1 Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215; and 2 Department of Surgery, University of Cincinnati Medical Center, Cincinnati, Ohio 45267
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ABSTRACT |
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Tumor necrosis factor (TNF) increases
epithelial permeability in many model systems. Protein kinase C (PKC)
isozymes regulate epithelial barrier function and alter ligand-receptor
interactions. We sought to define the impact of PKC on TNF-induced
barrier dysfunction in T84 intestinal epithelia. TNF induced a dose-
and time-dependent fall in transepithelial electrical resistance (TER)
and an increase in [3H]mannitol flux. The TNF-induced
fall in TER was not PKC mediated but was prevented by pretreatment with
bryostatin-1, a PKC agonist. As demonstrated by a pattern of
sensitivity to pharmacological inhibitors of PKC, this epithelial
barrier preservation was mediated by novel PKC isozymes. Bryostatin-1
reduced TNF receptor (TNF-R1) surface availability, as demonstrated by
radiolabeled TNF binding and cell surface biotinylation assays, and
increased TNF-R1 receptor shedding. The pattern of sensitivity to
isozyme-selective PKC inhibitors suggested that these effects were
mediated by activation of PKC-. In addition, after bryostatin-1
treatment, PKC-
and TNF-R1 became associated, as determined by
mutual coimmunoprecipitation assay, which has been shown to lead to
receptor desensitization in neutrophils. TNF-induced barrier
dysfunction occurs independently of PKC, but selective modulation of
novel PKC isozymes may regulate TNF-R1 signaling.
protein kinase C; tumor necrosis factor; epithelial barrier function
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INTRODUCTION |
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TUMOR NECROSIS FACTOR (TNF) is a 17-kDa cytokine that has been implicated in the pathogenesis of numerous ischemic and inflammatory diseases such as Crohn's disease, Behcet's disease, and rheumatoid arthritis (19, 49). Elevated mucosal levels of TNF are thought to contribute to the chronic inflammation, diarrhea, and increased mucosal permeability seen in patients with inflammatory bowel disease and may be important in initiating and perpetuating general inflammatory and ischemic states. Two high-affinity TNF receptors, p55 (type I, TNF-R1) and p75 (type II, TNF-R2), have been identified and are located on the basolateral surface of intestinal epithelia (16). TNF-R1 appears to be the dominant receptor involved in most of the known actions of TNF (14, 21).
An important property of intestinal epithelia is its ability to form a selectively permeable barrier that separates the internal and external environments. Barrier function, which is thought to reside in the tight junctional complexes between adjacent epithelial cells, is known to be perturbed under a variety of inflammatory, infectious, and ischemic enteropathies. TNF is one of several cytokines that has been shown to disrupt epithelial barrier function in a number of in vivo and in vitro models (14, 16, 17, 24, 32, 34, 42). However, the mechanism of TNF-induced barrier dysfunction remains incompletely understood.
Protein kinase C (PKC) represents a family of serine-threonine kinases
involved in the modulation of diverse cellular processes, including
epithelial barrier function (10, 11, 43, 45, 46). PKC is
thought to be involved in junction reformation after calcium switch
(2) by a mechanism that involves the regulated assembly
and subcellular localization of tight junction proteins (2,
28) as well as their phosphorylation states (2,
11). There are at least 11 different isozymes of PKC classified
into three broad groups. The classic PKC isozymes (,
I,
II,
and
) are both Ca2+ and diacylglycerol (DAG)
dependent. The novel PKC isozymes (
,
,
, and
) are
Ca2+ independent but DAG dependent. The atypical PKC
isozymes (
,
, and
) are neither Ca2+ nor DAG
dependent. Specificity of individual PKC isozyme action is thought to
be conferred by translocation to, and interaction with, membrane-bound
target proteins that recognize the distinct isozymes. A number of
instances have been identified in which specific isozymes affect a
given cell function in similar ways as well as in opposite ways.
PKC has been linked to a number of TNF-mediated processes (15,
18, 27, 31, 33, 50). PKC activity is elevated in colonic samples
of inflammatory bowel disease patients, where TNF levels are also known
to be elevated (39). In various model systems, PKC
isozymes have variously been found to be either protective against or
to directly mediate the pathological effects of TNF (22, 33,
50), suggesting that a counterregulatory relationship among PKC
isozymes could account for the divergent experimental results. Evidence
exists that activation or inhibition of specific PKC isozymes may
protect against inflammatory and ischemic injury (6, 9,
52). In the heart, the novel PKC- isozyme is activated by
ischemia and has been shown to protect against ischemic
insults through a process known as preconditioning (9);
PKC-
under these circumstances appears to exacerbate
ischemic damage (12). PKC-
also appears to
dampen the Cl
secretory response in a model of intestinal
epithelial ischemia (51).
In the present study, we used the T84 human intestinal epithelial cell
line to study the relationship between TNF and PKC on epithelial
barrier function. T84 cells have been widely adopted as a model for
studying the role of various inflammatory cytokines and enteric
bacteria on intestinal barrier function (7, 16, 24, 32),
and we and others (10, 20, 43, 46, 51) have previously
shown that activation of PKC regulates barrier function in T84 cells.
To examine the effects of TNF on PKC, we studied whether PKC activation
is related to TNF-induced barrier dysfunction and whether
pharmacological modulation of PKC alters the effects of TNF. Our
results suggest that the novel isozymes PKC- and -
interact with
TNF signaling at the level of its membrane receptor and may mitigate
the pathological effects of TNF on barrier function in model T84 epithelia.
