1 Division of Digestive Diseases, Department of Internal Medicine, and Departments of Pharmacology and Molecular Physiology, Rush University Medical Center, Chicago, Illinois 60612; and 2 Institute of Human Nutrition, Columbia University, New York, New York 10032
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ABSTRACT |
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Using monolayers of
human intestinal (Caco-2) cells, we found that oxidants and ethanol
damage the cytoskeleton and disrupt barrier integrity; epidermal growth
factor (EGF) prevents damage by enhancement of protein kinase C (PKC)
activity and translocation of the PKC-1 isoform. To see if PKC-
1
mediates EGF protection, cells were transfected to stably over- or
underexpress PKC-
1. Transfected monolayers were preincubated with
low or high doses of EGF (1 or 10 ng/ml) or
1-oleoyl-2-acetyl-sn-glycerol [OAG; a PKC activator (0.01 or 50 µM)] before treatment with oxidant (0.5 mM
H2O2). Only in monolayers overexpressing
PKC-
1 (3.1-fold) did low doses of EGF or OAG initiate
protection, increase tubulin polymerization (assessed by quantitative
immunoblotting) and microtubule architectural integrity (laser scanning
confocal microscopy), maintain normal barrier permeability (fluorescein
sulfonic acid clearance), and cause redistribution of PKC-
1 from
cytosolic pools into membrane and/or cytoskeletal fractions (assessed
by immunoblotting), thus indicating PKC-
1 activation. Antisense inhibition of PKC-
1 expression (
90%) prevented these changes and
abolished EGF protection. We conclude that EGF protection against
oxidants requires PKC-
1 isoform activation. This mechanism may be
useful for development of novel therapies for the treatment of
inflammatory gastrointestinal disorders including inflammatory bowel disease.
tubulin; cytoskeleton; growth factors; paracellular permeability; fluorescein sulfonic acid clearance; Caco-2 cells; protein kinase C transfection, epidermal growth factor
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INTRODUCTION |
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A CRITICAL CHARACTERISTIC of the epithelium of the gastrointestinal (GI) mucosa is its ability to maintain a highly selective permeability barrier. This barrier normally restricts the passage of harmful proinflammatory and toxic molecules (e.g., bacterial endotoxin) into the mucosa and systemic circulation. Loss of mucosal barrier integrity, on the other hand, is characteristic of inflammatory bowel disease (IBD), necrotizing enterocolitis, and a variety of other GI disorders (e.g., gastric mucosal injury induced by ethanol) as well as several systemic disorders (e.g., alcoholic liver disease) (28, 35, 36). These disorders are also associated with oxidative tissue injury. The latter is of significant clinical importance because conditions of oxidative stress are common; they can cause mucosal barrier dysfunction and the initiation and/or perpetuation of mucosal inflammation and injury and have been implicated in the GI disorders mentioned above (28, 35, 36).
Because enhancing our knowledge of endogenous GI barrier protective
mechanisms might provide insights into developing more effective
treatment regimens for these oxidative inflammatory disorders, we have
been investigating the protective mechanisms used by growth factors.
Using monolayers of human Caco-2 cells exposed to oxidants as a model
of barrier disruption, we previously found that epidermal growth factor
(EGF) and transforming growth factor- protect intestinal barrier
integrity by stabilizing the microtubule cytoskeleton
(5-7), in large part by increasing the activity of
protein kinase C (PKC) (7). In other studies
(5-10), we have shown that the stability of the
cytoskeleton is key in mucosal healing under in vivo as well as in
vitro conditions. Because PKC consists of a family of at least 12 different serine/threonine kinases of fundamental importance in signal
transduction (16, 57, 58), we investigated which isoforms
mediate EGF protection in intestinal cells. Intestinal epithelial
cells, including Caco-2 cells, express at least five of these isoforms:
PKC-
, PKC-
1, PKC-
2, PKC-
, and PKC-
(1, 7, 16, 42,
58, 64). These isoforms differ in their activation, tissue
expression, intracellular distribution, and substrate specificity,
suggesting that each isozyme has a unique, nonredundant role in signal
transduction (1, 30, 32, 41, 44, 46, 52, 54, 55). Our
recent observations of naive Caco-2 cells suggest that EGF induces the membrane association (activation) of an abundant isoform of PKC, PKC-
1 (7). In the current investigation, using
molecular biological approaches (i.e., transfection), we tested the
hypothesis that EGF-induced protection against oxidant injury to both
the microtubule cytoskeleton and the barrier integrity of epithelial
monolayers depends on translocation and activation of the PKC-
1 isoform.
