1 Departments of Internal Medicine (Division of Digestive Diseases), 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 showed that epidermal growth factor (EGF)
protects intestinal barrier integrity against oxidant injury by
protecting the microtubules and that protein kinase C (PKC) is
required. Because atypical PKC- isoform is abundant in wild-type
(WT) Caco-2 cells, we hypothesized that PKC-
mediates, at least in
part, EGF protection. Intestinal cells (Caco-2 or HT-29) were
transfected to stably over- or underexpress PKC-
. These clones were
preincubated with low or high doses of EGF or a PKC activator
[1-oleoyl-2-acetyl-sn-glycerol (OAG)] before oxidant (0.5 mM H2O2). Relative to WT cells exposed to
oxidant, only monolayers of transfected cells overexpressing PKC-
(2.9-fold) were protected against oxidant injury as indicated by
increases in polymerized tubulin and decreases in monomeric tubulin,
enhancement of architectural stability of the microtubule cytoskeleton,
and increases in monolayer barrier integrity toward control levels (62% less leakiness). Overexpression-induced protection was OAG independent and even EGF independent, but EGF significantly potentiated PKC-
protection. Most overexpressed PKC-
(92%) resided in
membrane and cytoskeletal fractions, indicating constitutive activation of PKC-
. Stably inhibiting PKC-
expression (95%) with antisense transfection substantially attenuated EGF protection as demonstrated by
reduced tubulin assembly and increased microtubule disassembly, disruption of the microtubule cytoskeleton, and loss of monolayer barrier integrity. We conclude that 1) activation of PKC-
is necessary for EGF-induced protection, 2) PKC-
appears
to be an endogenous stabilizer of the microtubule cytoskeleton and of
intestinal barrier function against oxidative injury, and 3)
we have identified a novel biological function (protection) among the
atypical isoforms of PKC.
cytoskeleton; growth factors; epidermal growth factor; Caco-2 cells; gut barrier; protection; transfection; protein kinase C isoforms; inflammatory bowel disease
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INTRODUCTION |
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A FUNDAMENTAL PROPERTY OF epithelial cells of the gastrointestinal (GI) tract is to function as a highly selective permeability barrier, permitting the absorption from the lumen of nutrients, water, and electrolytes but restricting passage of harmful proinflammatory and toxic molecules (e.g., immunoreactive antigens, endotoxin) into the mucosa or the systemic circulation. Loss of mucosal barrier integrity has been implicated in the pathogenesis of multiple organ system dysfunction, inflammatory bowel disease (IBD), necrotizing entercolitis, ethanol- and nonsteroidal anti-inflammatory drug (NSAID)-induced chemical injury, and a variety of other GI disorders as well as several systemic disorders (e.g., alcoholic liver disease) (5, 30, 37). Pathogenesis of mucosal barrier dysfunction in these disorders remains poorly understood, but several studies, including our own (5-7, 9, 15, 17, 37), have shown that chronic gut inflammation is associated with high levels of reactive oxygen metabolites and that oxidants appear to be a key underlying cause of injury (31, 39, 42, 59). Oxidative injury is of clinical importance not only because reactive oxygen metabolites are common in inflammation but also because they can cause mucosal barrier hyperpermeability and, in turn, lead to the initiation and/or perpetuation of mucosal inflammation and injury (30, 31, 37, 38). For example, increases in epithelial barrier permeability after the injection of bacterial endotoxin into the mucosa in animal models can initiate an oxidative and inflammatory condition similar to IBD (59). Similarly, genetically engineered mice with a leaky gut develop intestinal inflammation (29).
We have been investigating endogenous protective mechanisms (e.g.,
growth factor signaling) against oxidant-induced barrier dysfunction in
an effort to develop a rational basis for more effective treatment
regimens for inflammatory disorders of the GI tract. We recently
showed, using monolayers of human intestinal cells (Caco-2) as a model
of barrier function, that epidermal growth factor (EGF) or transforming
growth factor- protect intestinal barrier integrity by stabilizing
the microtubule cytoskeleton (5, 6, 8-11) in large
part through the activation of protein kinase C (PKC) (8, 10,
11). Because involvement in protective mechanisms by PKC was a
novel finding, we surmised that one or more specific isoforms of PKC
might mediate the protective actions of PKC.
