For experiments, cells were plated at a density of 5 x 104 cells/cm onto collagen-coated permeable supports (4.7 cm2), where they were fed every 2 days, maintained until steady-state TER was achieved, and used from days 7 to 20.

ATP Depletion. Confluent monolayers were equilibrated in HEPES-phosphate-buffered Ringer solution [HPBR; containing (in mM) 135 NaCl, 5 KCl, 3.33 NaHPO4, 1 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES at pH 7.4] for 30 min before any treatment. Monolayers were then washed in HPBR without glucose and then treated for varying periods of time in a severe depletion buffer (HPBR without glucose containing 1 µM oligomycin-A and 10 mM 2-deoxyglucose).

MTT Assay. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium (MTT) stock solution (5 mg/ml) obtained from Sigma was added to the apical and basal sides of monolayers at a 1:10 dilution with buffer for a 3-h incubation. The buffer was then removed, and the converted dye was solubilized with 0.1 N HCl in absolute isopropanol while monolayers were sonicated for 15 s. Absorbance of the converted dye was then measured at a 570-nm wavelength.

Subcellular fractionation. After undergoing treatment, monolayers were washed twice in ice-cold PBS. Cells were then scraped into 400 µl of homogenization buffer [HB; containing (in mM) 20 Tris (pH 7.5), 5 EDTA, 2 EGTA, 50 NaF, 100 Na3VO4, and 30 Na4P2O7, with 0.25 M sucrose and Complete protease inhibitor cocktail tablets]. The cells were homogenized on ice with 25 strokes of a glass tissue homogenizer. The resulting homogenate was ultracentrifuged at 86,000 g for 32 min at 4°C (TLA 45 rotor, TL-100 ultracentrifuge; Beckman). The pellet was resuspended in 400 µl of the HB containing 0.5% (vol/vol) TX-100 by brief sonication and incubated on ice for 30 min. At the end of the incubation period, the samples were centrifuged at 14,000 g for 20 min at 4°C. The resulting supernatant was designated the membrane fraction.

In vitro kinase assay. After undergoing treatment, monolayers were washed twice in ice-cold PBS. Cells were extracted during a 30 min incubation on ice with 500 µl of apical lysis buffer (LB) containing 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1% (vol/vol) Triton X-100 (TX-100), 2 mM EDTA, 1 mM EGTA, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, and Complete protease inhibitor cocktail tablets. Protein concentration was measured by Bradford assay, and samples were all equalized to a concentration of 500 µg/400 µl. Ten microliters of anti-PKC-{alpha} or -{delta}, or 20 µl of anti-PKC-{epsilon} antibody were added to each sample, and samples were gently rotated at 4°C overnight. Twenty microliters of protein A Sepharose beads were added to the samples that were then rotated for 2 h at 4°C. The samples were then spun at 14,000 g for 1 min. The supernatant was aspirated, and the beads were washed with LB three times and with kinase buffer (35 mM Tris·HCl, pH 7.5, 10 mM MgCl2, 0.5 mM EGTA, 60 µM cold ATP, and 1 mM Na3VO4) two times with spins at 14,000 g for 1 min after each wash. Twenty microliters of kinase buffer with 32P-ATP (kinase buffer with 10 µCi [{gamma}-32P]ATP ) were added to each sample. After a brief spin, samples were incubated for 30 min at 30°C with intermittent agitation. After 5 µl of a loading buffer (5x Laemmli sample buffer with 10% {beta}-mercaptoethanol) were added, samples were boiled for 5 min. Samples were then run on a 12% SDS-PAGE and were then dried for 40 min before exposure.