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MATERIALS AND METHODS |
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Cell culture. T84 cells obtained from the American Type Culture Collection (Manassas, VA) were grown in a 5% CO2 humidified incubator at 37° on 162-cm2 flasks (Corning Costar, Acton, MA) with media containing a 1:1 mixture of Ham's F-12 nutrient mixture and DMEM supplemented with 6% heat-inactivated fetal bovine serum, 15 mM HEPES, 14.3 mM NaHCO3, and antibiotics-antimycotics (100 U/ml aqueous penicillin G, 100 µg/ml streptomycin, 250 ng/ml amphotericin B) at a pH of 7.4. Cells were passaged weekly on reaching confluence. For experiments, cells were plated onto collagen-coated permeable supports, where they were fed every 3 days and were maintained until steady-state transepithelial resistance (TER) was achieved and were used from days 7 through 14.
Cytokine treatment and TER measurements.
Dual voltage-current clamp and apical and basolateral Ag-AgCl and
calomel electrodes interfaced with "chopstick" KCl-agar bridges
were used to assess TER in confluent monolayers grown on
collagen-coated 0.33-cm2 inserts as previously described
(30, 43). TER measurements have been used as a measure of
paracellular permeability and barrier function in confluent T84
monolayers (20, 43). Baseline levels of TER in confluent
T84 monolayers generally exceed 1,000 · cm2.
Surface biotinylation. Confluent T84 monolayers grown on 4.7-cm2 Transwell inserts were treated with bryostatin-1 for up to 1 h, followed by three washes with ice-cold PBS containing 0.1 mM CaCl2 and 1.0 mM MgCl2, then followed by two 20-min incubation periods with buffer containing N-hydroxysuccinimidyl (NHS)-biotin added to both apical and basolateral surfaces. This was followed by two washes and a subsequent 20-min incubation with PBS-Ca2+-Mg2+ supplemented with 100 mM glycine to serve as the NHS-biotin quenching solution. Samples were washed again with PBS-Ca2+-Mg2+ twice, and monolayers were solubilized with 1 ml lysis buffer added for 1 h on ice. Cells were scraped from the filter, lysates were centrifuged at 14,000 g for 10 min at 4°C, and then lysates were filtered. Protein concentrations were normalized by using the Bradford method. Samples were incubated with 100 µl streptavidin beads overnight at 4°C with end-over-end rotation. The beads were then washed three times with lysis buffer, followed by two washes with high-salt buffer and one wash with no-salt buffer. Proteins were eluted by the addition of 5× Laemmli sample buffer containing DTT. Supernatants were subjected to SDS-PAGE (8% gel) followed by blotting with monoclonal antibody to TNF-R1. Samples were also taken before the addition of streptavidin beads for parallel Western blotting experiments.
125I-labeled TNF binding. Confluent T84 monolayers grown on 1.13-cm2 Transwell inserts were equilibrated for 30 min in HPBR followed by treatment with bryostatin-1 (100 nM) for 60 min. Monolayers were washed three times with ice-cold PBS and subsequently incubated at 4°C in HPBR containing 125I-TNF (10 ng/ml) in the basolateral compartment in the presence or absence of 200-fold excess cold TNF. After a 1-h incubation, filters were cut out and placed in vials containing ScintiSafe 30% Advanced Safety LSC-Cocktail scintillation fluid (Fischer Scientific, Pittsburgh, PA), and samples were measured on a scintillation counter.
In vitro kinase assay.
As previously described (58), T84 monolayers grown to
confluence on 4.7-cm2 Transwell inserts were treated with
TNF (250 U/ml) for 30 min followed by two washes in ice-cold PBS.
Protein extraction occurred by a 30-min incubation with lysis buffer
containing (in mM) 50 Tris · HCl, pH 7.5, 140 EGTA, 30 sodium pyrophosphate, and 50 NaF with 100 µM
Na3VO4 and complete protease inhibitor cocktail tablets. Samples were normalized to a concentration of 1.25 mg/ml and
were incubated overnight at 4°C with polyclonal antibodies to PKC-
(2 µg), PKC-
(2 µg), and PKC-
(4 µg). Immune complexes were
precipitated by using protein A-agarose beads (2 h incubation) and were
washed three times and resuspended in 20 µl kinase buffer containing
35 mM Tris · HCl, pH 7.5, 10 mM
MgCl2, 0.5 mM EGTA, 10 µCi [
-32P]ATP, 60 µM cold ATP, and 1 mM Na3VO4 in the presence
of 10 µg myelin basic protein at 30°C for 30 min. Sample buffer
(5× Laemmli) was added to terminate the reaction, and samples were
boiled for 5 min. Supernatants were subjected to SDS-PAGE (12% gels),
and gels were dried and analyzed by autoradiography.
Immunoprecipitation.
