1 Departments of Internal Medicine (Section of Gastroenterology and Nutrition), 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 intestinal (Caco-2) monolayers,
we reported that inducible nitric oxide synthase (iNOS) activation is
key to oxidant-induced barrier disruption and that EGF protects against
this injury. PKC- was required for protection. We thus hypothesized
that PKC-
activation and iNOS inactivation are key in EGF
protection. Wild-type (WT) Caco-2 cells were exposed to
H2O2 (0.5 mM) ± EGF or PKC modulators. Other cells were transfected to overexpress PKC-
or to inhibit it
and then pretreated with EGF or a PKC activator (OAG) before oxidant. Relative to WT cells exposed to oxidant, pretreatment with EGF protected monolayers by 1) increasing PKC-
activity; 2) decreasing iNOS activity and protein, NO
levels, oxidative stress, tubulin oxidation, and nitration);
3) increasing polymerized tubulin; 4) maintaining
the cytoarchitecture of microtubules; and 5) enhancing
barrier integrity. Relative to WT cells exposed to oxidant, transfected
cells overexpressing PKC-
(+2.9-fold) were protected as
indicated by decreases in all measures of iNOS-driven pathways and
enhanced stability of microtubules and barrier function. Overexpression-induced inhibition of iNOS was OAG independent, but EGF
potentiated this protection. Antisense inhibition of PKC-
(
95%)
prevented all measures of EGF protection against iNOS upregulation. Thus EGF protects against oxidative disruption of the intestinal barrier by stabilizing the cytoskeleton in large part through the
activation of PKC-
and downregulation of iNOS. Activation of PKC-
is by itself required for cellular protection against oxidative stress
of iNOS. We have thus discovered novel biologic functions, suppression
of the iNOS-driven reactions and cytoskeletal oxidation, among the
atypical PKC isoforms.
tubulin cytoskeleton; growth factors; oxidative stress; nitrogen metabolites; molecular biology; inflammatory bowel disease
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INTRODUCTION |
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THE GASTROINTESTINAL (GI) epithelium is a highly selective permeability barrier that normally permits the absorption from the lumen of nutrients, water, and electrolytes but prevents the passage of harmful proinflammatory and toxic molecules into the mucosa and the systemic circulation. Disruption of GI barrier integrity, in contrast, can allow the penetration of normally excluded luminal substances (e.g., immunoreactive antigens, endotoxin) into the mucosa and can lead to the initiation or continuation of inflammatory processes and mucosal damage (4, 36, 43, 44). Indeed, 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-induced chemical injury, and a variety of other GI disorders as well as several systemic disorders (e.g., alcoholic liver disease) (4, 36, 43, 44). The underlying difficulty in managing these inflammatory disorders is due, in large part, to our limited understanding of their pathophysiology and lack of effective preventive strategies.
Although the pathophysiology of mucosal barrier dysfunction in these disorders remains poorly understood, several studies (4, 5, 10, 12, 17, 35, 36, 44, 45, 48), including our own, have shown that chronic gut inflammation is associated with oxidative stress and that this stress appears to be a key cause of injury. Oxidative stress is of substantial clinical importance not only because oxidants are common in inflammation, but also because they can lead to mucosal barrier hyperpermeability and, in turn, to the initiation and/or perpetuation of mucosal inflammation and injury (35, 36, 43, 44). A major advance in recent years in GI inflammation (IBD) research was recognition that a leaky gut can cause intestinal inflammation and that maintaining/protecting a normal barrier function is key to intestinal health. In animal models, for example, intestinal barrier hyperpermeability induced by the injection of bacterial endotoxin into the mucosa can elicit an oxidative and inflammatory condition similar to IBD (66). Moreover, transgenic mice with a leaky gut exhibit symptoms of intestinal inflammation (34). Accordingly, understanding how GI barrier integrity is protected under oxidative, proinflammatory conditions is of fundamental clinical and biologic importance.
We have been investigating the mechanisms underlying oxidant-induced
injurious pathways and growth factor-mediated protective pathways
against that injury (and the GI barrier dysfunction) to devise a
rational basis for potentially more effective treatment regimens for
inflammatory disorders of the GI tract. It was shown (4, 5,
7-11) that cytoskeletal disassembly and disruption are key
events in this oxidative injury and that growth factors [epidermal
growth factor (EGF) or transforming growth factor (TGF)-] prevent
damage by stabilizing the cytoskeleton in large part through the
activation of protein kinase C (PKC). For example, it was reported
(5, 7-9, 11) that maintaining an intact microtubule cytoskeleton is required for protection of intestinal barrier integrity
by EGF via PKC.