Immunofluorescence and microscopy. After treatment, monolayers were washed twice with ice-cold PBS. Cells were then fixed in 4% paraformaledehyde for 1 h at room temperature, washed with PBS twice, permeabilized with 0.1% (vol/vol) TX-100 in PBS for 7 min, and rinsed with PBS twice. Filter membranes were cut out in rectangular shapes from the Transwell plastic assembly, placed between 50 µl of blocking buffer (1% normal goat serum, 3% BSA in PBS) at both the top and bottom of the monolayers, and incubated for 30 min at room temperature. Polyclonal antibodies against either PKC-{epsilon} or MARCKS were diluted to 10 µg/ml in the blocking buffer containing 0.1% Triton X-100, and 50 µl of each antibody were placed at both the top and bottom of the monolayers. After overnight incubation in a moisture chamber at 4°C, monolayers were washed in PBS three times for 10 min and incubated in rhodamine-conjugated goat anti-rabbit polyclonal IgG (1:100 dilution) for 1 h at room temperature along with FITC-phalloidin for F-actin staining. Monolayers were then washed three times in PBS and mounted on the microscope slide with Vectashield mounting medium. Confocal images were acquired using a Zeiss inverted microscope.

Gel electrophoresis and Western blot analysis. Samples were loaded at equal concentrations as determined by Bradford assay after addition of Laemmli sample buffer containing 10% {beta}-mercaptoethanol and boiling for 5 min. Proteins were separated by electrophoresis on 8–12% gels and transblotted on nitrocellulose membranes, followed by 1-h incubation at room temperature in blocking buffer [containing 20 mM Tris (pH 7.5), 500 mM NaCl, 5% nonfat dry milk, and 0.2% Tween-20], a 1-h incubation either with blocking buffer or BSA blotting buffer [containing 20 mM Tris (pH 7.5), 500 mM NaCl, 5% BSA, and 0.2% Tween-20] containing primary antibody, a 30-min rinse in wash buffer [20 mM Tris (pH 7.5), 500 mM NaCl, and 0.2% Tween-20], a 1-h incubation with blocking buffer containing secondary antibody, and another 30-min rinse in wash buffer. Bands were detected with enhanced chemiluminescence (ECL) detection reagents.

Materials. Tissue culture reagents were purchased from Life Technologies and gel electrophoresis and Western blotting reagents were from Bio-Rad, with the exception of the ECL detection reagent, which was purchased from Amersham. Complete protease inhibitor cocktail tablets were from Boehringer-Mannheim, and FITC-phalloidin was from Molecular Probes. Anti-PKC-{epsilon}, anti-PKC-{alpha}, anti-PKC-{beta}1, anti-PKC-{beta}II, anti-PKC-µ, anti-PKC-{delta}, and anti-phospho-MARCKS antibodies were purchased from Santa Cruz Biotechnology. Secondary antibodies conjugated with various fluorescent dyes were from Jackson Laboratories, and Vecta-shield mounting medium was from Vector Laboratories. Secondary antibodies conjugated with horseradish peroxidase were obtained from Bio-Rad. Protein A Sepharose beads were from Invitrogen. The conventional and novel PKC inhibitor Gö6850 (5µM) was obtained from Calbiochem and was added 30 min before treatment. All other chemicals were obtained from Sigma.