Confluent T84 monolayers on 4.7-cm2 Transwell inserts were
treated with bryostatin-1 (100 nM) for up to 30 min followed by two
washes in ice-cold PBS. Protein extraction occurred by a 30-min incubation with lysis buffer containing (in mM) 50 Tris · HCl, pH 7.5, 140 EGTA, 30 sodium
pyrophosphate, and 50 NaF, with 100 µM Na3VO4
and complete protease inhibitor cocktail tablets. Samples were
normalized to a concentration of 1.25 mg/ml and were incubated overnight at 4°C in the presence of monoclonal antibody to TNF-R1. Immune complexes were precipitated by using protein A-agarose beads
(2-h incubation) and were washed three times, then Laemmli sample
buffer with 10% -mercaptoethanol was added and samples were boiled
for 5 min. Supernatants were subjected to SDS-PAGE (8% gels), and gels
were blotted with polyclonal antibody to PKC-
, -
, and -
.
[3H]mannitol flux. Confluent T84 monolayers grown on 4.7-cm2 Transwell inserts were incubated in HPBR for 30 min followed by treatment with basolaterally applied TNF (250 U/ml) in the presence of [3H]mannitol (5 µCi/ml) in the basolateral compartment. Apical solution was sampled every 30 min (to detect the presence of [3H]mannitol) and exchanged with fresh HPBR buffer solution for a total of 4 h. The collected apical samples were placed in vials containing 4 ml of ScintiSafe 30% Advanced Safety LSC-Cocktail scintillation fluid and analyzed for the presence of [3H]mannitol by scintillation counter.
Detection of soluble TNF-R1. Confluent T84 monolayers grown on 1.13-cm2 Transwell inserts were equilibrated for 30 min in HPBR buffer solution followed by treatment with bryostatin-1 (100 nM) for 1-4 h. Basolateral supernatant was sampled at regular time points, and amounts of soluble TNF-R1 present were evaluated with ELISA by using a human soluble TNF-R1 detection kit supplied by R&D Systems (Minneapolis, MN).
Gel electrophoresis and Western blotting.
Samples were loaded at equal concentrations as determined by the
Bradford assay after addition of Laemmli sample buffer containing 10%
-mercaptoethanol and after 5 min of boiling. Proteins were separated
by electrophoresis on 8-12% gels and transblotted on nitrocellulose membranes, followed by a 1-h incubation at room temperature in blocking buffer [containing 20 mM Tris (pH 7.5), 500 mM
NaCl, 5% nonfat dry milk, and 0.2% Tween 20], a 1-h incubation with
blocking buffer containing primary antibody, a 30-min rinse in wash
buffer (20 mM Tris, pH 7.5, 500 mM NaCl, and 0.2% Tween 20), a 1-h
incubation with blocking buffer containing secondary antibody, and
another 30-min rinse in wash buffer. Bands were detected by using
enhanced chemiluminescence detection reagents.
Cytotoxicity assay. Confluent T84 monolayers grown on 1.13-cm2 Transwell inserts were treated with TNF (1,000 U/ml) for 4 h, then washed twice with warm (37°C) PBS and incubated with PBS containing methylthiazoletetrazolium (MTT; thiazolyl blue, 5 mg/ml) for 20 min. This was followed by two washes with ice-cold PBS. Filters were cut out and placed into an Eppendorf tube containing 1 ml of 0.1 N HCl/isopropanol and sonicated for 30 s. Supernatant was removed, spun at 14,000 g for 10 min, and again removed and measured at 570 nM.
Materials.
TNF was purchased from Sigma (St. Louis, MO). Transwell inserts were
purchased from Corning Costar. Polyclonal antibodies to PKC-, -
,
and -
and the monoclonal TNF-R1 antibody were obtained from Santa
Cruz Biotechnologies (Santa Cruz, CA). Bryostatin-1 was obtained from
Biomol (Plymouth Meeting, PA). Tissue culture reagents and agarose
beads were purchased from Life Technologies (Gaithersburg, MD). Gel
electrophoresis and gel blotting reagents were purchased from Bio-Rad
(Hercules, CA). Enhanced chemiluminescence detection reagents were
purchased from Amersham (Piscataway, NJ). Complete protease inhibitor
tablets were purchased from Boehringer Mannheim (Indianapolis, IN). The
PKC inhibitors Gö-6850, Gö-6976, and röttlerin were
purchased from Calbiochem (San Diego, CA). [
-32P]ATP
(specific activity = 3,000 Ci/mmol), [3H]mannitol
(specific activity = 17 Ci/mmol), and 125I-TNF
(specific activity = 780 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). A Quantikine sTNF R1 immunoassay detection kit was purchased from R&D Systems.
Statistical analysis.
Data are expressed as means ± SE. Statistical analysis was
performed by Student's t-test and by one-way ANOVA with the
Bonferroni-Dunn post hoc test for comparison with control, with
P < 0.05 considered statistically significant. For all
means, n 3.
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RESULTS |
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TNF causes a dose- and time-dependent fall in TER in T84 cells.
TNF was applied to the basolateral membrane of confluent T84 monolayers
at varying concentrations (10-1,000 U/ml), and TER was measured
(Fig. 1A). There is a decrease
in TER after 4 h with increasing concentrations of TNF (10 units,
96 ± 1% control; 100 units, 81 ± 1% control; 500 units,
43 ± 3% control; 1,000 units, 37 ± 3% control). Basal
short-circuit current is unchanged with TNF treatment. TNF also leads
to a time-dependent fall in TER (Fig. 1B). With TNF
treatment (1,000 U/ml), there is an initial rise in TER after 1 h
(113 ± 2% control) followed by a steady decline that was maximal
at 4 h (37 ± 3% control). With lower concentrations of TNF,
the fall in TER is slightly delayed and less pronounced (at 2 h,
250 U/ml TNF = 77 ± 8% control vs. 1,000 U/ml TNF = 65 ± 3% control; at 4 h, 250 U/ml TNF = 46 ± 5%
control vs. 1,000 U/ml TNF = 37 ± 3% control). An MTT assay
was used to assess cell viability at 4 h, and there was no
difference between untreated and TNF-treated cells at 1,000 U/ml TNF,
suggesting that direct cytotoxicity did not account for the fall in
TER.