PKC consists of a family of serine and threonine-specific kinases. The
PKC family, which includes at least 12 known isoenzymes, can be
classified into three subfamilies on the basis of differences in
sequence homology and cofactor requirement (2, 4, 17, 18, 25, 32,
34, 41, 44, 46, 48, 49, 50, 51, 53, 57, 60, 61). The
conventional (or classic) PKC isoforms (,
1,
2,
) require calcium, diacylglycerol (DAG), and
phospholipid for their activation, whereas the novel PKC isoenzymes
(
,
,
,
, µ) are calcium independent but require DAG and
phospholipid. Activation of the third group, atypical PKC isoforms
(
,
,
), is independent of both calcium and DAG
(21). Intestinal epithelial cells, including Caco-2 cells,
express at least five of these isoforms: PKC-
, PKC-
1,
PKC-
2, PKC-
, and PKC-
(1, 8, 11, 16, 21, 43,
53, 58). 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, 16, 32, 41, 44, 46, 49, 50).
We (8) previously showed in wild-type Caco-2 intestinal
cells that EGF induces the membrane translocation of native
PKC-1 and PKC-
isoforms, and therefore we considered
each as a possible contributor to EGF-afforded protection. Using
transfected cells that either stably overexpressed PKC-
1
or could not express PKC-
1, we recently found (6,
8, 11) that PKC-
1, a conventional or classic,
DAG-dependent isoform of PKC, was necessary for a substantial fraction,
but not all, of EGF protection. We noted that protection mediated by
PKC-
1 was DAG dependent because neither PKC-
1 overexpression nor low doses of
1-oleoyl-2-acetyl-sn-glycerol (OAG) alone afforded
protection but together they led to protection. In the current report,
we have explored the role of the
-isoform of PKC because:
1) it is translocated to the membranes in wild-type Caco-2
cells by EGF; 2) unlike PKC-
1, it is an
"atypical" PKC isoform; 3) it is of clinical and
biological importance to more fully establish the idea that specific
isoforms of PKC play fundamental roles in endogenous protective
mechanisms of cells; and 4) a better understanding of the
pathophysiology of hyperpermeability of the intestinal barrier and its
prevention could lead to the development of novel therapeutics for
inflammatory diseases of the GI tract related to oxidative injury.
Accordingly, we studied the PKC- isoform utilizing targeted
molecular interventions (transfection) that enabled us to develop two
novel and stably transfected intestinal cell lines. In one, the
atypical isoform PKC-
was reliably overexpressed; in the other,
PKC-
expression was almost completely inhibited. Using these new
models, we tested the hypothesis that EGF-induced protection against
oxidant injury to both the microtubule cytoskeleton and intestinal
barrier depends on activation of the
-isoform but without the
requirement for DAG (OAG).
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MATERIALS AND METHODS |
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Cell culture. Both Caco-2 and HT-29 cells 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. Utility and characterization of these cell lines have been previously reported (4, 13, 24, 45).
Plasmids and stable transfection.
Sense and antisense plasmids of PKC- were constructed and then
stably transfected as we previously described (10, 11, 21). Expression was controlled by
-actin promoter. The
antisense PKC-
plasmid (p
-actin SP72-As-PKC-
) was constructed
by ligating the 2.3-kb EcoRI fragment of PKC-
cDNA from
pJ6-PKC-
(21) into the unique EcoRI sites of
the p
-actin SP72 vector. The antisense orientation of the plasmid
was confirmed by SamI restriction digestion (21).
Experimental design.