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MATERIALS AND METHODS |
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Cell culture. Caco-2 cells, which were obtained from the American Type Culture Collection (ATCC, Manassas, VA) at passage 15, were chosen because they form monolayers that morphologically resemble small intestinal cells, with defined apical brush borders, junctional complexes, and a highly organized microtubule network (10, 21, 50). Cells were maintained at 37°C in complete DMEM in an atmosphere of 5% CO2 with 100% relative humidity. Naive-type cells or transfected cells (see Stable transfection) were split at a ratio of 1:6 on reaching confluence and were set up in 6- or 24-well plates for experiments or in T-75 flasks for propagation. Cells grown for the barrier function experiments were split at a ratio of 1:2 and seeded at a density of 200,000 cells/cm2 into 0.4 µM BioCoat collagen I cell culture inserts (0.3-cm2 growth surface; Becton Dickinson Labware, Bedford, MA), and experiments were performed at least 7 days postconfluence. The medium was changed every 2 days. The utility and characterization of this cell line has been reported previously (10, 21, 50).
Plasmids.
The sense and antisense plasmids of PKC-1 were constructed as
previously described (16). Expression was controlled by
the
-actin promoter, which is known to be expressed in Caco-2 cells (50). The antisense PKC-
1 plasmid (p
-actin
SP72-As-PKC-
1) was constructed by ligating the 2.3-kb
EcoR I fragment of PKC-
1 cDNA from pJ6-PKC-
1
(16) into the unique EcoR I sites of the p
-actin SP72 vector. The antisense orientation of the plasmid was
confirmed by SamI restriction digestion (16).
Stable transfection.
Cultures of Caco-2 cells grown to 50-60% confluence were
cotransfected with G418 resistance plasmid and expression plasmids encoding either PKC-1 or antisense PKC-
1 with the use of
LIPOFECTIN (GIBCO). Control conditions included vector alone. Briefly,
cells were incubated for 16 h at 37°C with the plasmid DNA in
serum-free medium in the presence of LipofectAMINE (25 µl/25-cm2 flask). Subsequently, the DNA-containing
solution was removed and replaced with fresh medium containing 10%
fetal bovine serum (FBS) to relieve cells from the shock of exposure to
the serum-free medium. After transfection, cells were subjected to G418
selection (0.6 mg/ml) over 4 wk. Resistant cells were maintained in
DMEM-FBS and 0.2 mg/ml of G418 (selection medium). PKC protein
expression or the lack of it was verified by Western blot analysis of
cell lysates (see Fractionation and Western immunoblotting of
PKC). Multiple clones stably overexpressing PKC-
1 or lacking
PKC-
1 were assessed by immunoblotting, plated on Transwell cell
culture inserts, and allowed to form confluent monolayers that were
subsequently used for experiments.
Experimental design.
In the first series of experiments, postconfluent monolayers of naive
Caco-2 cells were preincubated with EGF (1 or 10 ng/ml) or isotonic
saline for 10 min and then exposed to oxidant (0.5 mM
H2O2) or vehicle (saline) for 30 min. As we
have previously shown (5-7),
H2O2 at 0.5 mM disrupts microtubules and
barrier integrity; EGF at 10 ng/ml (but not 1 ng/ml) prevents this
disruption. These experiments were then repeated with cell monolayers
that were either stably overexpressing or almost completely lacking PKC-1. Reagents were applied on the apical side of the monolayers unless otherwise indicated. Because our previous studies (6, 7) showed that regardless of whether apical or basolateral exposure of oxidants was used the results were qualitatively similar, all current studies used apical application. In all experiments, barrier function, microtubule cytoskeletal stability (cytoarchitecture, tubulin assembly/disassembly), and PKC-
1 subcellular distribution were assessed. Additionally, because our previous studies demonstrated that protection is observed only when the protective agent (e.g., EGF
or a PKC activator) is added before exposure to the damaging agent
(e.g., H2O2 or ethanol), all of the
current experiments followed this preincubation protocol
(5-7, 10).