    RESULTS
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 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Selective translocation of PKC-{epsilon} in response to chemical ischemia. Ischemic stress has been widely modeled in vitro by the use of substrate deprivation in the presence of chemical inhibitors of aerobic and anaerobic metabolism (1 µM oligomycin-A and 10 mM 2-deoxyglucose). We validated this approach in the T84 model with MTT assay demonstrating no change in cell survival in monolayers treated with glucose containing buffer versus 60 min of chemical ischemia [0.35 ± 0.1 counts/s (cps) vs. 0.29 ± 0.02 cps, n = 3; not significant (NS)], confirming data previously published using a lactose dehydrogenase assay (23). As previously published, ATP content in cells subjected to chemical ischemia is <5% of control by 60 min of treatment. In the same mansuscript, the authors noted that whereas transepithelial resistance does change initially due to adenosine-mediated chloride secretion, it remains stable for the remainder of an hour of chemical ischemia (22). T84 monolayers grown to confluence on permeable supports were subjected to chemical ischemia in this fashion, and PKC isozyme activation was assessed by subcellular fractionation and Western blot analysis. Translocation from one biological compartment (cytosolic) to another (membrane associated) is the hallmark for activation of a PKC isoform. Of the isoforms examined, only PKC-{epsilon} was observed to undergo cytosol-to-membrane translocation. PKC-{epsilon} increased in the membrane fraction within 15 min and was maximal after 30 min of chemical ischemia. Translocation of PKC-{epsilon} in response to chemical ischemia is similar to that observed with authentic hypoxia (unpublished observations). Subsequently, this level declined such that after 60 min, PKC-{epsilon} had returned to its original distribution (Fig. 1, A and B). There was no evidence of PKC degradation during this time period. None of the other isoforms examined (PKC-{alpha}, -{beta}1, -{beta}2, -µ, -{zeta}, and -{delta}) underwent translocation in response to chemical ischemia (Fig. 2). PKC-{beta}1 is constitutively located in the membrane fraction in T84 cells and remains in that compartment even after cells are exposed to chemical ischemia or phorbol ester (unpublished observation). In vitro kinase assay (IVKA) suggested that PKC-{epsilon} was active under these conditions (Fig. 3A), thus confirming that translocation of PKC-{epsilon} is also accompanied by evidence of kinase activation. Thus ischemic stress induces selective translocation/activation of PKC-{epsilon} in T84 cells. IVKA indicated that PKC-{delta} and, to a lesser extent, PKC-{alpha}, which did not undergo translocation in response to chemical ischemia, also showed evidence of in vitro activation (Fig. 3B). This finding is similar to the response of this isozyme to phorbol ester, but due to limitations inherent in the interpretation of IVKA in the context of cofactor-dependent kinases such as PKC, is of unclear significance (40). To confirm that ischemia-induced PKC-{epsilon} translocation also occurs in other intestinal epithelial cell types, Caco-2 colon carcinoma cells and the nontransformed colonic epithelial NCM-460 line were examined, and similar results were obtained (data not shown).



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Fig. 1. PKC-{epsilon} activation occurs in response to chemical ischemia in T84 cells by subcellular fractionation. A: T84 cells grown as monolayers on permeable supports were treated with chemical ischemia (1 µM oligomycin-A and 10 mM 2-deoxyglucose in glucose-free buffer for varying periods of time). Activation of PKC-{epsilon} was assessed by translocation of the protein from the cytosol (C) to the membrane (M) fraction. PKC-{epsilon} activation was maximal at 30 min with subsequent inactivation by 1 h. B: the amount of membrane fraction with relation to total protein is used to assess PKC activation. PKC-{epsilon} has a 0.300 (±0.038) membrane/total protein fraction under control (con) conditions that increases with translocation to 0.666 (±0.027; P < 0.0005, n = 10).

 


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Fig. 2. Multiple PKC isoforms do not translocate in response to chemical ischemia. Numerous other PKC isoforms other than PKC-{epsilon} were examined for activation by subcellular fractionation. Of the 6 additional isoforms studied, none of them translocated in response to chemical ischemia in T84 cells. PKC-{beta}1 is constitutively located in the membrane fraction in T84 cells and remains in that compartment even after cells are exposed to chemical ischemia.

 


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Fig. 3. PKC-{alpha}, -{delta}, and -{epsilon} activation in response to chemical ischemia by in vitro kinase assay (IVKA). A: T84 cells grown as monolayers on permeable supports were treated with chemical ischemia (1 µM oligomycin-A and 1 mM glucose buffer for varying periods of time). Activation of PKC-{epsilon} was assessed by IVKA with MBP as the substrate. PKC-{epsilon} activation was noted at the 30- and 60-min time points. B: the PKC-{alpha} and -{delta} isoforms were also assessed for activation by IVKA. Both isoforms had been activated by 30 min of chemical ischemia.

 
Subcellular localization of PKC-{epsilon} in response to chemical ischemia and pharmacological PKC activators. We have shown that PKC-{epsilon} undergoes translocation/activation in response to both pharmacological stimuli [the phorbol ester phorbol 12-myristate 13-acetate (PMA), the nonphorbol macrocyclic lactone bryostatin-1, and the acetylcholine analog carbachol] (38) as well as to chemical ischemia. The redistribution of PKC-{epsilon} induced by these stimuli was visualized by indirect immunofluorescence and confocal microscopy. We have shown that in unstimulated T84 cells, PKC-{epsilon} is located in a diffuse cytosolic location and that in response to PMA, PKC-{epsilon} progressively redistributes to a predominantly lateral membrane location (38). As shown in Fig. 4, we now show that stimulation with 100 µM carbachol induces redistribution of PKC-{epsilon} to a similar lateral membrane distribution. Although by subcellular fractionation and Western blot analysis chemical ischemia induces dramatic translocation of PKC-{epsilon}, the pattern of redistribution was strikingly different than that induced by either CCh or PMA. Over the course of 30 min, PKC-{epsilon} initially assumed a punctate distribution throughout the cell and then progressively moved toward the basal membrane (Fig. 5).



