The role of protein kinase C isozymes in TNF-alpha -induced cytotoxicity to a rat intestinal epithelial cell line

Q. Chang and B. L. Tepperman

Department of Physiology, University of Western Ontario, London, Ontario, Canada N6A 5C1


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor (TNF)-alpha can induce cytotoxicity and apoptosis in a number of cell types and has been implicated in the regulation of many inflammatory processes. It has been suggested that protein kinase C (PKC) is one of the intracellular mediators of the actions of TNF-alpha . In the present study, the role of PKC isoforms in TNF-alpha -mediated cytotoxicity and apoptosis in intestinal cells was investigated using the rat epithelial cell line, IEC-18. Cells were incubated with TNF-alpha in the presence or absence of the transcription inhibitor actinomycin D (AMD). The extent of cell damage was enhanced when AMD was added to incubation medium, suggesting that new protein synthesis plays a role in the cytotoxic action of TNF. TNF-alpha also induced the translocation of PKC-alpha , -delta , and -epsilon from cytosol to the membrane fraction of the intestinal cells. Furthermore, the cytotoxic and apoptotic effects of TNF were reduced by pretreating the cells with the PKC-epsilon translocation inhibitor, PKC-epsilon V1-2. In contrast, although cells incubated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) also displayed an increase in cell injury, the extent of cytotoxicity and apoptosis was not enhanced by AMD. Furthermore, PMA-induced cell damage was reduced by rottlerin, a PKC-delta inhibitor. Caspase-3, an enzyme implicated in the mediation of apoptosis, was activated in cells in response to either TNF-alpha or PMA stimulation, and its effects on this activity were reduced by selective inhibition of PKC-epsilon and -delta , respectively. Furthermore, inhibition of caspase-3 activity reduced apoptosis. These data suggest that activation of selective PKC isoforms mediate the effects of TNF-alpha on intestinal epithelial cell injury.

IEC-18 cells; apoptosis; caspase-3; isoform translocation; phorbol ester; tumor necrosis factor-alpha


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TUMOR NECROSIS FACTOR (TNF)-alpha is a cytokine that is a member of the family of proteins that comprises lymphotoxin-alpha and lymphotoxin-beta (3, 12). TNF-alpha is involved in the regulation of many inflammatory processes, including experimentally induced intestinal inflammation in animals and inflammatory bowel disease in humans. A single dose of TNF-alpha has been shown to cause significant small intestinal injury in rodents (10). Furthermore, studies of animals with experimentally induced intestinal inflammation indicate that mononuclear cells from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-treated mice produce 10- to 30-fold higher levels of TNF-alpha mRNA and protein than cells from control mice (31). Administration of antibodies to TNF-alpha to TNBS-treated mice has been shown to result in an improvement of the clinical and histopathological signs of the disease. Similarly, antibody neutralization of TNF results in a reduction in the mucosal inflammation induced by dextran sulfate sodium instillation into mouse colon (20), and TNF inhibition can ameliorate mucosal inflammation and abnormalities in colonic permeability in a model of graft vs. host disease in mice (5). In humans, TNF-alpha has been detected in the colonic mucosa of patients with Crohn's disease (1). In functional studies of mononuclear cells derived from the lamina propria of patients with inflammatory bowel disease, stimulation of cells from inflamed areas of the mucosa produced more TNF than did cells from noninflamed areas (35). Finally, in vitro studies using intestinal cell lines have demonstrated that TNF-alpha treatment either alone or in combination with other cytokines can impair epithelial barrier function (1, 8, 28, 41, 48).

It has been suggested that PKC is one of the intracellular signaling mediators of the actions of TNF-alpha , which include cytotoxicity and apoptosis in a variety of cell types (26, 32, 49). Protein kinase C (PKC) consists of a family of at least 12 isozymes differing in tissue distribution and activation requirements. There are three subclasses: classical PKC isozymes like -alpha , -beta 1, -beta 2, and -gamma , which require calcium and are activated by diacylglycerol and phorbol ester; the novel PKC isozymes like -delta , -epsilon , -eta , and -theta , which are activated by diacylglycerol and phorbol ester independently of calcium; and the atypical PKC isozymes like -lambda , -iota , and -zeta , which are calcium independent and not responsive to phorbol ester. PKC has been found to be elevated in colonic mucosal samples excised from patients with ulcerative colitis (38), and activation of luminal PKC via phorbol ester instillation has been shown to induce ileal and colonic inflammation in experimental animals (2, 9). Furthermore, PKC activity is elevated in mucosal samples taken from animals in which colitis was induced via instillation of TNBS (6).

