Division of General and Gastrointestinal Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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The phorbol ester phorbol
12-myristate 13-acetate (PMA) inhibits Cl secretion
(short-circuit current, Isc) and decreases
barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in
this response, we compared PMA with two non-phorbol activators of PKC
(bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme
selectivity profiles. PMA sequentially inhibited cAMP-stimulated
Isc and decreased TER, as measured by
voltage-current clamp. By subcellular fractionation and Western blot,
PMA (100 nM) induced sequential membrane translocation of the novel
PKC
followed by the conventional PKC
and activated both isozymes
by in vitro kinase assay. PKC
was activated by PMA but did not
translocate. By immunofluorescence, PKC
redistributed to the
basolateral domain in response to PMA, whereas PKC
moved apically.
Inhibition of Isc by PMA was prevented by the
conventional and novel PKC inhibitor Gö-6850 (5 µM) but not the
conventional isoform inhibitor Gö-6976 (5 µM) or the PKC
inhibitor rottlerin (10 µM), implicating PKC
in inhibition of
Cl
secretion. In contrast, both Gö-6976 and
Gö-6850 prevented the decline of TER, suggesting involvement of
PKC
. Bryostatin-1 (100 nM) translocated PKC
and PKC
and
inhibited cAMP-elicited Isc. However, unlike
PMA, bryostatin-1 downregulated PKC
protein, and the decrease in TER
was only transient. Carbachol (100 µM) translocated only PKC
and
inhibited Isc with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1
and carbachol inhibition of Isc. We conclude
that basolateral translocation of PKC
inhibits Cl
secretion, while apical translocation of PKC
decreases TER. These
data suggest that epithelial transport and barrier function can be
modulated by distinct PKC isoforms.
isoenzymes; intercellular junctions; cytoskeleton; colonic neoplasms; intestinal mucosa; chlorides; intestinal secretion; cystic fibrosis; endocytosis; cell membranes
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INTRODUCTION |
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PROTEIN KINASE C
(PKC) regulates a number of fundamental properties of epithelial cells
including vectorial transport and barrier function. PKC profoundly
alters epithelial Cl secretion, the physiological process
that accounts for mucosal surface hydration (13, 30),
through activation or inhibition of specific ion transporters and
channels (8, 9, 12). In previous reports, we and others
have shown that extended activation of PKC by phorbol 12-myristate
13-acetate (PMA) progressively inhibits cAMP-regulated Cl
secretion in human intestinal T84 cells, a well-characterized model of
epithelial Cl
secretion (1, 8, 20). In
addition, PKC influences the barrier function of epithelial cells. In
T84 cells, we have shown that PMA impairs junctional integrity and
slowly but profoundly decreases transepithelial electrical
resistance (TER) (7, 20). Hecht and coworkers
(10) showed that extended PMA treatment induces
disassembly of T84 epithelial monolayers and causes cellular multilayering.
The PKC family of serine/threonine kinases plays a crucial role in
diverse cellular responses such as membrane trafficking, cytoskeletal
organization, ion transport, cell growth, and differentiation. At least
11 isoforms of PKC are known, and these are usually categorized into
three distinct subtypes: conventional (cPKC) isozymes (,
I,
II, and
), novel (nPKC) isozymes (
,
,
, µ, and
),
and atypical (aPKC) isozymes (
and
/
)
(23, 31). These three subtypes vary in their sensitivity
to activators and cofactors: the cPKC isozymes are dependent on
phosphatidylserine and the second messengers diacylglycerol (DAG) and
Ca2+, and they can also be activated by PMA. The nPKC
isozymes are similar to the cPKC isozymes in sensitivity to activators,
except they obtain full catalytic activity in the absence of
Ca2+. The aPKC isozymes are independent of DAG or
Ca2+ and, as a general rule, cannot be directly activated
by PMA. The PKC isoforms are widely distributed to varying degrees in mammalian tissue- and cell-specific patterns. Moreover, PKC isoforms exhibit distinct subcellular localizations within individual cell types.