Fractionation and Western immunoblotting of PKC.
Differentiated cell monolayers grown in 75-cm2 flasks were
processed for the isolation of the cytosolic, membrane, and
cytoskeletal fractions as previously described by others and by us
(1, 7, 16). Briefly, after treatments, postconfluent
monolayers were scraped and ultrasonically homogenized in
Tris · HCl buffer [20 mM Tris · HCl (pH 7.5), 0.25 mM
sucrose, 2 mM EDTA, 10 mM EGTA, and 2 µg/ml each of aprotinin,
pepstatin, leupeptin, and phenylmethylsulfonyl fluoride (PMSF)]. The
homogenates were then ultracentrifuged (100,000 g for 40 min
at 4°C), and the supernatant was removed and used as a source of the
cytosolic fraction. Next, pellets were washed with 0.2 ml of
Tris · HCl buffer, resuspended in 0.8 ml of a buffer containing
0.3% Triton X-100, and maintained on ice for 1 h. The samples
were then centrifuged (100,000 g for 1 h at 4°C), and the supernatant was used as the source of the membrane fraction. To
this remaining pellet, 0.3 ml of cold (4°C) lysis buffer (150 mM
NaCl, 50 mM Tris · HCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, and 2 µg/ml each of aprotinin, pepstatin, leupeptin, and PMSF) were added. The samples were then placed on ice for 1 h and ultracentrifuged as above. The remainder of the lysate or Triton-insoluble cytoskeletal fraction was then removed. Protein content of the various cell fractions was assessed by
the Bradford method (15). For total PKC extraction, which provided the fraction used to assess total PKC-1 expression, scraped
monolayers were placed directly in 1.5 ml of cold lysis buffer and
subsequently ultracentrifuged as described above. The supernatant was
used for bulk protein determination.
Immunofluorescent staining and high-resolution LSCM of
microtubules.
Cells from monolayers were fixed in cytoskeletal stabilization buffer
and then postfixed in 95% ethanol at 20°C as we previously described (5-7, 9, 10). Cells were subsequently
processed for incubation with a primary antibody, monoclonal mouse
anti-
-tubulin antibody (IgG1, rat/human reactive; Sigma, St. Louis,
MO) at 1:200 dilution for 1 h at 37°C. Slides were washed three
times in Dulbecco's phosphate-buffered saline (D-PBS) and then
incubated with a secondary antibody (FITC-conjugated goat anti-mouse;
Sigma) at a 1:50 dilution for 1 h at room temperature. Slides were
washed thrice in D-PBS, once with deionized H2O, and
subsequently mounted in Aquamount. All antibodies were diluted with
D-PBS containing 0.1% BSA. After staining, cells were observed with an
argon laser (
= 488 nm) with a ×63 oil immersion
plan-apochromat objective (NA 1.4, Zeiss). Single cells and/or a clump
of two or three cells from desired areas of the monolayers were
processed with image processing software on a Zeiss ultra
high-resolution laser scanning confocal microscope to create "neat
black" areas surrounding the cells. The cytoskeletal elements were
examined in a blinded fashion for their overall morphology,
orientation, and disruption as we previously described (5-7,
9, 10, 66). The slides were decoded only after examination was complete.
Microtubule (tubulin) fractionation and quantitative
immunoblotting of tubulin assembly and disassembly.
Polymerized (S2) and monomeric (S1) fractions of tubulin were isolated
as we previously described (5-7, 10). Cells were gently scraped and pelleted with centrifugation at low speed (700 rpm,
7 min, 4°C) and resuspended in microtubule stabilization-extraction buffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% DMSO, 1 mM
MgSO4, 10 µg/ml of anti-protease cocktail, 1 mM EGTA, and
1% Triton X-100) at room temperature for 20 min. Tubulin fractions
were separated after a series of centrifugation and extraction steps.