Previous studies have demonstrated that TNF-alpha can induce apoptosis in a variety of cell types, including cells of the gastrointestinal tract (42, 43). Furthermore, distinct PKC isozymes have been shown to signal apoptosis in human colonic cells (47). The presence and activation of discrete PKC isozymes within cells might influence the susceptibility of those cells to apoptotic or necrotic challenges. Indeed, we have recently demonstrated that activation of distinct PKC isozymes can mediate necrotic cell damage in rat colonic epithelial cells (44). Therefore, in the present study we have examined the effect of TNF-alpha on intestinal epithelial cell apoptosis and injury and have identified the role of PKC and the PKC isozyme(s) mediating these responses.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Materials. Human recombinant TNF-alpha was purchased from Upstate Biotechnology (Lake Placid, NY). Affinity purified rabbit polyclonal antibodies against PKC isozymes-alpha , -delta , -epsilon , and -zeta and blocking peptides were purchased from Santa Cruz (Santa Cruz, CA). Goat-anti-rabbit antibodies conjugated to horseradish peroxidase and FITC were purchased from Jackson ImmunoResearch Laboratories. Protein molecular markers, enhanced chemiluminescence (ECL) nitrocellulose membrane, and ECL blotting kits were all purchased from Amersham. The phorbol ester phorbol 12-myristate 13-acetate (PMA), the selective pan-PKC isoform antagonist GF-109203X (5 µM), the inhibitor of calcium-requiring conventional PKC isoforms Gö-6979 (0.1 µM) (29), PKC-delta (rottlerin; 10 µM) (14), PKC-epsilon (myristoylated PKC-epsilon V1-2 translocation inhibitor; 4 µM) (17), and PKC-zeta (myristolated PKC-zeta pseudosubstrate; 5.2 µM) (24) were purchased from Biomol (Plymouth Meeting, PA). The concentrations of these inhibitors were chosen based on previous studies, cited above, demonstrating their effectiveness. Furthermore, we have done some preliminary studies utilizing a range of doses of each inhibitor. The dose chosen from these preliminary studies was found to provide the lowest degree of cytotoxicity and the optimal degree of PKC suppression for each of the inhibitor agents tested. All other reagents were purchased from Sigma Chemical (St. Louis, MO).

Cell culture and preparation. IEC-18 cells were obtained from American Type Culture Collection (Rockville, MD) and were maintained in Dulbecco's modified Eagle's medium (DMEM) with 4 mM glutamine, 0.01 mg/ml insulin, 5% heat-inactivated fetal bovine serum, 50 U/ml penicillin G, and 50 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. The medium was replaced every 3 days, and cultures were passaged before confluency. The IEC-18 cells used for these studies are derived from rat ileal crypt epithelium (33) and maintain many of the characteristics of proliferating crypt cells.

Usually 2 × 105 cells were seeded in a 60-mm dish. After the cells reached 85-90% confluency, they were transferred to serum-free DMEM medium for a 30-min treatment with PKC inhibitors followed by treatment with PMA (0.2 µM) and TNF-alpha (10 ng/ml) with or without addition of the transcription inhibitor actinomycin D (AMD; 2 µg/ml) for periods ranging from 1 to 48 h. It has been shown that TNF-alpha -induced cytotoxicity could be enhanced in a number of cell types by the addition of the transcription inhibitor AMD (25). Furthermore, it was shown, in preliminary experiments, that this treatment eliminated the necessity for cells to be grown to confluence for manifestation of an apoptotic response.