A hallmark of activation of PKC family members is translocation from one biological compartment in the inactive state (e.g., cytosol) to another in the activated state (e.g., plasma or organellar membrane). However, translocation is not an absolute requirement; examples of changes in kinase activity without changes in subcellular localization (and vice versa) are known. Because there is considerable overlap in substrate specificity of the individual PKC isozymes, the precise subcellular localization of inactive and active forms probably confers isozyme specificity in regulating biological processes (23, 31).
The role of specific PKC isoform(s) in the alteration of T84 cell
epithelial transport and barrier function by PMA has not been clearly
defined. While PMA can have non-PKC cellular targets (e.g.,
-chimaerin), PKC-inactive phorbol esters do not affect T84 monolayer
TER or Cl
secretion. Interestingly, we have found that
several non-phorbol PKC agonists exert some but not all of the effects
of PMA on epithelial phenotype. For example, bryostatin-1 has minimal
effect on barrier function despite its ability to inhibit
transepithelial Cl
secretion (7). In fact,
bryostatin-1 is able to partially antagonize the effect of PMA on
barrier function (7). We hypothesized that different PKC
agonists are capable of activating selective subsets of PKC isoforms
that differentially affect the cellular properties of transport and
barrier function in T84 epithelia. To approach this hypothesis, we
exploited the differential effects of three PKC agonists and three
isozyme-selective PKC inhibitors to elucidate the major isoforms
involved in downregulation of transport and barrier function in this
model system.
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METHODS |
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Cell culture. T84 human intestinal epithelial cells obtained from Dr. Kim Barrett (University of California, San Diego) were grown to confluence at pH 7.4 in 162-cm2 flasks (Corning Costar, MA) with a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture supplemented with 6% fetal bovine serum (FBS), 15 mM HEPES, 14.3 mM NaHCO3, and antibiotics/antimycotic. Flasks were passaged weekly and fed every 3 days. Cell monolayers for experiments were grown to confluence on collagen-coated Transwell inserts (Corning Costar). Monolayers were fed every 3 days and used after stable TER was achieved, ~7-14 days postplating.
Electrophysiology.
Under a wide variety of experimental conditions, short-circuit current
(Isc) has been shown to approximate net
Cl secretion in T84 monolayers bathed in
HEPES-phosphate-buffered Ringer solution (5).
Isc was therefore used as a general assay of
transepithelial transport function and was measured in confluent monolayers grown on 0.33-cm2 permeable supports using a
dual voltage-current clamp and Ag-AgCl and calomel electrodes
interfaced via "chopstick" KCl-agar bridges, as previously
described (20, 21). In the absence of agonist stimulation,
the basal Isc of T84 monolayers is near zero,
and the TER is generally in excess of 1,000
· cm2. Thus, in this "tight" model
epithelium, TER in electrically quiescent monolayers is a convenient
measure of paracellular permeability and barrier function
(5). It has previously been validated, for example, that
the decrease in TER evoked by long-term treatment of T84 monolayers
with PMA represents, specifically, an increase in paracellular
permeability (10). TER is calculated by Ohm's law from
the voltage deflection induced by a 25-µA external current pulse, as
previously described.
In vitro kinase assay.
Confluent T84 monolayers grown on 4.7-cm2 permeable
supports were treated with various PKC agonists and washed twice with
cold PBS. Proteins were extracted by 30-min incubation in ice with 500 µl of apical lysis buffer (LB) containing 50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1% (vol/vol) Triton X-100 (TX-100), 2 mM EDTA, 1 mM EGTA, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, and Complete protease inhibitor
cocktail tablets. The protein concentration of each sample was measured
and adjusted to contain 500 µg in 400 µl of LB. Polyclonal
antibodies against cPKC (2 µg), nPKC
(4 µg), or nPKC
(2 µg) were added to each sample for overnight rotation at 4°C. After
incubation, immune complexes were precipitated using protein A-agarose
beads, washed, resuspended in 20 µl of kinase buffer (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) with or without PKC inhibitors and
incubated with 10 µg of myelin basic protein as a substrate at 30°C
for 30 min. After incubation, the reaction was terminated by adding 5×
Laemmli sample buffer to the samples, and the samples were boiled for 5 min. The supernatants were subjected to SDS-PAGE (12% gels), and the
gel was dried and subjected to autoradiography.
