1Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215; and 2Department of Surgery, University of Cincinnati Medical Center, Cincinnati, Ohio 45267
Submitted 10 June 2002 ; accepted in final form 22 March 2003
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ABSTRACT |
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protein kinase C; epithelial barrier function
The tight junction, also known as the zonula occludens, is composed of
occludin, the claudin family, and ZO-1, -2, and -3. Occludin is a
tetra-spanning integral transmembrane protein that regulates paracellular
permeability (12).
Overexpression of occludin has been associated with increases in
transepithelial electrical resistance (TER)
(32). Highly phosphorylated
forms of occludin have been shown to selectively concentrate at tight
junctions (1,
4,
9,
32). The claudin family
consists of at least 23 different types of tetra-spanning 23-kDa
transmembrane proteins that are thought to confer unique permeability
characteristics on the epithelial surface of various organs
(10,
11,
13,
19,
27,
36,
41,
42). Together, claudins and
occludin form the major structural components of the tight junctional strands
that seal the intercellular space and delineate apical from basolateral
membrane domains (40).
Claudins interact as homo- and heterodimers both within and between epithelia
to form aqueous pores within the strands
(13,
41,
42).
The ZO family consists of three peripheral membrane proteins that are members of the membrane-associated guanylate kinase (MAGUK) family. ZO-1, -2, and -3 are located in the underlying tight junction plaque and link to the actin cytoskeleton. ZO-1 (220 kDa) and ZO-2 (160 kDa) appear to interact directly with each other, occludin, the claudins, and the actin-based cytoskeleton through their PDZ domains (2, 16). The organization of the tight junction into this complex structure allows for regulation of its function at multiple levels. A number of inflammatory and ischemic enteropathies are thought to disrupt the epithelial barrier through modification of these components of the tight junction complex (3, 14, 15, 23, 30, 36, 44).
Protein kinase C (PKC) is a family of serine-threonine kinases known to
regulate epithelial barrier function
(1,
3,
5,
6,
21,
22,
25,
37,
39,
43). PKC appears to regulate
both the subcellular localization and the phosphorylation states of several
tight junction-associated proteins, although isozyme specificity has not been
clearly elucidated (2,
6,
24,
31,
37,
39). There are at least 11
different isozymes of PKC classified into three broad groups. The classic
conventional (cPKC) isozymes (,
I,
II, and
) are
both Ca2+- and diacylglycerol (DAG) dependent. The novel
(nPKC) isozymes (
,
,
, and
) are
Ca2+ independent but DAG dependent. The atypical (aPKC)
isozymes (
,
, and
) are neither Ca2+
nor DAG dependent. PKC has been variably shown to induce junction assembly and
disassembly depending on the cell type and conditions of activation. It is now
recognized that specific PKC isozymes can affect the same biological function
in either a similar or opposite (counterregulatory) fashion. The pattern of
selectivity for target proteins may reflect association of the particular
isozyme with specific anchoring proteins or other protein-protein
interactions.
In the present study we used the human T84 intestinal cell line to study
the role of PKC in the regulation of barrier function and subcellular
distribution of tight junction proteins. The T84 cell line has been used
extensively to study the regulation of epithelial barrier function
(3,
21,
23,
28,
30,
31,
39,
44). T84 cells are polarized
intestinal epithelial crypt cells that display high TER and have
well-developed tight junction structures
(31). It was shown previously
that prolonged stimulation of PKC by the phorbol ester PMA reduces TER and
induces junction disassembly, an effect that appears to be due to sustained
activation of PKC- (5,
34). In the present study, we
found that bryostatin-1, a novel nonphorbol PKC agonist derived from a marine
sponge, instead increases TER. We found that bryostatin-1, likely through
PKC-
, induces occludin phosphorylation and biochemically defined
redistribution of several protein components of the tight junction.
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MATERIALS AND METHODS |
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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 (26,
34). TER measurements have
been used as a measure of paracellular permeability and barrier function in
confluent T84 monolayers (17,
34). Baseline levels of TER in
confluent T84 monolayers generally exceed 1,000 ·
cm2.