Separation of cultured IEC-18 cells into membrane and cytosolic fractions. After cells were treated with test components in DMEM, the medium was aspirated from the culture dish and the cells were washed once with ice-cold phosphate-buffered saline and 500 µl of homogenization buffer were added, which consisted of 50 mM Tris · HCl, 5 mM EDTA, 25 mM EGTA, 50 µg/ml phenylmethylsulfonyl fluoride, 10 mM benzamide, 25 µg/ml each of soybean trypsin inhibitor, leupeptin, and aprotinin and 5% mercaptoethanol. Cells were scraped into the medium to form a suspension and lysed by sonication for 10-15 s on ice. Cytosol protein was released into the medium. The resulting lysate was centrifuged at 100,000 g for 1 h at 4°C to pellet the membrane protein. The supernatant was collected as the cytosolic fraction. The resulting pellet was resuspended in 500 µl of homogenization buffer to which was added Triton X-100 (final concentration 0.5%) and incubated on ice for 1 h to extract soluble membrane proteins. Samples were centrifuged again at 100,000 g for 30 min at 4°C to remove insoluble membrane components. The supernatant containing the membrane protein was kept. The particulate and cytosolic fraction extracts were frozen at -80°C until use.

Immunoblot analysis of PKC isoforms. Particulate and cytosol samples (10-15 µg protein) were prepared for electrophoresis by boiling for 5 min in an equal volume of SDS sample buffer (125 mM Tris, pH 6.8, containing 20% glycerol and 10% mercaptoethanol). Samples containing equal amounts of protein were loaded in each lane of 10% SDS polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes at 100 V for 75 min. The membranes were blocked for 1 h at room temperature in PBS-Tween buffer [80 mM Na2HPO4, 10 mM NaCl, and 0.05% Tween-20 (pH 7.5)] containing 10% nonfat milk and then were icubated for 2 h with specific PKC-alpha antibody (1:1,000) and 3 h with PKC-delta , PKC-epsilon , and PKC-zeta antibodies (1:800) at room temperature followed by incubation with a 1:6,000 dilution of HRP-conjugated anti-rabbit IgG for 1 h at room temperature. ECL reagents were used to develop the blots. The densitometric assessment of the bands of the autoradiogram was done using Image Master VDS (Pharmacia Biotech). Band intensity was quantified by measurement of the absolute integrated optical intensity, which estimates the volume of the band in the lane profile.

PKC isoform translocation. To examine the translocation of the PKC isozymes examined in this study, IEC-18 cells were grown to subconfluence on sterile glass coverslips and treated as described above. The coverslips were then washed three times in cold PBS and fixed and permeabilized for 30 min in 1:1 cold methanol-acetone followed by two washes with cold PBS. Cells were then incubated for 50 min with 1% normal goat serum in PBS containing 0.1% Triton X-100 followed by an overnight incubation with the PKC isozyme- specific antibodies diluted 1:100-1:300 in PBS containing 2 mg/ml bovine serum albumin and 0.1% Triton X-100. Cells were next washed three times with PBS followed by a 2-h incubation with FITC-conjugated anti-rabbit IgA antibody at 1:500. The specificity of the staining obtained using the PKC antibodies was determined by preabsorption of the antibodies with the immunizing peptides. After the cells were washed three times with PBS and then twice with distilled water, the coverslips were mounted using Airvol (Doval, PA) and viewed with a Zeiss microscope equipped with appropriate optics and filter modules at ×63 oil-immersion objective. Images from the microscope were recorded by Sensican software with Sensicentral 402 and Adobe Photoshop image processing utilities.

Cell viability analysis. The effects of PMA or TNF-alpha with or without PKC isoform inhibitors on cell viability were determined by a formazan-based assay as described by Twentyman and Luscombe (45). This technique has been used previously to assess intestinal epithelial cell viability (7). Briefly, IEC-18 cells were plated onto 96-well plates at a density of 2 × 104 cells/well and left for 24 h at 37°C. After different test inhibitors were added to the wells, cells were incubated for 18 h with either TNF-alpha or PMA. Cells were then washed in PBS, and freshly prepared 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) solution was applied to each well at a concentration of 0.5 mg/ml for 2 h at 37°C followed by aspiration of the medium and addition of a solubilization medium (90% isopropanol, 0.01 N HCl, and 0.2% SDS) to dissolve the formazan crystals formed in the wells. Absorbance was read at 570 nm on a Spectrareader plate reader (SLT). The percent cytotoxicity was calculated as previously reported (13).