First, postconfluent monolayers of wild-type cells were preincubated
with EGF (1 or 10 ng/ml) or isotonic saline for 10 min and were then
exposed to oxidant (0.5 mM H2O2) or vehicle
(saline) for 30 min. As we have previously shown (5-7, 11,
13, 15), 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 using
cell monolayers either stably overexpressing or almost completely
lacking PKC-. Reagents were applied on the apical side of monolayers
unless otherwise indicated. In all experiments, barrier function,
microtubule cytoskeletal stability (cytoarchitecture, tubulin
assembly/disassembly), and PKC-
subcellular distribution were then assessed.
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, 8, 11). Protein content of the various cell fractions was assessed by the Bradford method (19). For total PKC extraction, scraped monolayers were placed directly into 1.5 ml of a standard cold lysis buffer (4°C) and subsequently ultracentrifuged. Supernatant was used for bulk protein determination.
For immunoblotting, samples (75 µg protein/lane) were added to SDS buffer (250 mM Tris · HCl, pH 6.8, 2% glycerol, 5% mercaptoethanol), boiled for 5 min, and then separated on 7.5% SDS-PAGE (8, 11). The immunoblotted proteins were incubated for 2 h in Tween 20, Tris-buffered saline, 1% BSA, and the primary mouse monoclonal anti-PKC-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, 6, 8, 12-14). Cells were subsequently
processed for incubation with a primary antibody, monoclonal mouse
anti-
-tubulin (Sigma, St. Louis, MO) at 1:200 dilution for 1 h
at 37°C, and were then incubated with a secondary antibody
(FITC-conjugated goat anti-mouse; Sigma) at 1:50 dilution for 1 h
at room temperature. Slides were washed three times in
D-PBS and subsequently mounted in aquamount. After being
stained, cells were observed with an argon laser (
= 488 nm)
using a ×63 oil immersion plan-apochromat objective, 1.4 numerical
aperture (Zeiss). Single cells and/or a clump of two to three cells
from desired areas of monolayers were processed using the image
processing software on a Zeiss ultra high-resolution LSCM so as 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 have described (5,
6, 8, 12, 13). Two hundred cells per slide were examined in four
different fields by LSCM, and the percentage of cells displaying normal
microtubules was determined. 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
after a series of centrifugation and extraction steps as we have
described (5, 6, 8, 13). Fractionated S1 and S2 samples
were then flash frozen in liquid N2 and stored at 70°C
until immunoblotting. For immunoblotting, samples (5 µg protein per
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, 6, 8,
13). To quantify the relative levels of tubulin, the optical
density of the bands corresponding to immunoradiolabeled tubulin were
measured with a laser densitometer.
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 fluorescent markers, such as fluorescein sulfonic acid (FSA, 200 µg/ml; 0.478 kDa), as we (5-8, 9-11, 15) and others (33, 35, 52, 56) have described. In select experiments, higher molecular mass fluorescein dextran (FD) probes such as the 4 kDa FD and 70 kDa FD (1 mg/ml) were also utilized. After treatments, fluorescent signals from samples were quantitated by a fluorescence multiplate reader (FL 600, BIO-TEK Instruments).
Statistical analysis. Data are presented as means ± SE. All experiments were carried out with a sample size of at least four to six observations per group. Statistical analysis comparing treatment groups was performed using ANOVA followed by Dunnett's multiple range test (27). Correlational analyses were done using the Pearson test for parametric analysis or, when applicable, the Spearman test for nonparametric analysis. P values <0.05 were deemed statistically significant.
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RESULTS |
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Stable overexpression of PKC- isoform.
Intestinal cells were cotransfected with complementary DNA (cDNA)
encoding both G-418 resistance (for selection) and PKC-
. Western
immunoblotting analysis of cell lysates of these transfected cells from
confluent monolayers demonstrates (Fig.
1A) the overexpression of the
PKC-
isoform (3 µg of DNA plasmid shown). The PKC-
isolated from transfected cells ran at the expected molecular mass of 72 kDa as
confirmed by a known positive control for PKC-
. Identity of the
PKC-
band was further ascertained by using the PKC-
blocking peptide in combination with the anti-PKC-
antibody that prevented the appearance of the corresponding major band
in the Western blots. Additionally, in the absence of the primary
antibody to PKC-
, no corresponding band for PKC-
was observed.