Confluent T84 monolayers were equilibrated in HEPES-phosphate-buffered Ringer solution [HPBR; containing (in mM) 135 NaCl, 5 KCl, 3.33 NaHPO4, 1 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES at pH 7.4] for 30 min before further treatment. Bryostatin-1 (100 nM) was applied to the basolateral compartment of confluent T84 monolayers, and TER was measured over a 4-h time course. Selective PKC inhibitors (Gö-6850, 5 µM; Gö-6976, 5 µM; röttlerin, 10 µM) were added 30 min before treatment with bryostatin-1.
Triton X-100-soluble and -insoluble fractions. Triton X-100-soluble and -insoluble fractions are operational definitions that have been used to biochemically define the localization of tight junction proteins, and this method has been used in a number of studies (1, 3, 30, 31, 44). Proteins found in the Triton X-100-insoluble fraction have been associated with the tight junction complex.
T84 monolayers grown on 4.7-cm2 Transwell inserts were washed twice in ice-cold PBS and then lysed with a 1% Triton X-100-based lysis buffer (1% Triton X-100, 50 mM Tris · HCl, pH 7.5, 140 mM EGTA, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, and complete protease inhibitor cocktail tablets). The supernatant was completely removed and was designated the Triton X-100-soluble fraction. The remaining filter-associated cellular residue was solubilized with heated (95°C) 1% SDS-based lysis buffer (1% SDS, 50 mM Tris · HCl, pH 7.5, 140 mM EGTA, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, and complete protease inhibitor cocktail tablets). The cells were scraped from the filter with a rubber policeman, collected, and heated at 95°C for 5 min followed by brief sonication. This was designated the Triton X-100-insoluble fraction. Protein concentrations were measured by the Lowry method, and protein concentrations in Triton X-100-soluble and -insoluble fractions were equalized separately.
Ca2+ switch. Confluent T84 monolayers grown on collagen-coated 0.33-cm2 Transwell inserts were equilibrated in HPBR. After a 30-min equilibration period, apical and basolateral buffer was switched to HPBR containing no Ca2+ with EGTA (2 mM). Buffer was later switched back to Ca2+-containing HPBR in the absence of EGTA. TER was measured over time. T84 monolayers grown on collagen-coated 4.7-cm2 Transwell inserts were used for parallel Western blotting experiments to assess occludin in both Triton X-100-soluble and -insoluble fractions.
Immunoprecipitation. Confluent T84 monolayers on
4.7-cm2 Transwell inserts were treated with bryostatin-1 (100 nM)
for up to 4 h followed by two washes in ice-cold PBS. Extraction of Triton
X-100-soluble proteins occurred by a 30-min incubation with lysis buffer
containing 1% Triton X-100, 50 mM Tris · HCl, pH 7.5, 140 mM EGTA, 30
mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4,
and complete protease inhibitor cocktail tablets. The resulting extract was
completely aspirated and constituted the Triton X-100-soluble fraction. The
remaining filter-associated cellular residue was next used for the extraction
of Triton X-100-insoluble proteins, which occurred by addition of heated
(95°C) lysis buffer containing 1% SDS, 50 mM Tris · HCl, pH 7.5,
140 mM EGTA, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM
Na3VO4, and complete protease inhibitor cocktail
tablets. The filters were scraped with a rubber policeman, heated at 95°C
for 5 min, and sonicated. 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 occludin. The Triton X-100-insoluble proteins were diluted 1:5
with 1% Triton X-100 lysis buffer to avoid degradation of antibody. Immune
complexes were precipitated with protein A agarose beads (2-h incubation) and
washed three times, Laemmli sample buffer with 10% -mercaptoethanol was
added, and samples were boiled for 5 min. Supernatants were subjected to
one-dimensional SDS-PAGE, and gels (8%) were blotted with phosphospecific
antibodies.