Microscopic determination of apoptosis. Cells were grown on glass coverslips to subconfluence and treated with test compounds in DMEM medium and then fixed in 4% paraformaldehyde. Nuclear condensation and fragmentation were visualized by fluorescent microscopy after a 15-min incubation with the cell-permeable flurochrome Hoechst 33258 (2.5 µg/ml) and were mounted onto slides using fluorescent mounting medium (Dako, Carpinteria, CA). The proportion of cells undergoing apoptosis 18 h after initiation of treatment was determined by counting the total number of cells and the cells exhibiting two or more membrane blebs and brightly stained condensed and fragmented chromatin per high-power field (×40 oil-immersion objective). Apoptotic index was calculated as the percentage of cells displaying the characteristics described above. Means and standard errors were calculated based on the results of a minimum of five different fields for each treatment. At least 300 cells were counted for each sample. All experiments were repeated at least three times to ensure reproducibility.

DNA fragmentation assay. Apoptosis was also estimated by DNA fragmentation using a cellular DNA fragmentation ELISA assay kit (Roche Diagnostics, Mannheim, Germany). This assay measures apoptotic cell death by detection of 5-bromodeoxyuridine (BrdU)-labeled DNA fragments in the cytoplasm of affected cells. Briefly, cells were incubated with the thymidine analog, BrdU, which is incorporated in the genomic DNA, and then were treated with TNF and varying concentrations of the PKC inhibitors or caspase-3 inhibitor for at least 18 h. The appearance of DNA fragments in the cytoplasm was detected and quantified by using an anti-BrdU-antibody-peroxidase conjugate. Absorbance was read at 450 nm on a Spectra plate reader. Means and standard errors were calculated from four identically treated wells for each cellular treatment. Experiments were repeated twice to ensure reproducibility.

Assay for caspase-3 activity. The activation of caspase-3 was detected using assay kits purchased from Biomol, which use N-Asp-Glu-Val-Asp-p-nitroaniline (Ac-DEVD-pNa) as a substrate. The assay was conducted in accordance with the supplier's protocol. Briefly, a sufficient quantity of cultured cells were harvested and washed with ice-cold phosphate buffered saline and then resuspended with cell lysis buffer containing 50 mM HEPES (pH 7.4), 0.1% CHAPS, 1 mM dithiothreitol, and 0.1 mM EDTA and incubated for 10 min on ice. This was followed by centrifugation for 20 min at 12,000 g at 4°C. The supernatant was used for assay by adding 200 µM AC-DEVD-pNA substrate to a final volume of 100 µl. Purified caspase-3 was used as positive control and standard for comparison with cellular supernatant samples. In some experiments, the caspase-3 inhibitor II Z-DVED-FMK [Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-FMK; Calbiochem] was used at a concentration of 10 µM in IEC-18 cells treated with TNF. This concentration has previously been demonstrated to be an effective, noncytotoxic inhibitory dose of this agent (37). The absorbance was read at 405 nm on a Spectrareader SLT plate reader, and substrate standard was recorded to calculate caspase activity.

Statistical calculation. All numerical data are means ± SE. The statistical differences within groups was determined using analysis of variance and Duncan's multiple-range test. Statistical differences between groups treated with different agents was determined by t-test for paired data. P < 0.05 was the accepted level of significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IEC-18 cells strongly express PKC-alpha , -delta , -epsilon , and -zeta proteins as determined by Western blot analysis (Fig. 1). Treatment of IEC-18 cells with TNF-alpha resulted in changes in the abundance of specific isoform protein (Fig. 1). The amount of PKC-alpha protein increased significantly both in cytosolic and particulate fractions by 3 h after treatment and continued up to 48 h after TNF-alpha addition. The expression of PKC-zeta protein increased mainly in the particulate fraction during the entire incubation period. PKC-delta and -epsilon proteins were observed to translocate from cytosolic to particulate fractions from 3 to 24 h after treatment, reflecting activation of these enzymes.


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Fig. 1.   Protein kinase C (PKC) isoform protein levels from IEC-18 cells challenged with tumor necrosis factor-alpha (TNF-alpha ). Cells were collected at times (T) indicated after challenge, membrane cytosolic fractions were obtained, and Western blotting was performed as described in MATERIALS AND METHODS using specific antibodies. Protein levels were quantified by densitometry and expressed relative to levels determined in untreated control (C) cells. Values are means ± SE of 3-4 separate experiments each performed in triplicate. Isoform protein distributions for PKC-alpha (A), PKC-delta (B), PKC-epsilon (C), and PKC-zeta (D) are displayed. IOD, integrated optical density. * P < 0.05 from respective control. + P < 0.05 from respective control.