Specifically, cell lysates were centrifuged at 105,000 g for
45 min at 4°C, and the supernatant containing the soluble monomeric
pool of tubulin (S1) was gently removed. The remaining pellet was then
resuspended in 0.3 ml of a Ca2+-containing depolymerization
buffer (0.1 M PIPES, pH 6.9, 1 mM MgSO4, 10 µg/ml of an
anti-protease cocktail, and 10 mM CaCl2) and incubated on
ice for 60 min. Subsequently, samples were centrifuged at 48,000 g for 15 min at 4°C, and the supernatant (S2 fraction or
cold Ca2+-soluble fraction) was removed. To ensure the
complete removal of the S2 fraction, the remaining pellet was treated
with the Ca2+-containing depolymerization buffer twice more
by resuspension and centrifugation. The "microtubules" were
recovered by separately incubating (at 37°C for 30 min) the S1 and S2
fractions with the stabilizing agents taxol (10 µM) and GTP (1 mM) in microtubule stabilization buffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% DMSO, 10 µg/ml of anti-protease cocktail, 1 mM
EGTA, 1 mM MgCl2, and 1 mM GTP) to promote the
polymerization of tubulin. Tubulin was then recovered by centrifugation
and resuspended in the stabilization buffer. Fractionated S1 and S2
samples were then flash-frozen in liquid N2 and stored at
70°C until being immunoblotted. For immunoblotting, samples (5 µg
protein/lane) were placed in a standard SDS sample buffer, boiled for 5 min, and then subjected to PAGE on 7.5% gels. Procedures for Western
blotting were performed as previously described (5-7,
10). To quantify the relative levels of tubulin, the optical
density of the bands corresponding to immunoradiolabeled tubulin were
measured with a laser densitometer.
Immunoprecipitation and Western blot analysis of tubulin
phosphorylation.
After the treatments, confluent cell monolayers were lysed by
incubation for 20 min in 500 µl of cold lysis buffer (20 mM Tris · HCl, pH 7.4, 150 mM NaCl, 10 µg/ml of the
anti-protease cocktail, 10% glycerol, 1 mM sodium orthovanadate, 5 mM
NaF, and 1% Triton X-100). The lysates were clarified by
centrifugation at 14,000 g for 10 min at 4°C. For
immunoprecipitation, the lysates were incubated for 4 h at 4°C
with monoclonal anti--tubulin (1:50 dilution, in excess). The
extracts were then incubated with protein G plus Sepharose 4B (Zymed,
South San Francisco, CA) for 2 h at 4°C. The immunocomplexes
were collected by centrifugation (2,500 g for 5 min) in a
microfuge tube and were washed three times with immunoprecipitation
buffer containing 5 mM Tris · HCl, pH 7.4, and 0.2% Triton
X-100. The resultant pellets were resuspended in a standard SDS sample
buffer and boiled at 95°C for 5 min before separation by PAGE as
previously described (5-7, 10). Gels were transferred
to nitrocellulose membranes, blocked with 1% BSA and 0.01% Tween 20 in PBS for blotting by polyclonal anti-phosphoserine (1:3,000 dilution;
Transduction Labs, Lexington, KY) and for detection of immune complexes
by horseradish peroxidase-conjugated secondary antibody, incubated with
chemiluminescence reagents, and autoradiographed for analysis by densitometry.
Determination of barrier permeability by fluorometry.
Barrier integrity was determined by a widely used and validated
technique that measures the apical-to-basolateral paracellular flux of
a fluorescent marker, fluorescein sulfonic acid (FSA; 200 µg/ml, 478 Da) as we (5-8) and others (11, 12, 22, 27, 31,
34, 39, 45, 47, 49, 57, 59-61) have described. This
fluorescent tracer is a lipophobic moiety and is known to be cell
membrane impermeant, moving instead through the paracellular space
(11, 19, 22, 27, 31, 34, 39, 42, 45, 57, 59-61). In
earlier permeability studies, we found that dextrans of higher
molecular mass (up to 70 kDa) yielded similar results. Briefly, fresh
phenol-free DMEM (800 µl) was placed into the lower (basolateral)
chamber, and phenol-free DMEM (300 µl) containing FSA was placed in
the upper (apical) chamber. Aliquots (50 µl) were obtained from the
upper and lower chambers at time 0 and at several subsequent
time points (e.g., 0, 10, 20, 30, and 40 min) and transferred to clear
96-well plates (clear bottom, Costar, Cambridge, MA). Fluorescent
signals from the samples were quantitated with a fluorescence
multiplate reader (FL 600, Bio-Tek Instruments). The excitation and
emission spectra for FSA were excitation = 485 nm and
emission = 530 nm. The paracellular permeability of monolayers was
expressed as clearance (Cl), calculated as the apical-to-basolateral
flux of the FSA probe divided by the concentration of probe in the
apical chamber. Clearance was expressed as nanoliters per hour per
square centimeter. To calculate Cl, we used the following formula: Cl
(nl · h1 · cm
2) = Fab/([FSA]a × S), where Fab is the
apical-to-basolateral flux of FSA (light units per hour),
[FSA]a is the concentration at baseline (light units per
nanoliter), and S is the surface area (0.3 cm2,
which is the growth surface for monolayers). Because both the basal
permeability of the monolayers and the magnitude of the effect of
oxidants and protective agents on permeability varied from experiment
to experiment, this variability was controlled by including
simultaneously run controls (e.g., vehicle/isotonic saline, oxidant,
and EGF-pretreated) with each experiment.