Immunoblotting assessment of PKC-
protein levels (Fig.
1B) showed that total levels of this overexpressed isoenzyme
were increased by ~2.9-fold compared with wild-type cells. Optical
densities (means ± SE) for these PKC-
overexpression
studies were 12,210 ± 118 vs. wild type 4,175 ± 79. Preliminary studies confirmed that overexpression of PKC-
did not
injure intestinal cells as indicated by a lack of change in viability
assessed by ethidium homodimer-1 probe (5, 6).
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Protective effects of the overexpressed PKC- isoform against
oxidant-induced injury.
Multiple clones of intestinal Caco-2 cells (Table
1) or HT-29 cells (Table
2) transfected with 1, 2, 3, 4, or 5 µg of PKC-
sense cDNA showed a dose-dependent protection of
barrier integrity in monolayers against oxidant-induced injury as
assessed by FSA clearance. In Caco-2 monolayers, the clone transfected
with 3 µg of PKC-
sense provided the maximum protection observed
(Table 1). This was comparable to 3 µg PKC-
sense, which also
provided the maximum protection in HT-29 cells (Table 2). Accordingly, we used the appropriate clones for overexpressing PKC-
in all subsequent experiments.
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Intracellular distribution and constitutive activation of the
overexpressed PKC- in transfected intestinal monolayers.
Western immunoblotting assessment of the cytosolic, membrane, and
cytoskeletal-associated fractions from transfected cells overexpressing
PKC-
showed that the
-isoform (72 kDa) is found mostly in the
membrane and cytoskeletal fractions of these transfected cells with
only a small distribution to the cytosolic fractions (Fig.
5A, Caco-2 shown). In
wild-type cells (Fig. 5B), in contrast, we found a mostly
cytosolic distribution of PKC-
with smaller pools in the membrane
and cytoskeletal (particulate) fractions, indicating inactivity. Figure
6 shows a graphic depiction of the intracellular distribution of the overexpressed PKC-
in various Caco-2 cell monolayer fractions as a fraction of total distribution (expressed in arbitrary units). Finding PKC-
in particulate pools indicates that the overexpressed PKC-
isoform is "constitutively active" because achieving this distribution by PKC-
did not
require EGF or OAG (a PKC activator). Pretreatment of transfected cells with EGF, however, further increased the fraction of PKC-
isoform into the membrane and cytoskeletal fractions, reaching near-total activation of PKC-
. Wild-type cells exposed to vehicle or oxidant show a mostly cytosolic distribution of PKC-
. In these wild-type cells, we noted rapid translocation of native PKC-
into particulate (membrane + cytoskeletal) fractions of cells only after exposure to high doses of EGF, which confirms our recent and preliminary findings (8).
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Stable antisense inhibition of PKC- to underexpress the
-isoform and its prevention of EGF-induced protective effects.
The above findings indicate that PKC-
might, by itself, play a key
role in cellular protection. To show that PKC-
specifically contributes to EGF-mediated protection, we utilized an antisense approach to stably decrease the steady-state levels of PKC-
protein. Figure 7A shows an immunoblot
of cell lysates of wild-type Caco-2 cells transfected with PKC-
antisense cDNA (3 µg) and plasmid encoding G-418 resistance. These
data show a substantial reduction (
95%) in the levels of PKC-
protein in these antisense-transfected cells.
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DISCUSSION |
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We have demonstrated that the -isoform of PKC
plays an important role in EGF-mediated protection against oxidant
damage to the microtubule cytoskeleton and to cell monolayer
integrity. This isoform of PKC also appears to be a critical
endogenous stabilizer of both cytoskeletal and barrier function.
Several lines of evidence in the current study support the
aforementioned findings.