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 boiling for 5
min. Proteins were separated by electrophoresis on 812% gels and
trans-blotted 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, 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, 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 with enhanced chemiluminescence (ECL) detection reagents.
Materials. Transwell inserts were purchased from Corning Costar. 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). ECL 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). Antibodies to ZO-2, occludin, phosphoserine, phosphothreonine, and claudin-1, -2, -3, and -5 were purchased from Zymed (San Francisco, CA). Anti-phosphotyrosine antibody and antibody to ZO-1 were purchased from Transduction Laboratories (Lexington, KY).
Statistical analysis. Data are expressed as means ± SE.
Statistical analysis was performed by Student's t-test, one-way
ANOVA, and Tukey pairwise multiple-comparison test, with P < 0.05
considered statistically significant. All n 3.
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RESULTS |
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Occludin exists in Triton X-100-soluble and -insoluble fractions. The assembly of structural proteins into the tight junctional complex is a dynamic process that involves changes in their association with components of the cytoskeleton. Biochemically, this association or assembly event can be operationally defined by changes in detergent (Triton X-100) solubility (1, 3, 30, 31, 44).
Occludin has been shown to partition into both Triton X-100-soluble and
-insoluble fractions (Refs. 1,
7,
30,
31;
Fig. 2A). The Triton
X-100-soluble fraction appears to represent cytoplasmic and basolaterally
associated forms of occludin, whereas the Triton X-100-insoluble fraction,
characterized by higher-molecular-weight (HMW) forms reflecting enhanced
occludin phosphorylation, is associated with the tight junction complex
(1,
4,
9,
32). Because of these
phosphorylated forms, occludin exhibits a molecular weight in the range of
6585 on Western blot. Occludin was immunoprecipitated from Triton
X-100-soluble and -insoluble fractions followed by Western blot analysis with
phosphospecific antibodies. By this method, HMW occludin appears to be
phosphorylated predominantly on threonine and tyrosine residues
(Fig. 2B).
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Bryostatin-1 treatment is associated with an increase in HMW forms of
occludin. Confluent T84 monolayers were treated with bryostatin-1 (100
nM) over 4 h, and occludin was assessed in both Triton X-100-soluble and
-insoluble fractions. Bryostatin-1 treatment of confluent T84 monolayers was
associated with an increase in HMW forms of occludin in both the Triton
X-100-soluble and -insoluble fractions
(Fig. 3A). This effect
was evident by 2 h, which correlated with the time course of increase in TER,
and was sustained over a 4-h time period. Bryostatin-1 did not affect total
protein levels of occludin as assessed by Western blot (data not shown),
suggesting that the new HMW forms seen after bryostatin-1 treatment occurred
through post-translational modification of occludin. The
bryostatin-1-associated increase in HMW occludin was selectively sensitive to
PKC inhibitors. These changes were attenuated by the cPKC and nPKC inhibitor
Gö-6850 (5 µM) but not by the cPKC inhibitor Gö-6976 (5 µM) or
by the PKC--specific inhibitor röttlerin (10 µM)
(Fig. 3B). This
pattern of inhibitor sensitivity suggests that PKC-
may be the key PKC
isozyme responsible for occludin phosphorylation.
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Bryostatin-1 recruits claudin-1 to Triton X-100-insoluble fraction. Confluent T84 monolayers were treated with bryostatin-1 (100 nM) for 4 h, and claudin-1, -2, -3, and -5 were assessed in both Triton X-100-soluble and -insoluble fractions. Within the claudin family of proteins we examined, bryostatin-1 selectively affected the biochemical localization of claudin-1 (Fig. 4A). Bryostatin-1 treatment led to a roughly 50% reduction in the amount of claudin-1 found in the Triton X-100-soluble fraction (bryostatin-1 treated = 54 ± 5% control; P = 0.0014; Fig. 5A), with a parallel increase of claudin-1 in the Triton X-100-insoluble fraction (174 ± 13% control; P = 0.0024; Fig. 5B). The distribution of claudin-2, -3, and -5 between Triton X-100-soluble and -insoluble fractions was unaffected by bryostatin-1 treatment.