Immunofluorescence and microscopy.
Monolayers grown on 0.33-cm2 permeable supports were
treated with PMA in medium for the specified time and washed three
times with cold PBS. Cells were then fixed in 4% paraformaldehyde for 1 h at room temperature, washed with PBS twice, permeabilized with
0.1% (vol/vol) TX-100 in PBS for 7 min, and rinsed with PBS twice.
Filter membranes were cut out in rectangular shape from the Transwell
plastic assembly, placed between 50 µl of blocking buffer (1% normal
goat serum, 3% BSA in PBS) at both the top and bottom of the
monolayers, and incubated for 30 min at room temperature. Polyclonal
antibodies against either PKC or PKC
were diluted to 10 µg/ml
in the blocking buffer containing 0.1% TX-100, and 50 µl of each
antibody were placed at both the top and bottom of the monolayers.
After overnight incubation in a moisture chamber at 4°C, monolayers
were washed in PBS three times for 10 min and incubated in
rhodamine-conjugated goat anti-rabbit polyclonal IgG (1:100 dilution)
for 1 h at room temperature along with FITC-phalloidin for F-actin
staining. Monolayers were then washed three times in PBS and mounted on
the microscope slide with Vectashield mounting medium. Confocal images
were acquired using a Zeiss inverted microscope equipped with MRC-1024
and Lasersharp software (Bio-Rad).
Subcellular fractionation. T84 cells grown to confluence on collagen-coated permeable supports (4.7 cm2) were washed with ice-cold PBS three times and scraped into 400 µl of the cold homogenization buffer (HB) containing 20 mM Tris · HCl, pH 7.5, 250 mM sucrose, 4 mM EDTA, 2 mM EGTA, and Complete protease inhibitor cocktail tablets. The cells were homogenized on ice with 25 strokes of a glass tissue homogenizer. The resulting homogenate was ultracentrifuged at 86,000 g for 50 min at 4°C (TLA 45 rotor, TL-100 Ultracentrifuge; Beckman). The supernatant was designated the cytosolic fraction. The pellet was resuspended in 400 µl of the HB containing 0.5% (vol/vol) TX-100 by brief sonication and incubated in ice for 30 min. At the end of the incubation period, the samples were centrifuged at 14,000 g for 20 min at 4°C. The resulting supernatant was designated the membrane fraction.
Gel electrophoresis and Western blotting.
Equal amounts (~50 µg/sample) of protein, as determined by the
Bradford assay, were combined with Laemmli's Sample Buffer containing
5% (vol/vol) -mercaptoethanol and boiled for 5 min. Proteins were
separated by electrophoresis on 7.5% SDS-PAGE gels and transblotted to
nitrocellulose membranes. The protein-bound nitrocellulose sheets were
first incubated for overnight at 4°C in a blocking buffer containing
20 mM Tris, pH 7.5, 500 mM NaCl, and 5% nonfat dry milk.
Nitrocellulose sheets were then incubated with the polyclonal
antibodies to different PKC isoforms diluted in the blocking buffer
(PKC
1:10,000, PKC
1:100, PKC
1:100, and PKC
1:100) for
1 h at room temperature and rinsed for 30 min with a wash buffer
containing 20 mM Tris, pH 7.5, 500 mM NaCl, and 0.2% Tween 20. Finally, the membranes were incubated with horseradish peroxidase
(HRP)-conjugated goat anti-rabbit IgG antibody (1:3,000 dilution) for
1 h at room temperature and washed for 30 min with agitation,
during which the wash buffer was changed every 5 min. PKC bands were
visualized with ECL (enhanced chemiluminescence) detection reagents.