The PKC family, which includes at least 12 known isoenzymes, can be
classified into three subfamilies based on differences in sequence
homology and cofactor requirement (2, 7-9, 37, 47, 50, 52,
54, 55). 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 (8, 25). Intestinal epithelial cells,
including Caco-2 cells, express at least six isoforms of PKC: PKC-
,
-
1, -
2, -
, -
, and -
as we and
others have reported (1, 7-9, 18, 25, 49, 65). 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 (37, 47, 50, 52, 54, 55).
The involvement in protective mechanisms by PKC in the GI epithelium as
it was originally reported was a novel finding (7, 9).
Banan et al. (9) showed using wild-type (WT) Caco-2 intestinal cells that EGF induces the membrane translocation of the
native PKC- isoform and therefore considered it as a possible contributor to EGF-mediated protection of the GI epithelial barrier. To
address this possibility, we developed several unique cell lines, some
clones stably overexpressing PKC-
, the other clones underexpressing
PKC-
. Banan et al. (8) found that PKC-
, an atypical,
DAG-independent isoform of PKC, is required for a substantial fraction
of EGF-mediated protection of the monolayer barrier function. Also,
PKC-
-mediated protection was DAG independent, because overexpression by itself afforded substantial protection. Despite the critical importance of the
-isoform of PKC, the fundamental mechanism for the
protection afforded by PKC-
(and EGF) remains unknown.
Other studies (10, 12) reported on the importance of the
inducible nitric oxide (NO) synthase (NOS; iNOS)-dependent mechanisms in the underlying cause of oxidant-induced intestinal cytoskeletal and
barrier disruption. The original concept was demonstrated that specific
iNOS and NO/ONOO-mediated nitration and oxidation of key
cytoskeletal protein subunits and the disruption that is caused to
cytoskeletal networks lead to the loss of GI barrier integrity. Indeed,
overproduction and uncontrolled generation of iNOS-derived reactive
nitrogen metabolites (e.g., NO, ONOO
) have been proposed
by several recent studies (16, 39, 42, 56, 60), including
our own, to be an important factor in tissue damage during
inflammation, including IBD. For example, Banan et al.
(16) and Keshavarzian et al. (42) showed that
a number of these oxidative reactions, including cytoskeletal nitration and oxidation, also occurs in intestinal mucosa from patients with IBD.
In view of the aforementioned considerations, we hypothesized that
PKC- not only prevents oxidant-induced iNOS upregulation and its
injurious consequences, but also it is key to EGF-mediated protection
of microtubule cytoskeleton and intestinal barrier integrity against
the oxidative stress of this upregulation. To this end, we used both
pharmacological and targeted molecular interventions employing several
unique transfected intestinal cell lines we have developed. In several
clones, the atypical isoform PKC-
was reliably overexpressed; in the
other clones, PKC-
expression was inhibited. Herein, we report novel
biological functions, i.e., prevention of the oxidative stress of iNOS
induction and of cytoskeletal protein oxidation, by the atypical
-isoform of PKC.
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MATERIALS AND METHODS |
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Cell culture. Caco-2 cells were obtained from ATCC (Rockville, MD) at passage 15. This cell line was chosen for our studies because they form monolayers that morphologically resemble small intestinal cells, with defined apical brush borders, tight junctions, and a highly organized microtubule network on differentiation (14, 29, 51). Caco-2 cells also form monolayers that can be studied for weeks rather than just days, as is typical of most fresh in vitro preparations (38, 41, 63). This allowed us to measure alterations in intestinal barrier integrity. In addition, Caco-2 cells closely resemble normal intestinal cells in that they express intestinal hydrolases such as sucrase-isomaltase and alkaline phosphatase. Furthermore, these cells are similar to native intestinal epithelial cells in that they have receptors for prostaglandins, growth factors, vasoactive intestinal peptide, low-density lipoprotein, insulin, and specific substrates such as dipeptides, fructose, glucose, hexoses, and vitamin B12 (14, 29, 51). Accordingly, this cell line provides a suitable in vitro model for our studies. The utility and characterization of this cell line has been previously reported (51).
Plasmids and stable transfection.