Activation of the PKC-zeta isoform was not observed after cells were treated with PMA. However, translocation of PKC-alpha , -delta , and -epsilon from cytosol to membrane fractions was apparent in IEC-18 cells (Fig. 2). The activation of these isoforms occurred within 10-15 min after PMA addition and appeared to reach stable levels of translocation 30-120 min after challenge with the phorbol ester.


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Fig. 2.   PKC isoform protein levels from IEC-18 cells challenged with phorbol 12-myristate 13-acetate (PMA). Cells were collected at times indicated after challenge, membrane and cytosolic fractions were obtained, and Western blotting was performed as described in MATERIALS AND METHODS using specific antibodies. Protein levels were quantified by densitometry and expressed relative to levels determined in untreated control cells. Values are means ± SE of 3 separate experiments each performed in triplicate. Isoform protein distributions for PKC-alpha (A), PKC-delta (B), and PKC-epsilon (C) are displayed. * P < 0.05 from respective control. + P < 0.05 from respective control.

Immunostaining for various PKC isoforms using specific antibodies to these isozyemes revealed the localization of PKC-alpha , -delta , and -epsilon in the cytosol of untreated control cells and faint staining for PKC-zeta around the periphery of these cells (Fig. 3). After exposure to TNF-alpha for 18 h, PKC-alpha staining appeared to be intensified within both cytosolic and membrane compartments. PKC-delta appeared more intense in the perinuclear region, whereas PKC-epsilon staining decreased within the cytosolic compartment and intensified within the cell periphery. Similarly, staining for PKC-zeta increased in the region of the cell membrane (Fig. 3). Immunostaining for PKC isoenzymes after exposure to PMA showed a similar translocation from cytosol to nuclear and cellular membrane for all of the isoforms detected here (Fig. 4). The PKC-epsilon translocation inhibitor PKC-epsilon V1-2 blocked the appearance of translocation of PKC-epsilon in response to TNF-alpha , whereas the PKC activity inhibitor GF-109203X was not effective in this regard (Fig. 5).


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Fig. 3.   Immunofluorescent localization of PKC isoforms in IEC-18 cells treated with TNF-alpha or untreated control cells. Cells were harvested 18 h after treatment. Control cells show primary cytosolic distribution of PKC-alpha , -delta , and -epsilon and faint peripheral staining for PKC-zeta . TNF-treated cells display intensified staining for PKC-alpha and -zeta in both cytosolic and membrane compartments. PKC-delta shows intense perinuclear staining while PKC-epsilon shows intensified peripheral staining. Increased staining is shown by arrows (magnification, ×815).



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Fig. 4.   Immunofluorescent staining of PKC isoforms in IEC-18 cells treated with PMA or untreated control cells. Cells were harvested 2 h after treatment. Control cells show primary cytosolic localization of PKC-alpha , -delta , and -epsilon . PMA-treated cells show staining translocation to cell periphery and nuclear membrane from the cytosolic compartments for each isoform detected. Increased staining within the cytosolic and nuclear compartments are shown by arrows (magnification ×815).



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Fig. 5.   Fluorescence micrographs of PKC-epsilon staining in IEC-18 cells treated with TNF-alpha in the presence or absense of actinomycin D (AMD). Some cells were also treated with the PKC inhibitor GF-109203X (GFX) or PKC-epsilon V1-2 (epsilon V1-2) as indicated in MATERIALS AND METHODS. Staining indicates a reduction in isoform translocation in response to epsilon V1-2 but not GFX. Enhancement of PKC-epsilon staining is shown by filled arrow while reduction of isoform translocation by epsilon V1-2 is shown by open arrows (magnification ×815).

The cytotoxic effects of PKC isoforms affected by TNF-alpha or PMA treatment are demonstrated in Fig. 6. IEC-18 cells were incubated with PMA or TNF-alpha for a period of 18 h. Cell injury was estimated by examining cellular metabolism using the MTT assay. PMA induced a significant increase in cell injury after 2 h of incubation, and this increased with increasing incubation times, whereas cytotoxicity in response to TNF-alpha was not evident until 3-6 h after addition of the cytokine and became maximum by 18 h after TNF addition (data not shown). Both TNF-alpha and PMA induced an increase in the extent of cytotoxicity (Fig. 6). The effect of TNF-alpha on the extent of cell injury was significantly increased over the level evident in control cells, and this effect was enhanced by addition of AMD to the incubation medium. In contrast, AMD treatment did not significantly increase cell injury in response to PMA. The cytotoxic effects of TNF-alpha and PMA were reduced by addition of the isoform nonselective PKC antagonist GF-109203X to the incubation medium. The PKC-epsilon translocation inhibitor PKC-epsilon V1-2 significantly reduced cell damage induced by TNF-alpha , whereas the PKC-delta selective antagonist rottlerin reduced cell injury in response to PMA treatment. None of the other inhibitors tested produced statistically significant effects in this regard.