Materials.
Tissue culture reagents and protein A-agarose beads were purchased from
Life Technologies, and gel electrophoresis and Western blotting
reagents were from Bio-Rad, with the exception of ECL detection
reagent, which was purchased from Amersham. Complete protease inhibitor
cocktail tablets were from Boehringer Mannheim, and FITC-phalloidin was
from Molecular Probes. Anti-PKC for Western blotting was obtained
from Sigma, and anti-PKC
, anti-PKC
, and anti-PKC
for
immunofluorescent staining and in vitro kinase assay were purchased
from Santa Cruz Biotechnology. Secondary antibodies conjugated with
various fluorescent dyes were from Jackson Laboratories, and
Vectashield mounting medium was from Vector Laboratories. Secondary antibodies conjugated with HRP were obtained from Bio-Rad. The PKC inhibitors Gö-6976, Gö-6850, and rottlerin were
obtained from Calbiochem. [
-32P]ATP with a specific
activity of 3,000 Ci/mmol was purchased from NEN. All other chemicals
were from Sigma.
Statistical analysis. Data are reported as means ± SE. Data were analyzed by one-way ANOVA with Bonferroni/Dunn's post hoc test for comparison with control. Statistical significance is indicated where P < 0.05.
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RESULTS |
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Effect of PMA on T84 cell transport function.
Our previous results (7, 8, 20, 22) and the results of
others (30) indicate that treatment of confluent T84
monolayers grown on permeable supports with PMA progressively inhibits
cAMP-elicited Cl secretion. Figure
1A shows the peak
Isc achieved in response to stimulation by 10 µM forskolin at varying times after exposure to 100 nM PMA. Under
control conditions, forskolin markedly stimulated Isc, reaching the peak (baseline
Isc = 4.0 ± 0.2 µA/cm2,
peak Isc = 135 ± 16 µA/cm2, P < 0.05) within 15 min.
Inhibition of peak Isc was seen well within
1 h of PMA exposure. The IC50 for
Isc inhibition by PMA was ~70 nM (Fig.
1B).
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Effect of PMA on PKC isozymes in T84 cells.
We previously showed that PMA increases total PKC activity in T84 cells
(20) and translocates both cPKC and nPKC
(35) but not nPKC
or aPKC
. To further delineate the
action of PMA on PKC isozymes in T84 cells, we performed an in vitro
kinase assay. The time course for kinase activation of PKC
and
PKC
by PMA closely correlated with membrane translocation of these isozymes (Figs. 2A and
6A). Unexpectedly, despite the absence of translocation in
Western blot experiments, PKC
kinase activity was increased within
30 min of exposure to PMA (Fig. 2A). Thus PMA increases the
kinase activity of PKC
, PKC
, and PKC
in T84 cells, but
translocation is only evident by our methods for PKC
and PKC
. We
next examined which of these isoforms was involved in regulation of
transport and barrier function.
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Effect of isozyme-selective PKC inhibitors on PMA inhibition
of T84 transport function.
To elucidate the PKC isoform involved in regulation of T84
transport function, we first examined the effect of three structurally unrelated PKC inhibitors, Gö-6976, Gö-6850, and rottlerin,
which have well-established but different activity profiles against cPKC and nPKC isoforms. The substituted indolocarbazole compound Gö-6976 has been shown to inhibit cPKC isoforms exclusively
(IC50 2 nM against PKC
in vitro)
(19), with no demonstrable in vitro inhibitory activity
against novel Ca2+-independent or aPKC isoforms even at
high micromolar concentrations. We examined this selectivity for T84
cells by in vitro kinase assay. As expected, 5 µM Gö-6976 had
no inhibitory effect on PKC
in T84 cells but clearly inhibited
activity of PKC
in vitro (Fig. 2B). However,
Gö-6976 did show some inhibitory activity against nPKC
at the
5 µM concentration in T84 cells. The bisindolmaleimide compound
Gö-6850 is known to inhibit both cPKC and nPKC isoforms (IC50
8 nM and 132 nM in vitro for PKC
and
PKC
, respectively) (19). In T84 cells, 5 µM
Gö-6850 completely inhibited PMA activation of cPKC
, nPKC
,
and nPKC
in the in vitro kinase assay as shown in Fig.