The sense and antisense plasmids of PKC- were constructed and then
stably transfected by Lipofectin reagent (GIBCO-BRL) as previously
described (7, 8, 25). Expression was controlled by
-actin promoter. The antisense PKC-
plasmid (p
-actin
SP-As-PKC-
) was constructed by ligating the 2.3-kb EcoRI
fragment of PKC-
cDNA from pJ6-PKC-
into the unique
EcoRI sites of the p
-actin SP vector (8,
25). The antisense orientation of the plasmid was confirmed by
SamI restriction digestion. Multiple clones stably overexpressing PKC-
or lacking PKC-
were assessed by
immunoblotting and were plated on Biocoat Collagen I cell culture
inserts (Becton Dickinson, Bedford, MA) and subsequently used for experiments.
Experimental design.
First, postconfluent monolayers of WT cells were preincubated with EGF
(1-10 ng/ml) or isotonic saline for 10 min and then exposed to
oxidant (H2O2, 0.5 mM) or vehicle (saline) for
30 min. As previously shown (4, 5, 7, 9, 12, 14, 17), H2O2 at 0.5 mM disrupts microtubules and
barrier integrity and upregulates iNOS. EGF at 10 ng/ml (but not 1 ng/ml) prevents both microtubule and barrier disruption. These
experiments were then repeated using transfected cells. In all
experiments, microtubule stability (cytoarchitecture, tubulin
assembly/disassembly), barrier integrity, PKC- subcellular
distribution, iNOS activity, NO levels, reactive nitrogen metabolites
(RNM)/RNM levels (e.g., ONOO
), oxidative stress
dichlorofluorescein fluorescence (DCF), tubulin nitration
(nitrotyrosination), and tubulin oxidation (carbonylation) were assessed.
Fractionation and 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 (7-9).
Protein content of the various cell fractions was assessed by the
Bradford method (21). For immunoblotting, samples (75 µg
protein/lane) were added to a standard SDS buffer, boiled, and then
separated on 7.5% SDS-PAGE (7, 8). The immunoblotted
proteins were incubated with the primary mouse monoclonal anti-PKC-
(Santa Cruz Biotechnology, Santa Cruz, CA) at 1:2,000 dilution. A
horseradish peroxidase-conjugated goat anti-mouse antibody (Molecular
Probes, Eugene, OR) was used as a secondary antibody at 1:4,000
dilution. Proteins were visualized by enhanced chemiluminescence (ECL;
Amersham, IL) and autoradiography (e.g., 1 h at
20°C) and
subsequently analyzed by densitometry. The exposure times were adjusted
to ensure linear responses. Under these immunological detection
conditions, the chemiluminescence assay was linear between 25 and 100 µg of total protein. The identity of the PKC-
band was confirmed
as previously described (8). We also confirmed that
overexpression of PKC-
or antisense inhibition of PKC-
expression
did not affect the relative expression levels of other PKC isoforms,
nor did it injure the Caco-2 cells.
Assay of NOS activity. Conversion of L-[3H]arginine (Amersham, Arlington Heights, IL) to L-[3H]citrulline was measured in the cell homogenates by scintillation counting. Experiments in the presence of NADPH, without Ca2+ and with 5 mM EGTA, determined Ca2+-independent NOS (iNOS) activity (10, 12).
Western blot of the level of iNOS. After treatments, the cells were washed once with cold PBS, scraped into 1 ml of cold PBS, and harvested in a standard antiprotease cocktail. For immunoblotting, samples (25 µg protein/lane) were separated on 7.5% SDS-PAGE. Membranes were visualized by ECL and autoradiography (10, 12).
Chemiluminescence Analysis of NO.
NO production was assessed by a unique chemiluminescence procedure
(10, 12). Briefly, cells were homogenized and the
endogenous nitrate (NO
Determination of cell oxidative stress.
Oxidative stress was assessed by measuring the conversion of a
nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (Molecular
Probes) into a fluorescent dye, dichlorofluorescein (4, 10,
12). Fluorescent signals from samples were quantitated using a
fluorescence multiplate (excitation = 485 nm, emission = 530 nm). The dependence of the assay on ROM production (e.g., H2O2 or · O
Immunofluorescent staining and high-resolution laser scanning
confocal microscopy of microtubules.
Cells from monolayers were fixed in cytoskeletal stabilization buffer
and then postfixed in 95% ethanol at 20°C as previously described
(4, 5, 9, 13-15). Cells were subsequently processed for incubation with a primary antibody, monoclonal mouse
anti-
-tubulin (Sigma) and then with a secondary antibody
(FITC-conjugated goat anti-mouse; Sigma). After being stained, cells
were observed with an argon laser (
= 488 nm) using a ×63 oil
immersion plan-apochromat objective, numerical aperature 1.4 (Zeiss,
Germany). The cytoskeletal elements were examined in a blinded fashion
for their overall morphology, orientation, and disruption (4, 5,
9, 13).