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Fig. 4. Carbachol results in the translocation of PKC-{epsilon} to the lateral membrane. T84 cells were treated for varying periods of time with carbachol (100 µM). The T84 cells were fixed and then stained with phalloidin as well as rhodamine-tagged secondary antibody to the primary PKC-{epsilon} antibody. The monolayers were then examined with confocal microscopy for PKC-{epsilon} translocation. The isoform translocates largely to the lateral membrane (arrow) by 30 min from its dispersed basal cytosolic state. PMA results in similar translocation of PKC-{epsilon} to the lateral membrane. AP, apical; BL, basolateral.

 


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Fig. 5. PKC-{epsilon} translocates to the basal membrane in response to chemical ischemia (Isch.). T84 cells were treated for varying periods of time with chemical ischemia. The T84 cells were fixed and then stained with phalloidin as well as rhodamine-tagged secondary antibody to the primary PKC-{epsilon} antibody. The monolayers were then examined with confocal microscopy for PKC-{epsilon} translocation. The majority of the isoform translocates to the basal membrane indicated by arrowheads (rather than the lateral membrane identified by arrows) by 30 min and near completely by 45 min. By 60 min, however, the isoform is moving out of the basal membrane location to its dispersed cytosolic state.

 
MARCKS is located at the lateral membrane and is a target for PKC-{epsilon} activated by pharmacological but not ischemic stimuli. We have previously shown that PMA and CCh induce remodeling of the basolateral actin cytoskeleton associated with redistribution of the F-actin cross-linker and PKC substrate MARCKS (39). Consistent with this finding, we observed that MARCKS is located at the lateral membrane of T84 during control conditions. Thirty and sixty minutes of ATP depletion have little effect on the location of MARCKS. MARCKS, however, translocates to a subapical cytosolic region in response to 30 min of CCh stimulation (Fig. 6). As shown in Fig. 7A, CCh and PMA also increased phosphorylation of MARCKS, an effect completely blocked by pretreatment with the cPKC and nPKC inhibitor Gö6850 (5 µM) but not by the selective cPKC inhibitor Gö6976 (5 µM), consistent with a role for the nPKC-{epsilon} (39). The increased phosphorylation of MARCKS is consistent with the translocation of PKC-{epsilon} in response to PMA and CCh to the vicinity of MARCKS at the lateral membrane (Fig. 6). However, MARCKS phosphorylation fails to increase in response to chemical ischemia (Fig. 7B), consistent with failure of the PKC-{epsilon} isozyme to redistribute in proximity to its lateral membrane association.



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Fig. 6. Myristoylated alanine-rich C kinase substrate (MARCKS) is located at the lateral membrane. T84 cells were fixed and then stained with FITC-phalloidin and rhodamine-tagged secondary antibody to the primary MARCKS antibody. The monolayers were then examined with confocal microscopy. In control and chemical ischemia treatment, MARCKS is located at the lateral membrane (identified with arrows). However, in response to carbachol, MARCKS translocates mostly to a subapical cytosolic location (arrowhead).

 


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Fig. 7. MARCKS is phosphorylated in response to phorbol 12-myristate 13-acetate (PMA) and carbachol (CCh), but not chemical ischemia. A: T84 cells grown on monolayers were treated with PMA (100 nM) or CCh (100 µM) for 30 min. MARCKS phosphorylation increased in both PMA and CCh-treated samples. B: T84 monolayers were treated for varying periods of time with chemical ischemia (1 µM oligomycin-A and 10 mM 2-deoxyglucose in glucose-free buffer) for varying periods of time. The control condition was overexposed to detect the minimal basal MARCKS phosphorylation. MARCKS phosphorylation not only failed to increase in response to chemical ischemia, but phosphorylation actually decreases as ATP levels drop.