First, overexpression of PKC- induces an EGF-like protection against
oxidant-induced disruption of barrier integrity. This protection
appears to require overexpression and constitutive activation of
PKC-
. In particular, protection is dependent on constitutive
activation through the distribution of PKC-
into the particulate
(cytoskeletal + membrane) fractions. Second, overexpression of
PKC-
induces stabilization of the microtubule cytoskeleton, a
protective phenomenon we have shown to be key in the maintenance of
cell monolayer integrity. Overexpression of PKC-
decreases the
unstable monomeric (S1) tubulin, increases the stability of polymerized
(S2) tubulin, and increases the percentage of Caco-2 cells displaying
normal microtubules. Third, a low, nonprotective concentration of EGF
potentiates all measures of PKC-
-induced protection. Fourth,
antisense inhibition of the expression of PKC-
reduces EGF
protection of barrier integrity by ~48 ± 8%, the remaining
52% of EGF protection apparently being PKC-
independent. In these
antisense-transfected clones, which expressed
-isoform at ~5% of
wild-type levels, EGF protection of S2 tubulin assembly and
microtubules was also significantly prevented. Fifth, increases in
expression of PKC-
quantitatively correlate with increases in
outcomes indicating protection (barrier integrity, tubulin polymerization, microtubule assembly, and integrity of microtubule cytoarchitecture).
Our findings are consistent with our previous reports that activation
of PKC in general is required for EGF protection (8) and
that specific isoforms mediate that protection (11). For example, PKC-1 isoform mediates a substantial portion
(60 ± 8%) of EGF protection (11). On the basis of
percent mediation of protection, it is reasonable to speculate that
activation of both
1 and
-isoforms of PKC can account for 100%
of EGF-induced protection.
Although PKC-1 and PKC-
share in common the ability
to protect, there appear to be differences in their mechanisms of
action. Protection by PKC-
, as shown herein, does not require the
presence of pharmacological activators of PKC (e.g., OAG or EGF),
whereas protection by PKC-
1 does require them (8,
11). This difference is fully consistent with the fact that
PKC-
is an "atypical" isoform of PKC, whereas
PKC-
1 is a "conventional" isoform. Indeed, our
findings on the atypical PKC-
are consistent with reports in non-GI
models in which the
-isoform activation was shown to be independent
of PKC activators (e.g., OAG or 12-O-tetradecanoylphorbol 13-acetate) (18, 25, 50). For example, the activation of PKC-
is not dependent on treatment with phorbol esters or DAG (OAG)
(25). Similarly, atypical PKC isozymes
and
do not respond to phorbol esters or OAG (21). In contrast, OAG
has been shown to induce activation of classic isoforms of PKC, such as
1, in non-GI cellular models (e.g., fibroblasts) (25)
as well as in GI cells (e.g., Caco-2 cells) as we recently reported (11).
Our findings regarding the subcellular distribution of PKC isoforms are
consistent with known biochemical properties of PKC isoforms. All PKCs
consist of NH2-terminal regulatory domains and
COOH-terminal catalytic domains (separated by a flexible hinge region)
(26). In resting cells, PKC is mainly found in an inactive conformation. In this inactive phase, PKC is mainly distributed in the
soluble (cytosolic) fraction and only loosely bound to membrane
components. Regulatory domains of PKC isoforms vary from one subfamily
to the next as well as among individual isoforms within a given
subfamily (21, 26, 47). For example, OAG (or DAG) binding
sites are present in the regulatory "zinc finger" domain of
PKC-1 but are absent from the regulatory domain of PKC-
. Not surprisingly, PKC-
has severalfold lower affinity for
OAG than has PKC-
1 at the zinc finger domains
(26). Additionally, in many wild-type cells,
PKC-
1 and PKC-
appear to be found in different
subcellular fractions (11, 21). Consistent with these
known facts, our previous and present studies collectively demonstrate
a novel concept that increased levels of these PKC isoforms in the
particulate fractions (i.e., activation) either by pharmacological
manipulation (OAG or EGF for PKC-
1) or by transfection
(constitutive activation for PKC-
) lead to enhanced cellular protection.
Although most cells express more than one type of PKC isoform,
differences among isozymes with respect to activation conditions and
subcellular locations suggest that individual PKC isoforms have
distinct activation mechanisms as well as mediate distinct biological
processes (1, 4, 16, 17, 32, 41, 44, 46, 49-51, 60).