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Evidence that the TNF-induced fall in TER is not mediated by PKC.
The effect of TNF (250 U/ml) on PKC isozyme activity was assessed by in
vitro kinase assay. After 30 min, TNF led to an increase in PKC-
activity (178 ± 25% control, P = 0.0364) and a
decrease in the activity of PKC-
(59 ± 10% control,
P = 0.0160) and PKC-
(51 ± 14% control,
P = 0.0259) (Fig.
2A).
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Bryostatin-1 pretreatment prevents the TNF-induced fall in TER.
Treatment of T84 monolayers with bryostatin-1 (100 nM) for at least 30 min before TNF exposure (250 U/ml) prevented the TNF-induced fall in
TER (at 2 h, TNF alone = 77 ± 8 vs. TNF + bryostatin-1 = 94 ± 2% control; at 3 h, TNF alone = 46 ± 7 vs. TNF + bryostatin-1 = 83 ± 1%
control, P = 0.0103; at 4 h, TNF alone = 46 ± 5 vs. TNF + bryostatin-1 = 81 ± 1% control,
P = 0.0020) (Fig. 3).
Bryostatin-1 alone increases TER in T84 cells (117 ± 4% control
after 4 h, n = 3 in triplicate, P < 0.05) through a mechanism that may be related to the regulated
assembly of tight junction proteins (51). To control for
the independent effects of bryostatin-1 on TER, the TER measurements of
the TNF and bryostatin-1-treated monolayers were normalized to
monolayers treated with bryostatin-1 alone. Subsequent data are
normalized to a bryostatin-1 control to account for the small but
significant independent effects of this agent on TER. Bryostatin-1 did
not attenuate the TNF effect on TER if administered simultaneously with
(Fig. 4) or subsequent to (not shown)
TNF.
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The effect of bryostatin-1 is mediated by a novel PKC isozyme.
We have previously shown by both subcellular fractionation and Western
blot as well as by in vitro kinase assay that bryostatin-1 induces
rapid and sustained activation of the novel PKC- and -
isozymes
in T84 cells (43). In contrast, activation of the conventional PKC-
isozyme occurs only after 2 h, followed by downregulation (43). Thus the most likely candidate
isozymes to mediate the effects of bryostatin-1 on the TNF response are novel PKC-
and -
. To confirm this, T84 monolayers were pretreated for 30 min with two different PKC inhibitors before treatment with
bryostatin-1 and TNF (250 U/ml). Gö-6850 (5 µM) inhibits conventional (
) and novel (
,
) PKC isozymes, whereas
Gö-6976 (5 µM) inhibits only the conventional (
) PKC
isozymes. Treatment with Gö-6850 blocked the bryostatin-1 effect
(at 4 h, TNF + bryostatin-1 = 81 ± 1, TNF + bryostatin-1 + Gö-6850 = 52 ± 3, and TNF
alone = 46 ± 5% control), whereas Gö-6976 did not
(TNF + bryostatin-1 + Gö-6976 = 83 ± 2%
control) (Fig. 5). This suggests that a
novel PKC isozyme (
or
) is responsible for the effects of
bryostatin-1 on preventing the TNF-induced fall in TER. This data is
also consistent with the finding that a 30-min pretreatment with
bryostatin-1 is sufficient time to prevent the fall in TER by TNF,
since only PKC-
and -
are activated by bryostatin-1 during this
time period (43).
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Bryostatin-1 induces PKC- coprecipitation with TNF-R1.
The association between TNF-R1 and PKC isozymes was assessed by
treating T84 cells with bryostatin-1 (100 nM) for up to 30 min,
followed by immunoprecipitation with TNF-R1 and subsequent Western
blotting using antibodies to various PKC isozymes. After bryostatin-1
treatment, PKC-
was found to coprecipitate with TNF-R1, whereas
PKC-
and -
did not (Fig.
6A). Coprecipitation of
PKC-
with TNF-R1 became statistically significant as early as 15 min
after bryostatin-1 treatment and was sustained over 30 min (Fig.
6B).
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PKC- activation by bryostatin-1 decreases surface TNF-R1.
Bryostatin-1 treatment led to a progressive decrease in the surface
expression of TNF-R1 as assessed by surface biotinylation and Western
blot (bryostatin-1 for 30 min = 61 ± 8 vs. for 60 min = 53 ± 27% control) (Fig.
7A). According to Western
blot, there was no decrease in total TNF-R1 after bryostatin-1
treatment (Fig. 7B), suggesting that the decrease in surface
expression of TNF-R1 was not due to a decrease in total TNF-R1 protein.
This data is consistent with 125I-TNF studies that used
radiolabeled TNF to assess cell surface binding. Bryostatin-1-treated
monolayers had reduced 125I-TNF binding (at 1 h,
71 ± 6% control, P = 0.022) (Fig.