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Fig. 6.   Effect of TNF-alpha (A) or PMA (B) treatment on IEC-18 cellular cytotoxicity in the presence or absence of AMD. Cellular integrity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and recorded by spectrophotometry as indicated in MATERIALS AND METHODS. Degree of cytotoxicity was percentage of total number of cells used in each group. Some groups of cells were also treated with PKC inhibitors as indicated. Each bar of histogram is mean ± SE from identically treated groups of cells. P < 0.05: + significant difference compared with control groups of cells; ++ significant differences compared with TNF alone; * significant effects of the PKC inhibitors compared with the TNF-AMD or PMA-AMD groups, respectively, as assessed by analysis of variance and Duncan's multiple-range test.

Apoptosis was estimated by Hoechst 33258 nuclear staining to determine the incidence of nuclear condensation and fragments after TNF-alpha or PMA treatment (18 h) in the presence or absence of added AMD (Figs. 7 and 8). Many cells were detached from the culture dish by 24 h. TNF-alpha alone resulted in a small degree of nuclear fragmentation, an effect that was augmented by the addition of AMD. In contrast, the effect of PMA was not influenced by AMD addition (Figs. 7 and 8). GF-109203X and inhibition of PKC-epsilon translocation blocked the appearance of apoptotic nuclear fragments in TNF-alpha -AMD treated cells. Furthermore, apoptosis was also inhibited by treating cells with Gö-6976, a specific inhibitor of the conventional PKC isoforms. In the case of cells incubated in the presence of PMA and actinomycin D, the extent of apoptosis was ameliorated by inhibition of either PKC-delta activity (rottlerin) or PKC-epsilon translocation (PKC-epsilon V1-2). Preincubation of the cells with Z-DEVD-FMK also resulted in inhibition of apoptotic nuclear appearance after treatment with TNF-alpha and AMD.


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Fig. 7.   Effect of TNF-alpha (A) or PMA (B) on apoptotic index of IEC-18 cells in the presence or absence of AMD. Some groups of cells were also treated with PKC inhibitors as indicated. Apoptotic index is a percentage of 300 cells displaying nuclear condensation and fragmentation. A total of at least 300 cells were counted in this procedure. Data are means ± SE of 3 separate experiments each performed in triplicate. Z-DEVD-FMK, Z-Asp-(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-FMK. P < 0.05: + significant differences compared with control groups of cells; ++ significant differences compared with TNF alone; * significant effects of the PKC inhibitors compared with TNF-AMD or PMA-AMD groups, respectively, as determined by analysis of variance and Duncan's multiple-range test.



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Fig. 8.   Fluorescent micrographs of apoptosis of IEC-18 cells induced by TNF-alpha or PMA in the presence or absence of AMD. TNF- or PMA (in combination with AMD)-treated cells were also incubated with the PKC antagonist GF-109203X, PKC-epsilon V1-2 (for TNF), or rottlerin (for PMA). Nuclear condensation and fragmentation were detected by staining with Hoescht 33258, and cells were observed under a high-power fluorescent microscope (×40) with optical filter. Representative apoptotic cells are marked by arrows (magnification ×450).

The presence of nuclear fragments was also confirmed via theDNA fragmentation assay in response to TNF-alpha (Fig. 9). DNA fragmentation increased markedly in the TNF-AMD treated group of cells. The increase in DNA fragmentation was effectively blocked by preincubation of cells with the PKC inhibitor GF-109203X, the PKC-alpha inhibitor Gö-6976, the PKC-epsilon translocation inhibitor epsilon V1-2, and the caspase-3 inhibitor Z-DEVD-FMK.