Microtubule (tubulin) fractionation and quantitative
immunoblotting of tubulin assembly and disassembly.
Polymerized (S2) and monomeric (S1) fractions of tubulin were isolated
using a series of extraction and ultracentrifugation steps as described
(4, 5, 9, 14). 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/lane) were
placed in a standard SDS sample buffer, boiled, and then subjected to
PAGE on 7.5% gels. Standard (purified) tubulin loading controls (5 µg/lane) were run concurrently with each run. To additionally verify
equal loading, blots were routinely stained with 0.1% India ink in
Tris-buffered saline-Tween 20 (TBST) buffer. To quantify the
relative levels of tubulin, the optical density of the bands corresponding to immunoradiolabeled tubulin were measured with a laser densitometer.
Immunoblotting determination of protein tubulin oxidation and tubulin nitration. Oxidation and nitration of the microtubule (tubulin) cytoskeleton were assessed, respectively, by measuring protein carbonyl and nitrotyrosine formation using a unique method (5, 10, 12). To avoid unwanted oxidation of tubulin samples, all buffers contained 0.5 mM DTT and 20 mM 4,5-dihydroxy-1,3-benzene sulfonic acid (Sigma). Processing and film exposure were as in a standard Western blot protocol (5, 10, 12). The relative levels of oxidized or nitrated tubulin were then quantified by measuring, with a laser densitometer, the optical density (OD) of the bands corresponding to anti-DNP (carbonylation) or antinitrotyrosine (nitration) immunoreactivity. Immunoreactivity was expressed as the percentage of carbonyl or nitrotyrosine formation (OD) in the treatment group compared with the maximally oxidized or nitrated tubulin standards, which also served as loading controls. These tubulin loading controls (5 µg/lane) were run concurrently with corresponding treatment groups. To further verify equal loading of lanes, blots were routinely stained with 0.1% India ink in TBST buffer.
Determination of barrier permeability by fluorometry. Status of the integrity of monolayer barrier function was confirmed 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 and others have described (4, 5, 7, 9-11, 38, 41, 58, 63). 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 six observations per treatment group. Statistical analysis comparing treatment groups was performed using analysis of variance followed by Dunnett's multiple-range test (32). 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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We initially confirmed our earlier preliminary finding that
intestinal cells cotransfected with cDNA encoding both G-418 resistance (for selection) and PKC- sense stably overexpress the
-isoform (72 kDa) of PKC (~2.9-fold compared with WT cells) and that this overexpression protects monolayer barrier integrity against exposure to
oxidant challenge (8). Overexpression of PKC-
at these levels caused no cellular toxicity (0% cell death assessed by ethidium
homodimer probe) (8). In the current investigation, using
both pharmacological and molecular biological interventions, we studied
the underlying mechanism by which PKC-
protects.
Stable overexpression of PKC- isoform protects against oxidative
damage to the cytoskeleton: inhibition of both tubulin nitration and
oxidation.
Because PKC-
protects against oxidant-induced injury, we surmised
that this protection may be due to the inhibition of oxidant-activated pathways. Thus, with the use of our WT and transfected cells, we
measured the "footprints" of RNM formation, nitrotyrosine moieties, under conditions of oxidant challenge. We also simultaneously measured
oxidation footprints by assessing the carbonylation levels. This was done by sequentially fractionating and purifying the 50-kDa
tubulin molecule, the structural protein of the microtubule cytoskeleton, from cell monolayers and subsequently immunoblotting these fractions. In WT cells (those not overexpressing PKC-
), oxidant H2O2 alone resulted in substantial
levels of nitration and oxidation of the tubulin cytoskeleton (Fig.