7C), which provides further evidence that there was a
decrease in the number of surface TNF receptors. The discrepancy
between the biotinylation data (53 ± 27% control) and the
125I-TNF data (71 ± 6% control) may be explained by
nonspecific binding of 125I-TNF to the cell surface or by
binding of 125I-TNF to the TNF-R2 receptor, whose surface
expression we have not characterized and which may not be
affected by bryostatin-1.
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PKC- activation by bryostatin-1 induces TNF receptor (TNF-R1)
shedding.
T84 cells were treated with bryostatin-1, and T84 cell culture
supernatant was collected from the basolateral compartment after
1-4 h and analyzed by ELISA. Bryostatin-1 increased the amount of
soluble TNF-R1 in the basolateral supernatant in a time-dependent fashion (Fig. 8).
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DISCUSSION |
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The proinflammatory cytokine TNF increases intestinal mucosal
permeability in vivo and in vitro. We used the T84 human
intestinal cell line as a reductionist model to study the effects of
TNF on epithelial permeability. TNF has been shown in various cell lines and natural tissue models to increase epithelial permeability, although the mechanism underlying this effect is not yet established. In pulmonary endothelial cells, TNF-induced barrier dysfunction appears
to be mediated by PKC- (15). Another study implicated the p38-MAPK pathway in TNF-induced endothelial permeability
(14). In the human intestinal cell line HT-29/B6,
TNF increased epithelial permeability but could be partially inhibited
by the tyrosine kinase inhibitor genistein and the protein kinase A
inhibitor H-8 (42).
Several signaling pathways are known to regulate epithelial permeability, and TNF signaling has been shown in a number of instances to interact with these pathways. These include p44/p42 ERKs (24), p38 MAPK (14), PLC (21), PKC (8, 15, 38), myosin light-chain kinase (MLCK) (47), phosphoinositol 3-kinase (PI3-kinase) (7), and tyrosine kinase (25, 42). It is unknown whether activation of these pathways in the T84 model contribute to the TNF-induced fall in TER; however, in our hands (unpublished data), neither the MEK inhibitor PD-98059 (50 µM), the p38 MAPK inhibitor SB-203580 (25 µM), the PLC inhibitor D-609 (10 µM), the MLCK inhibitor ML-7 (20 µM), the PI3-kinase inhibitor wortmannin (100 nM), nor the general tyrosine kinase inhibitor genistein (50 µM) attenuated the TNF-associated fall in TER. Although TNF induced minor changes in the activation state of various PKC isozymes, neither the general PKC inhibitor Gö-6850 (5 µM) nor the conventional PKC inhibitor Gö-6976 (5 µM) attenuated the fall in TER, making it unlikely that PKC directly mediates the effects of TNF on junctional permeability in the T84 model.
The mechanism of the TNF-induced increase in epithelial permeability
did not appear to involve gross disruption of junctional protein
integrity, based on biochemically-defined subcellular localization and
on immunohistochemical analysis (Fig.
10). Specifically, TNF did not appear
to alter the distribution of several key tight junction proteins
(claudin-1, -2, -3, and -5; occludin; ZO-1 and -2) between Triton
X-100-soluble and -insoluble fractions. Although TNF does induce
apoptosis in the T84 line (unpublished data), this effect lags
many hours behind the initial fall in TER and is unlikely to contribute
significantly to the observed changes over the experimental time course
examined.
|
In the present study, we have demonstrated that bryostatin-1 attenuates
TNF-associated barrier dysfunction, likely through activation of a
novel PKC isozyme. The observation that pretreatment with bryostatin-1
appears to be required in this regard suggests that the underlying
mechanism involves the regulation of an early signaling event, perhaps
occurring at the level of the TNF receptor and the initiation of
subsequent signaling events. Both the novel PKC- and -
isozymes
appeared to interact with TNF-R1, albeit in strikingly different ways
(Fig. 11). It is not yet certain
whether all of these effects are required to attenuate TNF-induced
barrier dysfunction.
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We have shown that on bryostatin-1 treatment PKC- coprecipitates
with TNF-R1 within 30 min (depicted schematically in Fig. 11,
1). TNF-R1 is a known substrate of PKC-
, although this
has not been previously demonstrated in an epithelial cell line. In neutrophils, TNF treatment leads to coprecipitation of PKC-
with TNF-R1 and is associated with serine phosphorylation of the receptor and subsequent receptor desensitization (23).
Two major effects of PKC- on TNF-R1 were observed (depicted
schematically in Fig. 11). The initial decrease in surface
TNF-R1, as evidenced by both surface biotinylation studies and
125I-TNF binding studies, is likely due to internalization
of surface receptors via endocytosis (Fig. 11, 2).
Activation of PKC-
has been shown to increase the rate of
fluid-phase endocytosis at the basolateral aspect of T84 cells
(44), and it is clear that endocytosis alters the surface
expression of a number of ion transporters and is involved in cell
surface receptor recycling (3, 13, 26). PKC activation has
been shown in a number of cell lines to downregulate TNF receptor
function either by decreasing receptor number or influencing receptor
affinity (1, 4, 22, 41, 48).