Statistical analysis. Data are presented as means ± SE. All experiments were carried out with a sample size of at least 4-6 observations/group. Statistical analysis comparing treatment groups was performed with ANOVA followed by Dunnett's multiple-range test (25). Correlational analyses were done with the Pearson test for parametric analysis or, when applicable, the Spearman test for nonparametric analysis. P values <0.05 were deemed statistically significant.
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RESULTS |
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Pretreatment of monolayers of naive-type Caco-2 cells with EGF and
PKC activators (e.g., OAG) protected the monolayer barrier against
H2O2-induced injury, a finding that confirmed
our previous observations (6, 7). We also confirmed in
these naive cells that EGF and PKC activators activated the PKC-1
isoform via its rapid translocation from the cytosol to membrane-bound
fractions (data not shown). In the current investigation, with
molecular interventions (transfection), the role of PKC-
1 was
further examined.
Stable overexpression of PKC-1 isoform after transfection of
intestinal cells.
Caco-2 cells were cotransfected with cDNA encoding both G418 resistance
(for selection) and PKC-
1. Cell lysates of confluent monolayers were
prepared from these transfected cells and then analyzed by Western
immunoblotting. Figure
1A shows
overexpression of the PKC-
1 isozyme in transfected cells (data for 4 µg of DNA plasmid shown). The PKC-
1 isolated from transfected
cells comigrated with a known standard (~78 kDa) of PKC-
1 from rat
brain lysates. The immunoblot shown in Fig. 1B demonstrates
that total PKC-
1 levels were elevated by 3.1-fold compared with
those in naive cells.
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Effects of EGF and PKC activator (OAG) on the intracellular
translocation and activation of overexpressed PKC-1 in transfected
Caco-2 monolayers.
Cytosol-, membrane- and cytoskeleton-associated fractions containing
PKC-
1 from transfected Caco-2 cells were isolated and assessed by
Western immunoblotting. After preexposure to low doses of EGF or OAG
(Fig. 6, A-E), there was
a rapid redistribution of PKC-
1 isoform from a mostly cytosolic
distribution into both the membrane and cytoskeletal fractions.
Translocation of the 78-kDa overexpressed PKC-
1 protein to these
particulate fractions (Fig. 6B) was readily observable as
early as 2.5 min after low doses of EGF and became undetectable in the
cytosolic fraction (Fig. 6A) by 10 min, indicating nearly
total activation of PKC-
1. OAG caused identical effects (particulate
fraction, Fig. 6D; cytosolic fraction, Fig. 6C).
We found rapid translocation of native PKC-
1 to particulate
fractions of naive cells only after higher doses of EGF or OAG (data
not shown), which confirmed our previous findings (7).
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Stable antisense inhibition of PKC-1 and its inhibitory effects
on EGF-mediated protection.
To demonstrate a key role for PKC-
1 in EGF-induced protection by an
independent method, naive Caco-2 cells were transfected with PKC-
1
antisense plasmid (4 µg shown in Fig.
8) and cDNA encoding G418
resistance. Figure 8A, which is an immunoblot of cell lysates, shows that this manipulation substantially and stably diminished the steady-state levels of PKC-
1 protein by ~90%.