 
PKC-{epsilon} activation during ATP depletion occurs independent of oxygen-derived free radical production. Monolayers of T84 cells were pretreated for 30 min either with 10,000 U/ml of superoxide dismutase or 100 U/ml of catalase before ATP depletion. Subcellular fractionation was performed to assess PKC-{epsilon} activation. Both superoxide dismutase and catalase failed to inhibit the activation of the PKC isoform compared with control monolayers that underwent ATP depletion as assessed by the ration of the isoform in the membrane fraction versus total [53% ± 4 vs. 48 ± 2%, n = 3 (NS), and 65 ± 2 vs. 64 ± 16%, n = 3 (NS); Fig. 8].



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Fig. 8. PKC-{epsilon} activation during ATP depletion occurs independent of oxygen-derived free radical production. A: T84 cells grown on monolayers were treated with 10,000 U/ml of superoxide dismutase (SOD) before ATP depletion. Subcellular fractionation was performed to assess the degree of PKC-{epsilon} activation. There was no difference in the amount of membrane-to-total protein ratio in cells pretreated with SOD vs. control monolayers that underwent ATP depletion without pretreatment [53 ± 4 vs. 48 ± 2%, n = 3, not significant (NS)]. This is a representative blot of 3 experiments. B: T84 cells grown on monolayers were treated with 100 U/ml of catalase before ATP depletion. Subcellular fractionation was performed to assess the degree of PKC-{epsilon} activation. There was no difference in the amount of membrane-to-total protein ratio in cells pretreated with catalase vs. control monolayers that underwent ATP depletion without pretreatment (65 ± 2 vs. 64 ± 16%, n = 3, NS). This is a representative blot of 3 experiments.

 

    DISCUSSION
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 METHODS
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 DISCUSSION
 REFERENCES
 
Epithelial cells are characterized by a polarized architecture in which apical and basolateral plasma membrane domains are delimited by the tight junctional complexes that link adjacent cells. This structural configuration is a prerequisite for fundamental epithelial functions including barrier formation and vectorial transport. Enterocytes are further distinguished from other epithelial cell types such as renal tubular cells and hepatocytes by their limited (3–5 day) lifetime during which they undergo maturation from secretory to absorptive phenotype as they exit the proliferative crypt compartment and progressively migrate toward the villus compartment, the zone of apoptotic cell death and extrusion (10, 15, 30). Previous reports have indicated that PKC is an important regulator of ion transport and barrier function in T84 cells as well as other model epithelial systems. We showed, for example, that activation of PKC-{epsilon} by PMA, bryostatin-1, or CCh negatively regulates electrogenic Cl secretion (the transport event of secretory diarrhea) (38). This effect was largely exerted at the level of the basolateral membrane, and in earlier reports and the present study, we have provided evidence that PKC-{epsilon} is targeted to the lateral membrane after pharmacological stimulation. PKC-{epsilon} also appears to be important in the maintenance of barrier function from certain inflammatory stimuli by an effect involving phosphorylation of tight junctional proteins (38). Given the identification of PKC-{epsilon} as an important regulator of both transport and barrier function, we were intrigued by the well-documented role of PKC-{epsilon} in the adaptive response to sublethal ischemic stress. We previously reported that chemical ischemia induces activation of electrogenic Cl secretion by an autocrine mechanism involving release of the ATP catabolite adenosine and postulated that this event recapitulated the known diarrheal response to intestinal ischemia (42). Subsequently, we found that inhibition of PKC-{epsilon} exaggerated (but prior pharmacological activation of PKC-{epsilon} dampened) this ischemia-induced secretory response (48).

The effects of ischemic stress on the distinct functional and structural characteristics of epithelial cells remain incompletely defined. The intestine is particularly sensitive to I/R injury, manifest by rapid loss of mature enterocytes and sloughing of villus tips. This is followed by a reparative phase consisting of migration of surviving crypt cells to cover denuded basement membrane as well as an increase in cell replication. An increase in epithelial cell apoptosis has also been observed (6). PKC has been shown to be activated during ischemic stress in a variety of cell types and organ systems, and, although its upstream activators and downstream targets are unclear, this pathway is thought to represent an adaptive response to enhance cell survival and prevent postischemic apoptosis (1, 2, 21, 32, 36, 43).