PKC- activation also leads to a time-dependent increase in TNF-R1
shedding (Fig. 11, 3). The concept of TNF receptor shedding has been studied most extensively in neutrophils (1, 4, 37, 41,
48), although there has been at least one report of TNF receptor
shedding in a colonic cell line (29). We confirm that
TNF-R1 shedding does occur in T84 intestinal epithelial cells in
response to bryostatin-1, as well as to other known activators of
shedding such as TNF and PMA (data not shown). Similar to its effects
in neutrophils, receptor shedding may be another important mechanism of
downregulation of TNF responses in epithelia. Soluble TNF-R1
proteolytically released from the basolateral membrane is known to bind
to exogenous TNF, thereby affecting TNF bioavailability (5,
36). TNF-R1 shedding may also decrease the number of TNF-R1
available on the cell surface, although the effect of PKC-
on
receptor synthesis or trafficking of intracellular stores in T84
epithelia is not known. Although PKC is known to be involved in TNF
receptor shedding in multiple cell lines (1, 4, 41, 48), a
specific isozyme has not been previously implicated. Our data suggest
that PKC-
may be the key isozyme involved in TNF-R1 shedding in T84
epithelia. Whether PKC-
also affects receptor affinity in this cell
line has not yet been examined.
Inhibition of TNF signaling at the receptor level has been used in the treatment of inflammatory gastrointestinal conditions associated with elevated levels of TNF. For example, monoclonal TNF antibody is used in the treatment of patients with refractory Crohn's disease (49). Soluble TNF receptor is currently in use in clinical trials for the treatment of Crohn's disease and rheumatoid arthritis (40). TNF receptor surface regulation, through either internalization or shedding of endogenous soluble receptor, appears to be important in mitigating the pathological actions of TNF on model T84 intestinal epithelia. The development of isozyme-selective PKC activators, which are currently being developed for the treatment of ischemic heart disease (12), may be an alternative approach to the treatment of inflammatory conditions characterized by elevated TNF.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48010, DK-51630, and T32 DK-007754 (to J. Yoo).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. B. Matthews, Univ. of Cincinnati Medical Center, P.O. Box 670558, 231 Albert Sabin Way, Cincinnati, OH 45267-0558 (E-mail: jeffrey.matthews{at}uc.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 December 27, 2002;10.1152/ajpgi.00214.2002
Received 6 June 2002; accepted in final form 16 December 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aggarwal, BB,
and
Eessalu TE.
Effect of phorbol esters on down-regulation and redistribution of cell surface receptors for tumor necrosis factor-.
J Biol Chem
262:
16450-16455,
1987
2.
Andreeva, AY,
Krause E,
Muller EC,
Blasig IE,
and
Utepbergenov DI.
Protein kinase C regulates the phosphorylation and cellular localization of occludin.
J Biol Chem
2376:
38480-38486,
2001.
3.
Beron, J,
Forster I,
Beguin P,
Geering K,
and
Verrey F.
Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area.
Mol Biol Cell
8:
387-398,
1997[Abstract].
4.
Bjornberg, F,
Lantz M,
Olsson I,
and
Gullberg U.
Mechanisms involved in the processing of the p55 and the p75 tumor necrosis factor (TNF) receptors to soluble receptor forms.
Lymphokine Cytokine Res
13:
203-211,
1994[ISI][Medline].
5.
Brakebusch, C,
Nophar Y,
Kemper O,
Engelmann H,
and
Wallach D.
Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signaling, but not induced shedding of the receptor.
EMBO J
11:
943-950,
1992[Abstract].
6.
Castillo, A,
Pennington DJ,
Otto F,
Parker PJ,
Owen MJ,
and
Bosca L.
Protein kinase C is required for macrophage activation and defense against bacterial infection.
J Exp Med
194:
1231-1242,
2001
7.
Ceponis, PJM,
Batelho F,
Richards CD,
and
McKay DM.
Interleukins 4 and 13 increase intestinal epithelial permeability by a phosphatidylinositol 3-kinase pathway.
J Biol Chem
275:
29132-29137,
2000
8.
Chang, Q,
and
Tepperman BL.
The role of protein kinase C isozymes in TNF--induced cytotoxicity to a rat intestinal epithelial cell line.
Am J Physiol Gastrointest Liver Physiol
280:
G572-G583,
2001
9.
Chen, L,
Hahn H,
Wu G,
Chen CH,
Liron T,
Schechtman D,
Cavallaro G,
Banci L,
Guo Y,
Bolli R,
Dorn GW,
and
Mochly-Rosen D.
Opposing cardioprotective actions and parallel hypertrophic effects of PKC and
PKC.
Proc Natl Acad Sci USA
98:
11114-11119,
2001
10.
Chen, ML,
Pothoulakis C,
and
LaMont JT.
Protein kinase C signaling regulates zo-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A.
J Biol Chem
277:
4247-4254,
2002
11.
Clarke, H,
Soler AP,
and
Mullin JM.
Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets.
J Cell Sci
113:
3187-3196,
2000
12.
Dorn, GW II,
and
Mochly-Rosen D.
Intracellular transport mechanisms of signal transducers.
Annu Rev Physiol
64:
407-429,
2002[ISI][Medline].
13.
Farokhzad, OC,
Sagar GDV,
Mun EC,
Sicklick JK,
Lotz M,
Smith JA,
Song JC,
O'Brien TC,
Sharma CP,
Kinane TB,
Hodin RA,
and
Matthews JB.
Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl cotransporter.
J Cell Physiol
181:
489-498,
1999[ISI][Medline].
14.