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To establish a time course for the shift in claudin-1, T84 cells were
treated with bryostatin-1 (100 nM) and claudin-1 was assessed in Triton
X-100-soluble and -insoluble fractions at 1, 2, and 4 h. The shift in
claudin-1 occurs somewhere between 2 and 4 h
(Fig. 4B), which, like
occludin, was consistent in time with the bryostatin-1-induced increase in
TER. Bryostatin-1 did not affect total protein levels of claudin-1 as assessed
by Western blot (data not shown). The bryostatin-1-induced decrease in Triton
X-100-soluble claudin-1 displayed the same pattern of sensitivity to PKC
inhibitors (Fig. 5A),
being attenuated by the cPKC and nPKC inhibitor Gö-6850 (5 µM;
bryostatin-1 + Gö-6850 = 87 ± 4% control, bryostatin-1 alone = 54
± 5% control; P = 0.0015) but not by the cPKC inhibitor
Gö-6976 (5 µM; bryostatin-1 + Gö-6976 = 41 ± 6% control)
or the PKC--specific inhibitor röttlerin (10 µM; bryostatin-1 +
röttlerin = 37 ± 7% control). Similarly, the parallel increase of
claudin-1 in the Triton X-100-insoluble fraction
(Fig. 5B) was also
blocked by Gö-6850 (bryostatin-1 + Gö-6850 = 98 ± 13%
control, bryostatin-1 alone = 174 ± 13% control; P = 0.0042)
but not by Gö-6976 (bryostatin-1 + Gö-6976 = 193 ± 22%
control) or röttlerin (bryostatin-1 + röttlerin = 214 ± 41%
control).
Bryostatin-1 treatment increases ZO-2 in Triton X-100-insoluble
fraction. Confluent T84 monolayers were treated with bryostatin-1 (100
nM) over 4 h, and the tight junction proteins ZO-1 and ZO-2 were assessed in
Triton X-100-soluble and -insoluble fractions. Bryostatin-1 did not appear to
alter ZO-1 in either Triton X-100-soluble or -insoluble fractions
(Fig. 6A).
Bryostatin-1 also did not appear to affect the amount of ZO-2 found in the
Triton X-100-soluble fraction over the 4-h time course (111 ± 3%
control at 4 h; Fig.
6A) but did increase the amount of ZO-2 found in the
Triton X-100-insoluble fraction (Fig.
6A) (At 4 h, bryostatin-1 treated = 182 ± 11%
control; P = 0.002). Similar to its effects on occludin and
claudin-1, this effect was evident after 2 h of bryostatin-1 treatment
(Fig. 6B) and was
sustained over 4 h, which again correlated in time with the
bryostatin-1-induced increase in TER. Similar to occludin and claudin-1, total
ZO-2 protein was not affected by bryostatin-1 as assessed by Western blot
(data not shown). The increase of ZO-2 in the Triton X-100-insoluble fraction
(Fig. 7B) was blocked
by the cPKC and nPKC inhibitor Gö-6850 (5 µM; bryostatin-1 +
Gö-6850 = 111 ± 7% control, bryostatin-1 alone = 182 ± 11%
control; P = 0.018) but not by the cPKC inhibitor Gö-6976 (5
µM; bryostatin-1 + Gö-6976 = 190 ± 11% control) or by the
PKC--specific inhibitor röttlerin (10 µM; bryostatin-1 +
röttlerin = 167 ± 5% control).
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DISCUSSION |
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Bryostatin-1 treatment led to time-dependent changes in the phosphorylation of occludin in both Triton X-100-soluble and -insoluble fractions. The phosphorylation of occludin regulates its association with the cytoskeleton or with detergent-insoluble glycolipid rafts (DIGs) (1, 31) and appears to be a key step in tight junction assembly (1, 4, 9, 32). This has been confirmed in Ca2+ switch experiments (data not shown), in which the presence of HMW forms of occludin correlated with increases in TER. Although we were able to show further increases in HMW occludin forms, we were unable to identify increases in phosphorylation from baseline levels on tyrosine or threonine residues with occludin immunoprecipitation and Western blot with phosphospecific antibodies. A more sensitive technique (e.g., metabolic labeling for phosphoamino acid analysis) is likely required to demonstrate these changes.