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DISCUSSION |
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With the use of monolayers of intestinal epithelial cells as a
model of GI barrier integrity, we conclude that the 1 isoform of PKC
is critical to EGF-induced protection against oxidant-induced damage to
the microtubule cytoskeleton and to barrier integrity. This suggests
that the PKC-
1 isoform is a key intracellular regulator of
epithelial barrier integrity. Several independent lines of evidence
support these conclusions.
Transfected cells that overexpress PKC-1 are severalfold more
sensitive to protection by EGF. They are equally more sensitive to the
PKC activator OAG. These observations are consistent with our previous
findings in naive cells (5-7) that showed that
increases in overall PKC activity mediate EGF-induced protection.
PKC-
1 overexpression synergized with the addition of EGF or OAG to
enhance the stability of polymerized tubulin, reduce the unstable
monomeric tubulin, maintain a significantly higher percentage of Caco-2 cells with intact microtubules, and stabilize monolayer barrier integrity at almost normal levels. This increased sensitivity appears
to require not only overexpression, which by itself is not protective,
but also activation/translocation of PKC-
1. As in naive cells,
protection required activation through the translocation of PKC-
1
from the cytosolic to the particulate fractions. The increased
sensitivity did not appear to be due to a decrease in the ability of
oxidants to cause damage because transfected cells (in the absence of
EGF or OAG) were damaged to the same extent as naive cells; they showed
similar loss of barrier integrity, as measured by FSA clearance, and
similar degrees of damage and disruption of the microtubule
cytoskeleton, as assessed by examination of microtubule
cytoarchitecture and tubulin polymerization.
Robust and statistically significant correlations between several
measures of outcome further supported our conclusion. These included associations between increasing PKC-1
translocation/activation on the one hand and, on the other hand,
increasing protection against barrier disruption, tubulin disassembly,
and microtubule disruption. These findings are consistent with our
model showing that increased translocation/activation of PKC-
1 leads
to increased tubulin assembly, which leads to increased microtubule
stability, which, in turn, leads to increased monolayer integrity. For
example, this conclusion is supported by a correlation
(r = 0.91; P < 0.05) that shows that
increases in PKC-
1 translocation decrease FSA clearance (Fig.
6F). The same conclusion is reached when either tubulin
assembly (r = 0.92; P < 0.05) or the
percentage of normal microtubules (r = 0.95;
P < 0.05) is used as a marker of integrity. The high
strength of these correlations, which explains 80-95% of the
variance, suggests that PKC-
1 activation is critical to the
protective effects of EGF (and OAG) on microtubule and barrier function. These results also confirmed our earlier reports
(5-7) in naive cells in which we found a significant
correlation between the integrity of barrier permeability and
microtubule stability (r = 0.98; P < 0.05) and between the integrity of barrier permeability and total PKC
activation (r = 0.94; P < 0.05). This
mechanism is also consistent with previous studies that have documented that PKC translocation/activation is necessary for the observed effects
of specific PKC isotypes (13, 14, 23, 42, 58, 64).
Another major result that corroborates our conclusion is our finding
that cells that are transfected with antisense to PKC-1 and that
underexpress PKC-
1 (at 10% of normal levels) were rendered severalfold less sensitive to the protective effects of EGF and OAG. In
these cells, EGF and OAG were unable to enhance tubulin assembly,
stabilize the microtubule cytoskeleton, or maintain monolayer barrier
integrity. The reduced sensitivity did not appear to be a result of a
loss of the ability of oxidants to cause damage because transfected
cells (without EGF or OAG) were damaged to the same extent as naive cells.