Ferrero, E,
Zocchi MR,
Magni E,
Panzeri MC,
Curnis F,
Rugarli C,
Ferrero ME,
and
Corti A.
Roles of tumor necrosis factor p55 and p75 receptors in TNF--induced vascular permeability.
Am J Physiol Cell Physiol
281:
C1173-C1179,
2001
15.
Ferro, T,
Neumann P,
Gertzberg N,
Clements R,
and
Johnson A.
Protein kinase C- mediates endothelial barrier dysfunction induced by TNF-
.
Am J Physiol Lung Cell Mol Physiol
278:
L1107-L1117,
2000
16.
Fish, SM,
Proujansky R,
and
Reenstra WW.
Synergistic effects of interferon and tumour necrosis factor
on T84 cell function.
Gut
45:
191-198,
1999
17.
Grotjohann, I,
Schmitz H,
Fromm M,
and
Schulzke JD.
Effect of TNF and IFN
on epithelial barrier function in rat rectum in vitro.
Ann NY Acad Sci
915:
282-286,
2000
18.
Guo, YL,
Kang B,
and
Williamson JR.
Resistance to TNF- cytotoxicity can be achieved through different signaling pathways in rat mesangial cells.
Am J Physiol Cell Physiol
276:
C435-C441,
1999
19.
Hassard, PV,
Binder SW,
Nelson V,
and
Vasiliauskas EA.
Anti-tumor necrosis factor monoclonal antibody therapy for gastrointestinal Behcet's disease: a case report.
Gastroenterology
120:
995-999,
2001[ISI][Medline].
20.
Hecht, G,
Robinson B,
and
Koutsouris A.
Reversible disassembly of an intestinal epithelial monolayer by prolonged exposure to phorbol ester.
Am J Physiol Gastrointest Liver Physiol
266:
G214-G221,
1994
21.
Heller, RA,
and
Kronke M.
Tumor necrosis factor receptor-mediated signaling pathways.
J Biol Chem
126:
5-9,
1994.
22.
Johnson, SE,
and
Baglioni C.
Tumor necrosis factor receptors and cytocidal activity are down-regulated by activators of protein kinase C.
J Biol Chem
263:
5686-5692,
1988
23.
Kilpatrick, LE,
Song YH,
Rossi MW,
and
Korchak HM.
Serine phosphorylation of p60 tumor necrosis factor receptor by PKC- in TNF-
-activated neutrophils.
Am J Physiol Cell Physiol
279:
C2011-C2018,
2000
24.
Kinugasa, T,
Sakaguchi T,
Gu X,
and
Reinecker HC.
Claudins regulate the intestinal barrier in response to immune mediators.
Gastroenterology
118:
1001-1011,
2000[ISI][Medline].
25.
Koukouritaki, SB,
Vardaki EA,
Papakonstanti EA,
Lianos E,
Stournaras C,
and
Emmanouel DS.
TNF-alpha induced actin cytoskeleton reorganization in glomerular epithelial cells involving tyrosine phosphorylation of paxillin and focal adhesion kinase.
Mol Med
5:
382-392,
1999[ISI][Medline].
26.
Le, TL,
Joseph SR,
Yap AS,
and
Stow JL.
Protein kinase C regulates the endocytosis and recycling of E-cadherin.
Am J Physiol Cell Physiol
283:
C489-C499,
2002
27.
Lee, JY,
Hannun YA,
and
Obeid LM.
Functional dichotomy of protein kinase C (PKC) in tumor necrosis factor-alpha (TNF-alpha) signal transduction in L929 cells. Translocation and inactivation of PKC by TNF-alpha.
J Biol Chem
275:
29290-29298,
2000
28.
Lippoldt, A,
Liebner S,
Andbjer B,
Kalbacher H,
Wolburg H,
Haller H,
and
Fuxe K.
Organization of choroids plexus epithelial and endothelial cell tight junctions and regulation of claudin-1, -2, and -5 expression by protein kinase C.
Neuroreport
11:
1427-1431,
2000[ISI][Medline].
29.
Lombard, MA,
Wallace TL,
Kubicek MF,
Petzold GL,
Michell MA,
Hendges SK,
and
Wilks JW.
Synthetic matrix metalloproteinases inhibitors and tissue inhibitor of metalloproteinases (TIMP)-2, but not TIMP-1, inhibit shedding of tumor necrosis factor- receptors in a human colon adenocarcinoma (Colo 205) cell line.
Cancer Res
58:
4001-4007,
1998[Abstract].
30.
Matthews, JB,
Autrey CS,
Thompson R,
Hung T,
Tally KJ,
and
Madara JL.
Na/K/2Cl cotransport and Cl secretion evoked by heat-stable enterotoxin is microfilament-dependent in T84 cells.
Am J Physiol Gastrointest Liver Physiol
265:
G370-G378,
1993
31.
Mayne, GL,
and
Murray AW.
Evidence that protein kinase C mediates phorbol ester inhibition of calphostin C and tumor necrosis factor
induced apoptosis in U937 histiocytic lymphoma cells.
J Biol Chem
273:
24115-24121,
1998
32.
McKay, DM,
and
Singh PK.
Superantigen activation of immune cells evokes epithelial (T84) transport and barrier abnormalities via IFN- and TNF
.
J Immunol
159:
2382-2390,
1997[Abstract].
33.
Meng, XW,
Heldebrant MP,
and
Kaufmann SH.
