Department of Physiology, University of Western Ontario, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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Tumor necrosis factor
(TNF)- 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-
.
In the present study, the role of PKC isoforms in TNF-
-mediated
cytotoxicity and apoptosis in intestinal cells was investigated
using the rat epithelial cell line, IEC-18. Cells were incubated with
TNF-
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-
also induced the
translocation of PKC-
, -
, and -
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-
translocation inhibitor, PKC-
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-
inhibitor. Caspase-3, an enzyme implicated in
the mediation of apoptosis, was activated in cells in response
to either TNF-
or PMA stimulation, and its effects on this activity
were reduced by selective inhibition of PKC-
and -
, respectively.
Furthermore, inhibition of caspase-3 activity reduced
apoptosis. These data suggest that activation of selective PKC
isoforms mediate the effects of TNF-
on intestinal epithelial cell injury.
IEC-18 cells; apoptosis; caspase-3; isoform translocation; phorbol ester; tumor necrosis factor-
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INTRODUCTION |
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TUMOR NECROSIS
FACTOR (TNF)- is a cytokine that is a member of the family of
proteins that comprises lymphotoxin-
and lymphotoxin-
(3,
12). TNF-
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-
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-
mRNA and protein than cells from control mice
(31). Administration of antibodies to TNF-
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-
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-
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-, 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
-
, -
1, -
2, and -
, which require calcium and are activated
by diacylglycerol and phorbol ester; the novel PKC isozymes like -
,
-
, -
, and -
, which are activated by diacylglycerol and phorbol
ester independently of calcium; and the atypical PKC isozymes like
-
, -
, and -
, 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- 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-
on intestinal epithelial cell apoptosis and injury and have
identified the role of PKC and the PKC isozyme(s) mediating these responses.
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MATERIALS AND METHODS |
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Materials.
Human recombinant TNF- was purchased from Upstate Biotechnology
(Lake Placid, NY). Affinity purified rabbit polyclonal antibodies against PKC isozymes-
, -
, -
, and -
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-
(rottlerin; 10 µM) (14),
PKC-
(myristoylated PKC-
V1-2 translocation inhibitor; 4 µM) (17), and PKC-
(myristolated PKC-
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-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- antibody
(1:1,000) and 3 h with PKC-
, PKC-
, and PKC-
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- 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-
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.
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RESULTS |
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IEC-18 cells strongly express PKC-, -
, -
, and -
proteins as determined by Western blot analysis (Fig.
1). Treatment of IEC-18 cells with
TNF-
resulted in changes in the abundance of specific isoform
protein (Fig. 1). The amount of PKC-
protein increased significantly
both in cytosolic and particulate fractions by 3 h after treatment
and continued up to 48 h after TNF-
addition. The expression of
PKC-
protein increased mainly in the particulate fraction during the
entire incubation period. PKC-
and -
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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Activation of the PKC- isoform was not observed after cells were
treated with PMA. However, translocation of PKC-
, -
, and -
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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Immunostaining for various PKC isoforms using specific antibodies to
these isozyemes revealed the localization of PKC-, -
, and -
in
the cytosol of untreated control cells and faint staining for PKC-
around the periphery of these cells (Fig.
3). After exposure to TNF-
for 18 h, PKC-
staining appeared to be intensified within both cytosolic
and membrane compartments. PKC-
appeared more intense in the
perinuclear region, whereas PKC-
staining decreased within the
cytosolic compartment and intensified within the cell periphery.
Similarly, staining for PKC-
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-
translocation inhibitor
PKC-
V1-2 blocked the appearance of translocation of PKC-
in
response to TNF-
, whereas the PKC activity inhibitor GF-109203X was
not effective in this regard (Fig. 5).
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The cytotoxic effects of PKC isoforms affected by TNF- or PMA
treatment are demonstrated in Fig. 6.
IEC-18 cells were incubated with PMA or TNF-
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-
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-
and PMA
induced an increase in the extent of cytotoxicity (Fig. 6). The effect
of TNF-
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-
and PMA were reduced by addition of the
isoform nonselective PKC antagonist GF-109203X to the incubation
medium. The PKC-
translocation inhibitor PKC-
V1-2
significantly reduced cell damage induced by TNF-
, whereas the
PKC-
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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Apoptosis was estimated by Hoechst 33258 nuclear staining to
determine the incidence of nuclear condensation and fragments after
TNF- 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-
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-
translocation blocked the appearance of apoptotic nuclear fragments in
TNF-
-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-
activity (rottlerin) or
PKC-
translocation (PKC-
V1-2). Preincubation of the cells
with Z-DEVD-FMK also resulted in inhibition of apoptotic nuclear
appearance after treatment with TNF-
and AMD.
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The presence of nuclear fragments was also confirmed via theDNA
fragmentation assay in response to TNF- (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-
inhibitor Gö-6976, the
PKC-
translocation inhibitor
V1-2, and the caspase-3 inhibitor Z-DEVD-FMK.
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Caspases, a family of cysteine proteases, play a central role in
initiating, amplifying, and executing apoptosis. We have examined the effect of TNF- 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-
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-
translocation inhibitor while the effects
of PMA were significantly reduced by the PKC-
inhibitor rottlerin
(Fig. 10). By themselves, none of the inhibitors tested here displayed
any effects on cell viabiltiy or apoptosis.
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DISCUSSION |
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TNF- 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-
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-
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-
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- 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-
. The identity of
the protein(s) that play a role in determining the sensitivity of
intestinal epithelial cells to TNF-
-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--mediated processes has been documented in a number of cell types (26, 32, 49). However, its role in the regulation of
TNF-
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-
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--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-
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-
from cytosol to membrane, whereas in other cell types the
translocation of PKC-
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-
treatment resulted in the
translocation of both PKC-
and -
from cytosol to membrane. This
confirms previous findings in which TNF-
has been shown to induce
translocation of PKC-
and PKC-
in HL-60 cells (40).
Furthermore, we have also observed that the isoform selective
translocation inhibitor PKC-
V1-2 significantly attenuated the
apoptotic and cytotoxic effects TNF-
on IEC-18 cells. In
contrast to TNF, phorbol ester activation of intestinal epithelial
cells resulted in the translocation of PKC-
, PKC-
, and PKC-
.
However, inhibition of the PKC-
isoform reduced the extent of cell
injury as assessed by the MTT assay, whereas inhibition of PKC-
translocation reduced the extent of apoptosis. These data
suggest that the PKC-
and -
isoforms are important in the
maintenance of cell integrity and in the regulation of
apoptosis by different mechanisms as PKC-
translocated
primarily to the nuclear membrane and PKC-
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-
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- 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-
or PKC-
inhibition, respectively. It has
been demonstrated that caspase-3 may be a direct or indirect target of
activated PKC-
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-
pathway in the intestinal cellular apoptosis. It has also been
reported that the activation of PKC-
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-
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-
(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-- 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.
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ACKNOWLEDGEMENTS |
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This work was funded by the Medical Research Council of Canada Grant MT 6426.
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FOOTNOTES |
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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.
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