1A). In
contrast, overexpression of PKC-
by itself afforded protection
against oxidant-induced tubulin nitration and tubulin carbonylation
compared with those in WT cells. Indeed, only cells stably
overexpressing PKC-
were protected against oxidant-induced nitration
and oxidation injuries. Protection did not require the presence of
growth factor, EGF, in the cell culture media. Although 1 ng/ml EGF did
not afford significant protection against tubulin nitration or
oxidation in WT cells, this concentration did potentiate the protection observed in cells overexpressing PKC-
. In WT cells, higher doses of
EGF (10 ng/ml) were required for protection (Fig. 1A). As
expected, transfection of only the vector alone did not confer
protection against oxidation and nitration. For instance, the
percentage of tubulin that was nitrated was 0% for vector-transfected
cells exposed to vehicle, 0.72 ± 0.05% for vector-transfected
cells exposed to H2O2 alone, and 0.06 ± 0.08% for PKC-
sense transfected cells incubated in
H2O2. Similarly, the percentage of tubulin that
was carbonylated was 0% for vector-transfected cells exposed to
vehicle, 0.64 ± 0.04% for vector-transfected cells exposed to
H2O2 alone, and 0.11 ± 0.06% for PKC-
sense transfected cells incubated in H2O2 (all
ratios were normalized to a nitrated or oxidized tubulin standard
loading control run concurrently). These oxidative alterations did not
appear to be caused by changes in the ability of oxidants to cause
oxidation/nitration as vector-transfected cells and WT cells responded
in a similar fashion to H2O2, exhibiting comparable tubulin oxidation.
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PKC--induced protection involves downregulation of iNOS and
iNOS-driven reactions: inhibition of iNOS, NO,
ONOO
, and oxidative stress.
We next probed possible mechanisms by which PKC-
overexpression
reduces nitration and oxidation of cytoskeletal proteins. Because we
already showed that oxidants such as H2O2
upregulate iNOS and increase the levels of RNM and of oxidation and
nitration of cytoskeletal proteins (10, 12, 42), we
hypothesized that inhibition of iNOS-driven pathways might be a key
mechanism for PKC-
-induced protection.
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Suppression of oxidative stress in transfected cells protects the
cytoarchitecture of the microtubule cytoskeleton.
Because PKC- overexpression inhibited several measures of oxidative
stress, including tubulin oxidation, in our intestinal model, we
assessed microtubule cytoskeletal assembly. We initially confirmed the
preliminary finding (8) that PKC-
overexpression confers protection to both polymerized and monomeric forms of tubulin
(not shown) as well as the cytoarchitecture of the microtubule cytoskeleton (Fig. 6,
A-C). For example, Caco-2 cells overexpressing PKC-
exhibit an intact cytoskeleton even after exposure to oxidant (Fig.
6C) as indicated by the normal appearance of a stellate architecture of the microtubule cytoskeleton originating from the
perinuclear region. Without PKC-
overexpression, WT cells challenged
with H2O2 show instability, disruption, and
collapse of the microtubules. Untreated (control) cells also
exhibit a normal architecture of the microtubule cytoskeleton. The
appearance of the microtubules in these untreated (and normal) cells
was indistinguishable from that of transfected PKC-
-overexpressing cells exposed to oxidant that also showed a preserved microtubule cytoarchitecture. These findings on the protection of the microtubule cytoarchitecture by PKC-
parallel our findings on the protective effects of PKC-
overexpression against the injurious nitration and
oxidation of the tubulin backbone of this structural element.
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Intracellular distribution and constitutive activation of the
overexpressed PKC- in transfected intestinal cells correlates with
five different indexes of oxidative stress in monolayers.
We initially confirmed our preliminary findings in transfected,
PKC-
-overexpressing, intestinal cells (Table
2) that the
-isoform (72 kDa) is found
mostly in the particulate fractions of these transfected cells with
only a minor distribution to the cytosolic fractions, indicating its
constitutive activation (8). Finding PKC-
in
particulate pools (particulate = membrane + cytoskeletal fractions) indicates that the overexpressed PKC-
isoform is
"constitutively active," because achieving this intracellular
PKC-
distribution did not require EGF or OAG (a PKC activator).
Pretreatment of these transfected cells with EGF, nonetheless, further
increased the fraction of PKC-
isoform into the membrane and
cytoskeletal pools, reaching near-total activation of PKC-
. In WT
cells, in contrast, PKC-
is found in a mostly cytosolic distribution
(indicating inactivity) with smaller pools in the particulate
fractions. In these WT cells, there was rapid translocation (i.e.,
activation) of native PKC-
into particulate fractions only after
exposure to high doses of EGF.
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Stable antisense inhibition of PKC- and its prevention of
EGF-induced protection against oxidative stress of iNOS upregulation.
The above findings indicate that PKC-
might, by itself, play an
essential role in cellular protection against oxidative stress of
iNOS-driven reactions. To specifically investigate a possible role for
PKC-
in EGF-mediated protection against iNOS-pathway upregulation
and consequent RNM-driven oxidative stress, we used Caco-2 cells that
were transfected with PKC-
antisense plasmid and cDNA-encoding G-418
resistance. We confirmed earlier reports that this manipulation
substantially and stably reduces (~95%) the steady-state levels of
PKC-
protein as well as attenuates EGF protection of intestinal
monolayer barrier integrity (8).