Phorbol 12-myristate 13-acetate inhibits death receptor-mediated apoptosis in Jurkat cells by disrupting recruitment of Fas-associated polypeptide with death domain.
J Biol Chem
277:
3776-3783,
2002
34.
Mullin, JM,
and
Snock KV.
Effect of tumor necrosis factor on epithelial tight junctions and transepithelial permeability.
Cancer Res
50:
2172-2176,
1990[Abstract].
35.
Nishiyama, R,
Sakaguchi T,
Kinugasa T,
Gu X,
MacDermott RP,
Podolsky DK,
and
Reinecker HC.
Interleukin-2 receptor subunit-dependent and -independent regulation of intestinal epithelial tight junctions.
J Biol Chem
276:
35571-35580,
2001
36.
Noguchi, M,
Hiwatashi N,
Liu Z,
and
Toyota T.
Secretion imbalance between tumour necrosis factor and its inhibitor in inflammatory bowel disease.
Gut
43:
203-209,
1998
37.
Porteu, F,
and
Nathan CF.
Mobilizable intracellular pool of p55 (type I) tumor necrosis factor receptors in human neutrophils.
J Leukoc Biol
52:
122-124,
1992[Abstract].
38.
Rosson, D,
O'Brien TG,
Kampherstein JA,
Szallasi Z,
Bogi K,
Blumberg PM,
and
Mullin JM.
Protein kinase C- activity modulates transepithelial permeability and cell junctions in the LLC-PK1 epithelial cell line.
J Biol Chem
272:
14950-14953,
1997
39.
Sakanoue, Y,
Hatada T,
Horai T,
Shoji Y,
Kusumoki M,
and
Utsunomiya J.
Protein kinase C activity of colonic mucosa in ulcerative colitis.
Scand J Gastroenterol
27:
275-280,
1992[ISI][Medline].
40.
Sandborn, WJ,
Hanauer SB,
Katz S,
Safdi M,
Wolf DG,
Baerg RD,
Tremaine WJ,
Johnson T,
Diehl NN,
and
Zinsmeister AR.
Etanercept for active Crohn's disease: a randomized, double-blind, placebo-controlled trial.
Gastroenterology
121:
1242-1246,
2001[ISI][Medline].
41.
Scheurich, P,
Kobrich G,
and
Pfizenmaier K.
Antagonistic control of tumor necrosis factor receptors by protein kinases A and C.
J Exp Med
170:
947-958,
1989[Abstract].
42.
Schmitz, H,
Fromm M,
Bentzel CJ,
Scholz P,
Detjen K,
Mankertz J,
Bode H,
Epple HJ,
Riecken EO,
and
Schulzke JD.
Tumor necrosis factor-alpha (TNF) regulates the epithelial barrier in the human intestinal cell line HT-29/B6.
J Cell Sci
112:
137-146,
1999
43.
Song, JC,
Hanson CM,
Tsai V,
Farokhzad OC,
Lotz M,
and
Matthews JB.
Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms.
Am J Physiol Cell Physiol
281:
C649-C661,
2001
44.
Song, JC,
Hrnjez BJ,
Farokhzad OC,
and
Matthews JB.
PKC- regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS.
Am J Physiol Cell Physiol
277:
C1239-C1249,
1999
45.
Stuart, RO,
and
Nigam SK.
Regulated assembly of tight junctions by protein kinase C.
Proc Natl Acad Sci USA
92:
6072-6076,
1995
46.
Tai, YH,
Flick J,
Levine SA,
Madara JL,
Sharp GWG,
and
Donowitz M.
Regulation of tight junction resistance in T84 monolayers by elevation in intracellular Ca2+: a protein kinase C effect.
J Membr Biol
150:
71-79,
1996.
47.
Turner, JR,
Angle JM,
Black ED,
Joyal JL,
Sacks DB,
and
Madara JL.
PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase.
Am J Physiol Cell Physiol
277:
C554-C562,
1999
48.
Unglaub, R,
Maxeiner B,
Thoma B,
Pfizenmaier K,
and
Scheurich P.
Downregulation of tumor necrosis factor (TNF) sensitivity via modulation of TNF binding capacity by protein kinase C activators.
J Exp Med
166:
1788-1797,
1987[Abstract].
49.
Van Dullemen, HM,
Van Deventer SJH,
Hommes DW,
Bijl HA,
Jansen J,
Tytgat GNJ,
and
Woody J.
Treatment of Crohn's disease with anti-tumor necrosis factor chimeric monoclonal antibody (cA2).
Gastroenterology
109:
129-135,
1995[ISI][Medline].
50.
Wyatt, TA,
Ito H,
Veys TJ,
and
Spurzem JR.
Stimulation of protein kinase C activity by tumor necrosis factor- in bovine bronchial epithelial cells.
Am J Physiol Lung Cell Mol Physiol
273:
L1007-L1012,
1997
51.
Yoo, J,
Nichols A,
Song JC,
Cuppoletti J,
Matlin K,
and
Matthews JB.
PKC enhances barrier function in T84 epithelia through the regulation of tight junction proteins (Abstract).
Gastroenterology
122:
A55,
2002.
52.
Yoo, J,
Nichols A,
Song JC,
Mun EC,
and
Matthews JB.
Protein kinase epsilon dampens the secretory response of model intestinal epithelial during ischemia.
Surgery
130:
310-318,
2001[ISI][Medline].