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Fig. 9.   Effect of TNF-alpha in the presence or absence of AMD on DNA fragmentation of IEC-18 cells determined by ELISA for detection of 5-bromodeoxyuridine-labeled DNA fragments in cell culture supernatant as described in MATERIALS AND METHODS. Absorbance (Abs) values were measured at 450 nm. Various PKC and caspace-3 inhibitors were assessed against effect of TNF and AMD. Means ± SE absorbance values are from 4 identically treated cell groups. P < 0.05: ++ significant differences compared with control groups of cells; * significant effects of PKC or caspase inhibitors compared with the TNF- and AMD-treated group as determined by analysis of variance and Duncan's multiple-range test.

Caspases, a family of cysteine proteases, play a central role in initiating, amplifying, and executing apoptosis. We have examined the effect of TNF-alpha and phorbol ester on caspase-3 activity as well as the effects of PKC inhibition on this activity (Fig. 10). Consistent with the effects on nuclear fragmentation, AMD augmented the effects of TNF-alpha but not PMA on caspase-3 activation. The effects of TNF in combination with AMD on caspase activation were significantly reversed by pretreating cells with GF-109203X and PKC-epsilon translocation inhibitor while the effects of PMA were significantly reduced by the PKC-delta inhibitor rottlerin (Fig. 10). By themselves, none of the inhibitors tested here displayed any effects on cell viabiltiy or apoptosis.


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Fig. 10.   Effect of TNF-alpha (A) or PMA (B) treatement on specific activity of caspase-3 in IEC-18 cells in the presence or absence of AMD. Some groups of cells were also treated with PKC inhibitors as indicated. Caspase activity is pmol · µg cellular protein-1 · h-1. Values are means ± SE of 3 separate experiments each performed in triplicate. P < 0.05: + significant differences compared with control (untreated) cells; ++ significant differences compared with TNF- alone, * significant effects of the PKC inhibitors compared with TNF-AMD or PMA-AMD groups, respectively, as determined by analysis of variance and Duncan's multiple-range test.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TNF-alpha is a polypeptide cytokine that is considered to play a role in the pathogenesis of inflammatory bowel disease (2, 35). Elevated levels of TNF have been detected in mucosal biopsies from patients with inflammatory bowel disease (27, 35). Neutralization of TNF-alpha with specific monoclonal antibody has been shown to reduce the extent of inflammation in patients with Crohn's disease (46). Furthermore, evidence also indicates that TNF-alpha can also induce cell injury via necrosis or apoptosis in cells of the gastrointestinal tract (19, 48). In the present study, we have similarly demonstrated that TNF-alpha could induce cellular injury as assessed by a decrease in cellular metabolism and an enhancement of nuclear condensation and fragmentation, an index of apoptosis.

The present data also indicate that, by itself, TNF-alpha induced a small degree of cell injury but that level of damage could be enhanced by coincubation with the transcription inhibitor AMD. Previous studies have similarly indicated that cytokines, including TNF, by themselves were relatively ineffective in inducing cytotoxicity in intestinal epithelial cell lines (48). This confirms findings in other cell types treated with the cytokine and AMD (25) and suggests that the complete intracellular machinery needed for TNF to mediate apoptosis preexists in these cells and that new protein synthesis may play a role in determining the susceptibility of some cell types to the cytotoxic effects of TNF-alpha . The identity of the protein(s) that play a role in determining the sensitivity of intestinal epithelial cells to TNF-alpha -mediated challenge is unknown, although a number of these proteins have been associated with the susceptibility of cells such as hepatocytes and fibroblasts to TNF challenge and are believed to interfere with the steps in the signaling pathway leading from receptor activation to apoptosis (34).

The involvement of protein kinase C in the regulation of TNF-alpha -mediated processes has been documented in a number of cell types (26, 32, 49). However, its role in the regulation of TNF-alpha toxicity is uncertain and has been associated with cell injury as well as resistance of the cell to cytokine challenge (15). The data from this and previous studies suggest, however, that PKC activation in response to TNF-alpha is cytotoxic to intestinal epithelial cell lines. This confirms and extends our laboratory's previous findings in which direct activation of PKC activity in cells isolated from rat colonic mucosa resulted in cellular damage (44).