Other quantitative considerations further support our conclusion that
activation of the 1 isoform of PKC can explain, at least in large
part, EGF-induced protection. First, the maximum protection afforded by
EGF in PKC-
1-overexpressing cells (90%) is essentially the same as
the maximum protection afforded by EGF in naive cells (94%). Second,
as we found in a previous study (7), OAG was slightly less
effective as a protective agent (84%). Third, pretreatment with a
different PKC activator [30 nM 12-O-tetradecanoylphorbol
13-acetate (TPA), a phorbol ester] elicited approximately the
same level of protection (85%) (7). Fourth, protection of
a similar magnitude was observed when protection of the microtubule
cytoskeleton was the measure of outcome. When microtubule integrity was
measured as the percentage of cells with normal microtubules,
protection by EGF and OAG in PKC-
1-overexpressing cells was,
respectively, 88 and 83%. This is comparable to protection of
microtubules by high doses of EGF (10 ng/ml) and OAG (50 µM) in naive
cells, which was 89 and 82%, respectively. A similar parallelism was
found when tubulin assembly [(S2)/(S2 + S1)] was the measure of
outcome. These data indicate that a significantly large portion of
protection against oxidant insult is mediated through PKC-
1. It
should be noted, however, that other PKC isoforms may also contribute
to the protective effects of EGF, a question that merits further study.
Our previous studies showed that protection against damage and
disruption to the cytoskeleton protects barrier integrity. However, the
mechanism through which PKC-1 protects the cytoskeleton is not
known. Our previous and present studies suggest three possibilities: 1) decreasing oxidative stress, 2) normalizing
cytosolic calcium, and 3) phosphorylation of tubulin.
Activation of the
1 isoform of PKC may trigger one or more of these
mechanisms. Regarding the first mechanism, we have reported that
EGF/PKC prevents oxidant-induced upregulation of an inducible nitric
oxide synthase-driven pathway (5-8). Pretreatments
such as EGF and OAG that increase overall PKC activity are associated
with 1) inhibition of the ability of oxidants to upregulate
this inducible nitric oxide synthase pathway and its reaction products,
nitric oxide and peroxynitrite; and 2) prevention of
nitration, carbonylation, and disassembly of tubulin, three oxidative
mechanisms that are required for oxidant-induced disruption of
microtubules and monolayer barrier integrity (5-8). Regarding the second mechanism, we have shown that activation of PKC by
known PKC activators (e.g., OAG and TPA) attenuates oxidant-induced
increases in cytosolic calcium through stimulation of calcium efflux.
This, in turn, leads to the normalization of intracellular calcium and
prevention of oxidant-induced loss of cytoskeletal and barrier
integrity (7). These effects on cell calcium trafficking
are likely to be important because we (7) and others
(2, 40) have shown that the cytoskeleton is exquisitely sensitive to alterations in intracellular calcium homeostasis and that
it can be extensively disrupted by oxidant-induced increases in
intracellular calcium.
Our current data suggest a possible third mechanism: protein
phosphorylation of tubulin. These data show that EGF and the PKC
activator OAG cause an increase in serine phosphorylation of the
tubulin subunits of the microtubules. This increase was prevented by
PKC-1 antisense DNA, suggesting that PKC may be acting
directly or indirectly on these cytoskeletal protein subunits. This
phosphorylation mechanism is consistent with previous studies. For
example, PKC has been implicated in rearrangement of the cytoskeleton (2, 17, 23, 26), although it is not clearly known which PKC isoforms are key in this process. Recent reports have proposed that
PKC is capable of phosphorylating the cytoskeletal proteins talin and
vinculin (23). Furthermore, a major specific substrate for
PKC, myristoylated alanine-rich PKC substrate (MARCKS), has been
proposed as an actin cytoskeletal remodeler (26).
Specifically, the actin cytoskeletal organizing activity of MARCKS is
inhibited by PKC-mediated phosphorylation. Alternatively, PKC-
1 may
phosphorylate one of the tubulin-associated capping proteins (e.g.,
microtubule-associated proteins). Further studies will be
needed to explore the nature of the interactions between PKC-
1 and
the cytoskeleton in GI epithelial cells.
Our findings on PKC-1 are consistent with previous reports by other
investigators. EGF activates constitutive PKC in many naive
nonepithelial and epithelial cell types (4, 13, 56, 64,
65), including canine and human gastric cells (64,
65) and human colonic epithelial cells (4, 7).
Additionally, the translocation of PKC isoforms from the cytosolic to
the particulate fraction of the cell is a key step in their activation
(13, 14, 23, 42, 58, 64). Furthermore, OAG induces
activation of constitutively expressed PKC-
1 in non-GI cellular
models such as fibroblasts (23).