Subcellular targeting of activated PKC is tightly regulated, determined by protein-protein interactions with intracellular receptors known as receptors for activated C kinase (RACKs) that are distinct for each isozyme. RACK-1, for example, binds PKC-{alpha} and PKC-{beta}II but not nPKCs, whereas F-actin and {beta}'-COP may be PKC-{epsilon} specific (8, 35, 41). RACKs serve to concentrate activated PKC isozymes in specific subcellular domains in close proximity to certain substrate targets and, possibly, away from others. PKC-RACK interactions are thought to account for substrate specificity despite otherwise limited differences in catalytic activity and activation requirements among the isozymes.

In the present study, we found that chemical ischemia induced the selective translocation and activation of PKC-{epsilon} in T84 and other epithelial cell lines. However, by indirect immunofluorescence and confocal microscopy, we found that the subcellular redistribution of PKC-{epsilon} after an ischemic stimulus differed markedly from that induced by pharmacological agonists. PMA and CCh induced lateral membrane localization of PKC associated with an increase in protein phosphorylation of the target protein MARCKS, which shares this lateral membrane distribution. In contrast, although ischemic stress induced activation of PKC-{epsilon} (assessed by traditional operationally defined criteria based on membrane translocation and IVKA), this isozyme redistributed to a basal location, and MARCKS phosphorylation failed to increase. It is important to recognize that despite the depletion of cellular energy stores induced by chemical ischemia, it is unlikely that ATP supply becomes rate limiting for phosphorylation reactions until levels fall below ~10% of baseline conditions (5, 12). Certain kinases, such as AMP-activated kinase (AMPK), are in fact activated under low-ATP (high AMP) conditions (11, 20). We have found that during chemical ischemia, phosphorylation of various proteins including mitogen-activated kinases including ERK1/2 and AMPK is increased (unpublished observations). Thus failure of translocated/activated PKC-{epsilon} to phosphorylate MARCKS is more likely to reflect altered targeting rather than unavailability of ATP. With regard to PKC-{alpha} and -{delta}, which appear to be activated as determined by IVKA, it is important to note that neither isoform underwent translocation as detected by subcellular fractionation. The use of IVKA, by itself, is inadequate as a determinant of PKC isoform activation with translocation being a necessary component (9). It is important to concede, however, that translocation within a compartment may occur that cannot be detected by our biochemical technique.

Attempts were made to study the upstream players involved in the activation of PKC-{epsilon} during chemical ischemia in intestinal epithelia. To study the possible involvement of reactive oxygen species in leading to the activation of PKC-{epsilon} during ATP depletion, we pretreated and cotreated T84 monolayers either with catalase or superoxide dismutase. Neither agent was able to alter ATP depletion-induced translocation of PKC-{epsilon} in T84 cells. Authentic hypoxia also leads to the activation of PKC-{epsilon}, albeit with a different time course (in hours; unpublished observations).

One of the earliest features of ischemic epithelial injury is disruption of the cytoskeleton. Severing of membrane protein-cytoskeletal linkages leads to loss of cell polarity and membrane protein dysfunction. It is unclear whether PKC activation during epithelial ischemia contributes to these changes. The differential redistribution of PKC-{epsilon} in response to ischemic stress compared with pharmacological activation may represent mistargeting or a regulated adaptive response. Preliminary data suggest that "preconditioning" does occur in intestinal epithelial cell lines and native tissue and may be at least partially PKC dependent (unpublished observations), but there remains considerable controversy as to whether activation of PKC during intestinal inflammation is beneficial or harmful and what role distinct isozymes may play in the setting. The potential for disrupted PKC signaling to contribute to disease pathogenesis and for targeted manipulation of these events to be of therapeutic value are important areas for future investigation.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. B. Matthews, Dept. of Surgery, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, PO Box 670558, Cincinnati, OH 45267–0558 (E-mail: Jeffrey.Matthews{at}uc.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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