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DISCUSSION |
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Exploring the role of the -isoform of PKC in the suppression of
oxidative stress of iNOS-driven reactions in cells, as we have done in
the current investigation, is critical because 1) it is of
significant clinical and biological importance to fully establish the
idea that specific isoforms of PKC play fundamental roles in endogenous
protective mechanisms of cells against oxidative stress to essential
cellular proteins required for the maintenance of GI integrity and
2) a better understanding of effectively preventing (e.g.,
by PKC-
) the hyperpermeability of the intestinal barrier under
conditions of oxidative stress could lead to the development of novel
therapeutics for inflammatory diseases of the GI tract that are related
to oxidative injury due to iNOS and NO upregulation.
In the current study, we demonstrated that the -isoform of PKC is
required for EGF-mediated protection against oxidant-induced iNOS
upregulation and the consequent oxidative stress injury to the
integrity of the microtubule (tubulin based) cytoskeleton and the
intestinal epithelial barrier. A second conclusion, and also a novel
finding, is that PKC-
by itself appears to be key in the protection
of cells against this oxidative stress injury. To our knowledge, this
is the first time this mechanism has been ascribed to the defense and
repair of epithelial cells. These conclusions are based on several
independent lines of evidence as discussed below.
First, overexpression of PKC-, which we previously reported to
prevent H2O2-induced barrier
hyperpermeability, induces an EGF-like protection against
oxidant-induced iNOS upregulation. PKC-
evokes a cascade of
alterations that are consistent with the proposed mechanism, including
downregulation of iNOS activation, normalization of NO levels,
reduction of RNM footprints, and decreases in oxidative stress (DCF
fluorescence). This protection appears to require overexpression and
constitutive activation of the PKC-
. Second, overexpression of
PKC-
inhibits the footprints of oxidative injury (i.e., RNM
formation) to the tubulin (50 kDa) protein of the microtubule
cytoskeleton. Overexpression of PKC-
decreases the nitration
(nitrotyrosination) of tubulin, reduces the oxidation (carbonylation)
of the tubulin, and maintains normal-appearing microtubule
cytoskeleton. These new findings expand on the preliminary observations
we made in which the overexpression of PKC-
decreased the unstable
monomeric (S1) tubulin and increased the stability of polymerized (S2)
tubulin and enhanced monolayer barrier integrity (8).
Third, a low, nonprotective concentration of EGF potentiates all
measures of PKC-
-mediated protection against oxidative stress. Fourth, antisense to PKC-
, which leads to expression of PKC-
at
only 5% of WT levels, substantially interfered with EGF-mediated suppression of the iNOS upregulation (by ~92%), of nitration and carbonylation of tubulin, and of the instability of microtubules. Additionally, EGF was unable to normalize NO levels or reduce DCF
fluorescence in these antisense transfected cells. Finally, PKC-
activation quantitatively correlates with decreases in all outcomes
indicating protection against oxidative stress.
Our previous work in WT intestinal cells showed significant
correlations between protection of the integrity of monolayer barrier
permeability and microtubule stability and between the integrity of the
intestinal barrier and native PKC in general (r = 0.94;
P < 0.05) (9, 14). In the present study,
with the use both of transfected clones and WT cells, we show new and robust correlations between PKC--isoform activation and protection against microtubule cytoskeletal oxidation (r = 0.93;
P < 0.05) as well as several other parameters of
oxidative stress and microtubule integrity. These include protection
against tubulin nitration (RNM footprint) and PKC-
activation
(r = 0.92; P < 0.05), protection against tubulin carbonylation (oxidation) and PKC-
activation (r = 0.93; P < 0.05), and protection
against tubulin disassembly (increase S1 monomer pool) and PKC-
activation (r = 0.92; P < 0.05).