It should be noted, however, that the effects of PKC activation in
cellular models can sometimes be complex and may vary with different
experimental settings and cell types (3, 20, 38, 48). For
instance, a previous study reported that overexpression of PKC-
caused disruption of pig kidney epithelial (LLC-PK1) monolayers
(48). To the best of our knowledge, our current findings are the first to report that a specific PKC isoform plays an essential role in protection of GI cells.
Although our studies were designed to investigate possible beneficial
effects of EGF, PKC, and PKC-1 activation on protection of the GI
tract and our findings are consistent with many other published
studies, there do exist reports that PKC may have other effects that
are not beneficial. These include the suspected role of PKC and tumor
promoters (e.g., phorbol esters) in carcinogenesis (20, 33, 48,
63) as well as in barrier hyperpermeability (48).
The existence of a wide array of effects of PKC, some noxious, is one
reason that we focused our studies on specific PKC isoforms that
mediate EGF protection because it is possible, as our data suggest,
that activating or mimicking just one or a few PKC isoforms will have a
much higher beneficial/therapeutic index than activating and/or
mimicking total PKC activity.
It should be noted that in the current investigation we chose to use well-established and widely used fluorescent probes such as FSA for assessing intestinal monolayer permeability for several reasons. Fluorescently labeled dextrans (DF) and sulfonic acid (FSA) compounds (or radiolabeled agents) offer appropriate choices for epithelial flux assays. First, these probes are convenient because they come in a range of sizes, are nontoxic to cells, are membrane impermeable, and are relatively inexpensive (5-8, 11, 22, 27, 31, 34, 39, 45, 47, 57, 59-62). Second, both in vivo and in vitro studies have revealed that these probes move through the paracellular space (47, 57, 59, 60, 62). Third, the probes are nontoxic lipophobic moieties and are considered to be cell membrane impermeable so that their permeation of cell monolayers must be via the paracellular route. Indeed, our current data (Fig. 2D) support the aforementioned points. Accordingly, these probes can and have been used as tracers to examine many aspects of endothelial and epithelial permeability (5-8, 11, 22, 27, 31, 34, 39, 45, 47, 57, 59-62). Indeed, it is not surprising that the FSA and DF probes have been widely used by numerous groups studying GI inflammation (5-8, 18, 24, 33, 51, 62). For example, these probes were used to assess barrier integrity against lipopolysaccharide-induced mucosal inflammation (62). Similarly, in other studies, these fluorescent probes were used to assess Caco-2 monolayer hyperpermeability (5-8, 22, 31, 34, 45, 59-61).
We recognize that Caco-2 cells are a transformed cell line and that
tumor cells may respond differently to PKC-1 than do nontransformed
cells, including enterocytes, in native tissue. Nonetheless, our
findings now provide a rationale for conducting studies that test
whether these same protective mechanisms occur in vivo in animal models
and humans. They also provide a rationale for considering a strategy in
which agents that target PKC-
1 (e.g., PKC-
1 mimetics or targeted
gene therapy such as the delivery of sense vector for the PKC-
1
isoform) might lead to development of novel and improved therapies for
GI disorders related to free radical damage, such as IBD. These
approaches might maintain epithelial integrity under conditions of
oxidative stress and prevent the episodic attacks that recur under
proinflammatory oxidative conditions in ulcerative colitis (28,
29, 36, 37, 43, 53). Further studies will be needed to assess
the importance of PKC-
1 in GI tissues as well as in other cell models.
In summary, our data provide strong support for the idea that PKC-1
can influence the dynamics of microtubule cytoskeleton assembly and
intestinal barrier integrity and that it is key for the occurrence of
growth factor protection under in vitro conditions in Caco-2 monolayers.
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ACKNOWLEDGEMENTS |
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This work was supported, in part, by a grant from the Department of Internal Medicine, Rush University Medical Center, and by a grant from the American College of Gastroenterology.
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FOOTNOTES |
---|
This work was presented, in part, at the annual meeting of the American Gastroenterological Association, May 2001.
Address for reprint requests and other correspondence: A. Banan, Rush Univ. Medical Center, Div. of Digestive Diseases, 1725 W. Harrison, Suite 206, Chicago, IL 60612 (E-mail: ali_banan{at}rush.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 December 2000; accepted in final form 21 April 2001.
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