Although a role for PKC in the regulation of occludin phosphorylation has
been suggested on the basis of earlier studies
(4), there have been
contradictory findings regarding the phosphorylation state and its
relationship to TER (1,
7,
25,
34). It is likely that these
seemingly paradoxical findings are due to the simultaneous activation of
multiple PKC isozymes that may have opposing effects on tight junction
structure and function. It is increasingly well established that different PKC
isozymes can exert opposing effects on the same biological function. For
example, we previously demonstrated
(34) that PKC- and
PKC-
have opposing effects on Cl- secretion and cytoskeletal
structure in the T84 cell line.
The present study used bryostatin-1 to demonstrate an increase in TER
associated with occludin phosphorylation and suggests a role for PKC- in
this response. In contrast, previous work suggests that activation of
PKC-
decreases TER. On the basis of these observations, we propose that
PKC-
and PKC-
exert opposing actions on the macroscopic property
of junctional permeability (6,
21,
25,
34,
35). Although bryostatin-1
does activate PKC-
, it does so only transiently and after an extended
incubation (
4 h) and is followed by rapid downregulation of this isozyme
(34). Bryostatin-1, in
contrast to PMA, does not induce the long-term junctional disassembly seen
with phorbol ester (17) and in
fact antagonizes this action, presumably because of its ability to
downregulate PKC-
(5).
Although the precise mechanism of this PMA/PKC-
effect remains to be
determined, we suspect that it is probably unrelated to acute changes in
occludin phosphorylation because PMA induces changes in occludin
phosphorylation similar to those found here with bryostatin-1 (unpublished
observations) and, as in the case for bryostatin-1, this phosphorylation is
not sensitive to Gö-6976 (although the PMA-induced fall in TER is
completely blocked by this agent; Ref.
34).
Bryostatin-1 treatment also shifted claudin-1 from the Triton X-100-soluble to the insoluble fraction in a parallel fashion. This apparent shift in the biochemically defined localization of claudin-1 suggests that a cytoplasmic or membrane-associated pool (Triton X-100-soluble fraction) was being mobilized. Claudin-1 contributes to epithelial barrier function (11, 19, 41), and its subcellular distribution can be regulated by PKC (24). Overexpression of claudin-1 in MDCK cells increased TER and decreased paracellular flux to FITC-dextrans of different molecular sizes (19). In this MDCK model, claudin-1 and occludin colocalized in the tight junction and in cytoplasmic vesicles, suggesting that these proteins may be processed and targeted to the tight junction together.
Bryostatin-1 also increased the recruitment of ZO-2 to the Triton X-100-insoluble fraction. ZO-2 has also been demonstrated to be a target of phosphorylation by PKC (2). Unlike occludin, phosphorylated ZO-2 may not be entirely targeted to the tight junction but may also appear in non-junction-associated locations (2). It is not known whether bryostatin-1 treatment additionally affects the phosphorylation state of ZO-2 in our system.
Although PKC- activation appears to be associated with changes in the
subcellular localization and phosphorylation state of several tight junction
proteins, the precise mechanism whereby PKC-
exerts this effect remains
unclear. Although our data suggest that the novel isozyme PKC-
is the
key isozyme involved in these changes, this conclusion must be considered
tentative because it is largely based on sensitivity to pharmacological
inhibitors. A PKC-
-specific translocation inhibitor peptide failed to
reliably block bryostatin-1-induced PKC-
translocation in T84 cells;
anti-sense oligonucleotide downregulation of PKC-
was also unsuccessful
in this cell line. Our results suggest, but do not prove, that barrier
function of epithelia may be dynamically regulated by PKC-
. aPKC
isozymes such as PKC-
have been identified as potential regulators of
barrier function because they colocalize with tight junctions in a number of
cell lines (MDCK, LLC-PK1, Caco-2, and T84)
(6,
8,
38,
43). However, there is no
evidence to suggest that PKC-
is involved in bryostatin-1-induced tight
junction protein regulation in our model system. Bryostatin-1 activates the
,
, and
PKC isozymes but does not activate PKC-
in
our T84 cell line (34).