Similar correlations are reached when either tubulin assembly (increase
S2 polymer pool) and PKC-
activation (r = 0.92;
P < 0.05) or percent normal microtubules and PKC-
activation (r = 0.95; P < 0.05) are
used. Furthermore, protection against oxidant-induced iNOS upregulation
and PKC-
activation (r = 0.91; P < 0.05), NO levels and PKC-
activation (r = 0.88;
P < 0.05), and DCF fluorescence levels and PKC-
activation (r = 0.90; P < 0.05)
provide other robust correlations. The high strength of these
correlations, which explains 85-95% of the variance, indicates
that PKC-
activation is essential to the protection against iNOS
upregulation and consequent oxidative stress to the assembly of the
tubulin cytoskeleton and intestinal barrier function. In this view,
activation of PKC-
leads to the normalization of NO levels and
protection of the microtubule cytoskeleton and barrier against
oxidative injury induced by iNOS. Overall, our studies on the
-isoform are consistent with a model in which enhanced translocation
and activation of PKC-
results in downregulation of iNOS, reduction
of both NO and RNM levels, decreases in both tubulin nitration and
oxidation, increased assembly of polymerized tubulin pools, and
concomitant reduction in monomeric tubulin pools and subsequently leads
to enhanced stability of the microtubule cytoskeleton and monolayer barrier integrity under proinflammatory conditions of oxidative stress.
Although other PKC isoforms may also be involved in protection of
intestinal barrier integrity and microtubules against oxidative stress
of iNOS-driven reactions, our findings indicate that such protection is
mediated, in large part, through the
-isoform.
The findings of this report using targeted molecular interventions are
consistent not only with our own previous studies, but also with the
findings of others using pharmacology. Our current findings on the
atypical PKC- are also consistent with reports in non-GI models in
which
-activation was shown to be independent of PKC activators
(e.g., OAG or TPA) (7-9, 20, 30, 55). For example,
the activation of PKC-
is not dependent on treatment with phorbol
esters or DAG (30). Similarly, atypical PKC isozymes
and
do not respond to phorbol esters or OAG (25). 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) as well as in GI cells (e.g., Caco-2 cells) (7,
30). Our findings using molecular biological interventions are
also consistent with other previous pharmacological reports (9,
19, 20, 49, 57, 59, 61, 62, 65) in which general PKC activation (translocation) was shown to be necessary for the observed effects of
PKC. For instance, EGF activates "constitutively" expressed PKC in
several naive cells types (19, 65). Moreover, with the use
of these WT cellular models and pharmacological approaches, PKC in
general has been suggested to be a potential mediator of EGF-induced
alterations in the actin component of the cytoskeleton in HeLa and
corneal endothelial cells and of EGF inhibition of canine parietal cell
function (26, 40, 65). Our current findings on the
-isoform of PKC using specific and targeted molecular approaches
further expand on these previous reports and, we believe, now establish
a novel biological function: protection against oxidative stress of RNM
upregulation and of cytoskeletal oxidation, among the atypical
subfamily of PKC isoforms in cells.
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 (which are separated by a flexible
hinge region) (31). 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 is 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 (7, 25, 31, 53). For example, OAG
(or DAG)-binding sites are absent from the regulatory ("zinc
finger") domain of PKC- . Not surprisingly, PKC-
has very low
affinity for OAG (8, 31).
Despite the fundamental importance of PKC signal transduction, as our
studies indicate, the role of PKC isoforms in cell function, especially
in epithelial cells, has remained poorly understood. Most cells express
more than one type of PKC isoform, and the differences among these
isozymes with respect to activation conditions and subcellular
locations suggest that individual isoforms of PKC mediate distinct
biological functions (1, 7, 8, 17, 18, 37, 47, 50, 52,
55). For example, we recently reported (7, 17) that
the 1 (78 kDa)-isoform of PKC is required for
EGF-induced protective effects on the normalization of Ca2+
homeostasis, indicating that this isoform performs a unique protective task in cells. We further note that our series of studies on PKC to
date were designed to investigate beneficial effects of PKC isoforms in
the GI tract. Indeed, novel findings of our laboratory are that
1) PKC activation in general can be protective to cells, 2) specific PKC isoforms mediate this protection, and
3) each protective isoform of PKC works through a specific
downstream mechanism (7-9). For example, our previous
study linked the protective effects of activation of the classic
PKC-
1 isoform to normalization of intracellular
Ca2+ levels via the enhancement of Ca2+ efflux
(7, 17), whereas the current study links the protective effects of activation of the atypical PKC-
isoform with prevention of iNOS upregulation and normalization of NO levels.
We further note that there do exist reports that PKC may also have
other effects that are not beneficial. These include the suspected role
of PKC and tumor promoters (e.g., phorbol esters) in carcinogenesis
(18, 37, 47, 50, 52, 53). For example, a recent
immunofluorescent study suggested that PKC- appears to be involved
in TNF-
-induced injury in intestinal (IEC-18) cells
(23). Also, PKC-
causes disruption of pig kidney cell monolayers (LLC-PK1) (53). Thus it appears that activating
or mimicking just different isoforms of PKC will have distinct
beneficial (or damaging) effects on the GI epithelium.