TNF-alpha -mediated challenge to various types of cells has been associated with changes in the activities of different PKC isoforms (23, 30, 32). Several lines of evidence suggest that individual PKC isozymes play distinct regulatory roles in cell growth, differentiation, and apoptosis in the intestine. In transformed nonepithelial cell lines, overexpression of constitutively active catalytic fragments of PKC-delta causes apoptosis (11). Weller et al. (47) have also demonstrated in colonic epithelial cells that concentrations of PMA that induced apoptosis also resulted in the translocation of PKC-delta from cytosol to membrane, whereas in other cell types the translocation of PKC-epsilon has been associated with cytotoxicity (32). Activation of PKC is associated with the translocation of enzymes from the cytosol to the cell particulate fraction. In the present study, TNF-alpha treatment resulted in the translocation of both PKC-delta and -epsilon from cytosol to membrane. This confirms previous findings in which TNF-alpha has been shown to induce translocation of PKC-delta and PKC-epsilon in HL-60 cells (40). Furthermore, we have also observed that the isoform selective translocation inhibitor PKC-epsilon V1-2 significantly attenuated the apoptotic and cytotoxic effects TNF-alpha on IEC-18 cells. In contrast to TNF, phorbol ester activation of intestinal epithelial cells resulted in the translocation of PKC-alpha , PKC-delta , and PKC-epsilon . However, inhibition of the PKC-delta isoform reduced the extent of cell injury as assessed by the MTT assay, whereas inhibition of PKC-epsilon translocation reduced the extent of apoptosis. These data suggest that the PKC-delta and -epsilon isoforms are important in the maintenance of cell integrity and in the regulation of apoptosis by different mechanisms as PKC-delta translocated primarily to the nuclear membrane and PKC-epsilon mainly to the cytoplasmic membrane. These isoforms of PKC have also been shown to play similar roles in the regulation of the integrity of other cell types, including hepatocytes and fibroblasts (18, 32). In contrast, PKC-epsilon has been associated with antiapoptotic effects in some studies (30). These differences in the effects of PKC isoforms on cell integrity have been attributed to cell type, cellular environment, and mechanism of apoptosis induction (16).

In the present study, we found that caspase-3 activity is evident in intestinal epithelial cells. This confirms and extends the findings of others (22). Caspase activation is required for the execution of cell death in an apoptotic manner (39). Similarly, in intestinal epithelial cells, TNF-alpha stimulation has only been associated with small or insignificant increases in caspase-3 activity (37, 48). However, we have observed that these levels are augmented in the presence of a protein synthesis inhibitor, suggesting that TNF-mediated increases in caspase-3 are under the regulation of an inhibitory protein. In contrast, protein synthesis inhibition did not augment the caspase-3 activity in response to PMA treatment of these cells. Furthermore, caspase-3 activation by TNF and PMA was reduced by PKC-epsilon or PKC-delta inhibition, respectively. It has been demonstrated that caspase-3 may be a direct or indirect target of activated PKC-delta in some cell types (36). This is confirmed in the present study by the demonstration that a selective inhibitor of this PKC isoform also reduced caspase-3 activation in response to PMA and suggests the importance of a caspase-3-PKC-delta pathway in the intestinal cellular apoptosis. It has also been reported that the activation of PKC-delta during apoptosis inhibited the activation of DNA-protein kinase, which is essential for repair of DNA and hence promotes DNA damage (4). Our data demonstrating PKC-delta translocation to the perinuclear membrane support this notion. Similarly, it was demonstrated that apoptosis in myeloid leukemia cells is associated with caspase-3 activation and changes in PKC-epsilon (21). Thus, whereas TNF and PMA appear to induce apoptosis via activation of different PKC isoforms, both agents appear to induce changes in the activation of caspase-3. This may represent a common mediatory route in the process of apoptosis.

In summary, our results indicate that TNF-alpha - and PMA-induced apoptosis in intestinal epithelial cells may be mediated by differential regulation of PKC isoforms and that these processes may occur through a caspase-dependent pathway. Further studies are needed to identify the factors that mediate processes downstream of PKC in caspase activation and apoptosis in these cells and in a human intestinal cell line to verify a generalized feature of host epithelial cell responses to TNF.


    ACKNOWLEDGEMENTS

This work was funded by the Medical Research Council of Canada Grant MT 6426.


    FOOTNOTES

Address for reprint requests and other correspondence: B. L. Tepperman, Dept. of Physiology, Medical Science Bldg., Rm. M226, University of Western Ontario, London Ontario, Canada N6A 5C1 (E-mail: btepperm{at}med.uwo.ca).

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.

Received 19 June 2000; accepted in final form 14 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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