Similarly, the PKC inhibitors used in this study have no inhibitory effect on
the aPKC isozymes. Therefore, the specific effects of bryostatin-1 on TER and
tight junction protein regulation are not likely to be due to PKC-
.
There is some evidence that PKC- may also localize to the tight
junction in intestinal epithelia
(33). We previously
demonstrated (35) that in the
T84 cell line bryostatin-1-induced activation of PKC-
leads to its
translocation to the basolateral membrane, but we have not yet established
colocalization with the tight junction complex. Preliminary colocalization and
coimmunoprecipitation studies for PKC-
with the tight junction proteins
occludin and ZO-2 after bryostatin-1 treatment have failed to provide evidence
of a direct interaction (unpublished observations). Thus it is unclear whether
PKC-
interacts indirectly via an intermediate kinase or phosphatase or
another target. The intermediate signaling pathways following PKC-
activation have not yet been identified, although the bryostatin-1-induced
increase in TER was not inhibited by PD-98059 (50 µM), an inhibitor of the
p42/p44 MAPK pathway (data not shown). It is possible that PKC-
acts on
tight junction proteins either directly at the tight junction complex or on
cytoplasmic and/or membrane-associated pools, which are then targeted to the
tight junction complex.
The observation that bryostatin-1 specifically affects occludin, claudin-1, and ZO-2 (and not other protein components) in concert suggests that a specific protein stoichiometry may be required for junctional assembly and proper function. Imbalances in protein components may affect the integrity of this complex, as has been suggested both in overexpression models (27) and with functional loss of one component of the tight junction complex (30). Conversely, in disease states in which tight junction components are disturbed, accumulation and stabilization of one part of this complex may attenuate the degree of tight junction disruption. This was demonstrated in the MDCK line, where constitutively active Rho, which regulates the actin cytoskeleton as well as the phosphorylation state of occludin (15, 18), partially prevented tight junction disruption during ATP depletion (15). Maintaining the molecular organization of both the tight junction strands and the link via ZO family proteins to the actin cytoskeleton is likely to be critical for the functional properties of the tight junction complex.
Most studies concerning the regulation of tight junction proteins have
generally fallen into three main categories. Tight junction assembly and
disassembly has been studied most extensively with the
Ca2+ switch model
(1,
22,
32,
37). The relative importance
of specific tight junction proteins has been studied through deletion,
transfected overexpression, or incorporation of a structurally modified
protein into the tight junction complex
(11,
27,
29). Finally, the modification
of tight junction proteins by known toxins or enteropathogenic bacteria has
been used to identify important structural regulators of epithelial
permeability (3,
23,
30,
36,
44). For example, the
Clostridium perfringens enterotoxin has been shown to remove
claudin-4 from tight junction strands, which correlated in time with a
decrease in tight junction strand number, a decrease in TER, and an increase
in paracellular flux (36). Our
approach in the present study is novel in that it involves the augmentation of
TER in an already confluent epithelial monolayer. Furthermore, although PKC as
a broad family of proteins has been identified as an important regulator of
epithelial permeability, this is the first study to suggest that a novel
isozymeprobably PKC-may play a role in the steady-state
regulation of tight junction integrity. We speculate that PKC-
activation can augment barrier function by enhancing the recruitment of key
proteins to the tight junction complex. Also, similar to their mutually
antagonistic effects on other biological functions, PKC-
and PKC-
may also exert opposing effects on epithelial barrier function.
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FOOTNOTES |
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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.
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