Our conclusions are potentially relevant for developing new treatment
strategies for IBD. They suggest a novel antioxidative defensive
mechanism that might, if it occurred in vivo, protect against oxidative
stress and either prevent initiation or manifestation of the acute IBD
attack. This defensive mechanism is seen in the ability of PKC- to
prevent oxidant-induced cellular injury and barrier disruption through
suppression of oxidation. The potential therapeutic use of this
antioxidative mechanism would be consistent with the current
characterizations of the pathophysiology of IBD (e.g., 12, 17, 42, 44, 45, 48, 56, 60). This is especially true for the transition from the
inactive to active (flare up) phases of inflammation in IBD in which
intestinal oxidants and proinflammatory molecules periodically create a
vicious cycle that leads to sustained oxidative stress,
hyperpermeability, inflammation, and tissue damage. The current study
extends our previous investigation into the role of damaging oxidants
in the pathophysiological mechanisms of this disease (5, 12, 17,
42, 44). In our intestinal model, oxidants induce barrier
hyperpermeability (5, 9, 17), and this oxidative stress is
prevented by the atypical PKC-
as shown herein. We previously traced
the in vitro cause of this monolayer hyperpermeability to disruption of
the functioning and architectural integrity of the cytoskeletal
filaments and iNOS upregulation (5, 9, 10, 12, 17). We
also established the original concept that the biochemical cause of
injury to the cytoskeletal networks and the GI barrier is the oxidation
of essential cytoskeletal protein subunits, especially tubulin. We
recently confirmed some of these findings on the cytoskeleton in vivo
by showing that mucosa of patients with active IBD exhibits increases in tissue nitrotyrosination and oxidation of key cytoskeletal proteins
as well as upregulation of iNOS and NO (16, 42). Consistent with these findings, tissue nitration, which was detected by
immunofluorescent staining of nitrotyrosine, has been associated with
the inflamed human mucosa in IBD and was linked with the upregulation
of iNOS (45, 56, 60). It remains to be seen whether
PKC-
isoform confers protection against oxidative stress in IBD
animal models.
The mechanism through which PKC- isoform suppresses iNOS
upregulation remains unclear. On the basis of the known regulatory mechanisms for iNOS (22, 24, 28), we propose two
mechanisms by which the iNOS upregulation might be prevented:
inactivation of NF-
B, a proinflammatory transcription factor, and
inactivation of the iNOS enzyme molecules by any of several well-known
cellular mechanisms such as phosphorylation or dephosphorylation
(22, 24, 28). Studies are underway in our laboratory to
determine to what extent PKC-
protection is mediated by either of
these mechanisms in GI epithelial cells.
Finally, with the use of another intestinal cell line, HT-29, we
recently reported (8) that both Caco-2 cells and HT-29 cells behaved in an almost identical manner in terms of their responses
to PKC--isoform transfection including activation, translocation,
and over- and underexpression. Other similar responses included several barrier function-related outcomes such as FSA clearance, cytoskeletal assembly/integrity, and tubulin pools (8). Not surprisingly, they also responded similarly to
EGF and oxidant treatment regimens. We recognize that Caco-2 cells are a transformed cell line and that tumor cells may not perfectly model barrier integrity and NO pathways in nontransformed cells, including enterocytes in native tissue. Nonetheless, our findings, using the first GI cells stably over- or underexpressing "protective PKC isoforms," demonstrate an original concept that PKC-
appears to be responsible for a substantial portion of protection of the intestinal mucosal epithelium against oxidative stress induced by iNOS
upregulation and perhaps is key in preventing amplification and
perpetuation of an uncontrolled, oxidant-induced, inflammatory stress
cascade in IBD, one that can be ignited by free radicals and other
oxidants present in the GI tract.
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ACKNOWLEDGEMENTS |
---|
This work was supported, in part, by a grant from Rush Univ. Medical Center, Dept. of Internal Medicine and by a grant from the American College of Gastroenterology.
![]() |
FOOTNOTES |
---|
Portions of this work were presented as a "Poster of Distinction" at the annual meeting of the American Gastroenterological Association, May 2002.
Address for reprint requests and other correspondence: A. Banan, Rush Univ. Medical Center, Div. of Digestive Diseases, Section of Gastroenterology and Nutrition, 1725 W. Harrison, Ste. 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.
June 5, 2002;10.1152/ajpgi.00143.2002
Received 12 April 2002; accepted in final form 31 May 2002.
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