In resting cells (in the absence of lipid cofactors), most PKCs assume
an inactive structural conformation. This is maintained by an
intramolecular interaction between an autoinhibitory sequence
(i.e., the pseudosubstrate on the NH2 terminus) in the regulatory domain and the substrate-binding region of the catalytic domain (26). Moreover, accumulated evidence suggests that
modifications of the regulatory domain of a PKC isoform can lead to the
activation of that isoform (21, 26). For example, binding
of PKC- isoform to phospholipids, especially anionic
phosphotidylserine, in membranes (i.e., translocation to
membranes) is thought to be necessary to cause conformational
changes to its regulatory domain, before activation. Specifically,
structural studies (26) demonstrate that in the presence
of inducers (e.g., overexpression by transfection), this regulatory
domain (especially the zinc finger portion) forms an automatic
hairpin-like hydrophobic structure that mediates PKC interaction with
the membrane lipids and subsequent conformational changes within
the regulatory domain, leading to autoactivation. Thus overexpression
of PKC-
may promote a conformational change that releases the
inherent autoinhibition present and triggers kinase catalytic activity.
For DAG-dependent PKC isoforms such as PKC-
1 (11,
21), on the other hand, the inhibitory function of the
regulatory domain can be overcome by pharmacological agents that mimic
DAG (e.g., OAG or 12-O-tetradecanoylphorbol 13-acetate), thus producing conformational changes within this key domain and, in
turn, resulting in an activated form of the PKC isoform.
Our findings that activation of PKC in general (8) and
PKC-1 (11) and PKC-
in particular are
involved in protection of intestinal cells are supported by two recent
pharmacological studies (54, 55). For example, Terres et
al. (54) using intestinal T-84 cells showed that
Helicobacter pylori-associated decreases in monolayer
barrier resistance was inhibited in the presence of a PKC activator
12-O-tetradecanoylphorbol 13-acetate, thus suggesting a
possible role for PKC in protection against bacterial-induced damage.
Additionally, our findings on the
1-isoform of PKC
(11) and
-isoform (current report) utilizing more
specific and targeted molecular approaches further expand on these
previous pharmacological reports and, we believe, now establish a novel biological function (protection) among the isoforms of PKC subfamilies. Furthermore, we have more recently identified activation of EGF receptor tyrosine kinase and then phospholipase C-
1 as the upstream signal for EGF-induced, PKC-mediated protection of intestinal barrier
and cytoskeletal integrity (10).
Our series of studies on PKC, to date, were designed to investigate
possible beneficial effects of PKC isoform activation in the GI tract.
Although our findings are consistent with other published
pharmacological studies, several reports (3, 23, 47) have
shown that activation of PKC in cellular models may lead to
nonprotective effects and these may vary with different experimental conditions and cell types. For example,
overexpression of PKC- leads to the disruption of pig kidney
epithelial (LLC-PK1) cell monolayers (47). A recent
pharmacological study also suggested that PKC-
and PKC-
appear to
be involved in tumor necrosis factor-
-induced injury in intestinal
(IEC-18) cells (59).
Our findings show that stable antisense inhibition of PKC- prevents
all measures of EGF-induced protection in our intestinal cell model.
Whereas the mechanism for this inhibitory effect on EGF protection
needs to be fully established, this attenuation is consistent with our
data showing substantial downregulation of PKC-
expression by its
antisense inhibition as well as by parallel inhibition of three
separate EGF-related protective variables: barrier integrity, tubulin
assembly, and microtubule stability. A question that remains to be
answered is how antisense to PKC-
prevents EGF protection. We now
suggest a mechanism by which the PKC-
downregulation prevents
EGF-induced protection: protein phosphorylation by PKC-
by any of
several well-known cellular mechanisms, such as cytoskeletal
phosphorylation/dephosphorylation. This mechanism is consistent with
our previous reports that EGF protection is mediated through
stabilization of the assembly of the tubulin-based cytoskeleton
(6, 8) and that EGF-induction causes PKC to phosphorylate
tubulin and enhance tubulin assembly, which correlates significantly
(r = 0.90 and 0.88, respectively; P < 0.05 for each) with EGF protection of microtubule stability and of
barrier integrity (11). This proposed mechanism is further consistent with several reports in non-GI models that PKC activation phosphorylates and stabilizes cytoskeletal proteins (25,
28). Because cytoskeletal assembly and stability is critical in
cellular protection, it follows that downregulation of PKC-
(by
antisense) can prevent essential protein phosphorylation, thereby
preventing EGF protection of cytoskeletal integrity. For example, we
recently reported (11) that EGF or the PKC activator OAG
led to an enhancement of serine phosphorylation of the tubulin (50 kDa)
subunit protein of the microtubules. This increase was substantially
attenuated by antisense inhibition of PKC-
1, suggesting
that PKC may be acting, directly or indirectly, on the tubulin-based
cytoskeleton. It is possible, therefore, that activated
PKC-
1 or activated PKC-
phosphorylates the same or
similar cytoskeletal or membrane targets. This proposed mechanism in
our GI model is consistent with several previous studies in non-GI
models. For instance, PKC has been shown to be involved in remodeling
of the cytoskeletal filaments (2, 22, 25, 28, 40),
although it has not been clearly established which PKC isoforms are
essential in these processes. PKC can phosphorylate the cytoskeletal
proteins talin and vinculin (25). Also, a specific
substrate for PKC, myristoylated, alanine-rich PKC substrate protein
(MARCKS), has been suggested to be an actin cytoskeletal reorganizer
(28). In particular, MARCKS activity is abolished by
PKC-induced phosphorylation. It is also possible that PKC isozymes can
phosphorylate tubulin-associated capping proteins (e.g., microtubule
associated proteins).
Evidence exists for other possible mechanisms for protection by PKC. Our previous reports showed that certain antioxidants (5, 7, 9) or agents that normalize intracellular calcium homeostasis (8) prevent oxidative damage in our model. Therefore, enhancement of either of these mechanisms could conceivably underlie PKC protective effects. Studies are underway in our laboratory to determine to what extent PKC protection is mediated by either of these mechanisms.
In summary, it appears that PKC- is responsible for a substantial
portion of normal protection of the GI mucosal epithelium and perhaps
is key to preventing amplification and perpetuation of an uncontrolled,
oxidant-induced, inflammatory cascade that can be ignited by free
radicals and other oxidants present in the GI tract. By creating the
first GI cells stably overexpressing "protective PKC isoforms," our
laboratory has discovered that these PKC isoforms possess critical
functions in protecting cells against oxidative stress. This new
knowledge may prove useful because increasing the activity of
protective PKC isoforms through activation of endogenous PKC or using
PKC mimetics may lead to novel therapeutic strategies for the treatment
of a wide variety of oxidant-induced inflammatory disorders of the GI
tract, including IBD.
Finally, our proposed mechanism of protection against oxidative stress has laid the groundwork for future "translational research" in humans and animals. We envision that these in vitro experiments will lead to highly focused studies that will test the clinical relevance of these potentially key biochemical pathways in IBD. For example, we (A. Keshavarzian, A. Banan, S. Kommandori, Y. Zhang, and J. Z. Fields, unpublished observations) have shown that a number of these oxidative reactions also occur in intestinal mucosa from patients with IBD. An important question that remains to be answered is whether modulation of PKC activity in vivo might also prevent oxidative damage.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Gail Hecht (University of Illinois) for her generous donation of HT-29 cells.
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FOOTNOTES |
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This work was supported, in part, by a grant from Rush University Medical Center, Department of Internal Medicine, and by a grant from the American College of Gastroenterology.
Portions of this work will be presented at the Annual Meeting of the American Gastroenterological Association, May 2002.
Address for reprint requests and other correspondence: A. Banan, Rush Univ. Medical Center, Division 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.
First published January 9, 2002;10.1152/ajpgi.00284.2001
Received 28 June 2001; accepted in final form 19 December 2001.
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