2B. At 10 µM, rottlerin is rather specific for the nPKC
isoform (IC50
3-6 µM), weakly active
against cPKC isoforms (IC50
30 µM), and inactive
against PKC
(IC50
100 µM) (46).
We also confirmed this for T84 cells. Rottlerin showed a selective inhibition of PKC
in vitro at 10 µM concentration without
affecting PKC
while weakly inhibiting PKC
in T84 cells (Fig.
2B).
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Effect of PMA and isozyme-selective inhibitors on T84 cell-barrier
function.
Figure 4A shows that treatment
of T84 monolayers with 100 nM PMA results in a decrease in TER to
28 ± 5% of control within 4 h (baseline TER 1,142 ± 21 · cm2 vs. 4-h PMA TER 318 ± 32
· cm2). However, closer examination of the time
course of this change indicates that the earliest evidence of a change
in basal TER occurs well after the observed inhibition of
cAMP-stimulated Isc, which was prominently
evident within 1 h. The IC50 of PMA for inhibition of
TER after 2 h exposure was 300-500 nM as shown in Fig.
4B. In contrast to results with cAMP-stimulated
Isc, both Gö-6976 and Gö-6850 at 5 µM inhibited the PMA-induced decline of the TER (Fig.
5A). IC50 values
for Gö-6976 and Gö-6850 in vivo were ~2 and ~1 µM,
respectively (Fig. 5B). Rottlerin (10 µM) partially
reversed the effect of PMA. This partial inhibition could be due to the
ability of rottlerin to weakly inhibit PKC
at 10 µM (Fig.
2C). Rottlerin at a higher concentration inhibited both
cPKC
and nPKC
(data not shown) and thus is not a valid tool for
the inhibitor study. The strong sensitivity to Gö-6976 and
partial inhibition by rottlerin suggest that activation of the
conventional isoform PKC
is associated with downregulation of
junctional integrity.
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Spatiotemporal characteristics of PKC and PKC
translocation
in response to PMA.
Given the finding from the inhibitors study that nPKC
and cPKC
are the major isoforms responsible for regulation of transport and
barrier function in T84 cells, we further characterized the time course
for their membrane translocation upon PMA addition to substantiate the
finding. With Western blot analysis, both PKC
and PKC
were found
predominantly associated with the cytosolic fraction in control
monolayers (Fig. 6A). Upon PMA
addition, PKC
promptly translocated to the membrane as early as 15 min after PMA addition. Translocation of PKC
, however, did not begin
to become evident until 60 min after treatment. Activation of both PKC
and PKC
continued for at least 4 h without significant
degradation. The sequential activation of PKC
followed by PKC
is
consistent with our functional data obtained using selective PKC
isoform inhibitors that correlate early activation of PKC
with
Isc inhibition (Fig. 1A) and later
activation of PKC
with inhibition of TER (Fig. 4A).
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Effect of bryostatin-1 and carbachol on T84 cell transport and
barrier function.
Like PMA, the non-phorbol ester PKC agonist bryostatin-1 rapidly
translocated PKC to the membrane fraction of T84 cells (Fig. 9A). PKC
in this fraction
was sustained for at least 4 h after bryostatin-1 addition without
any evidence of downregulation (degradation) of total PKC
protein
(Fig. 9B). We also confirmed similar activation of PKC
by
the in vitro kinase assay (data not shown). In parallel experiments,
the peak Isc elicited by forskolin was
significantly inhibited by 100 nM bryostatin-1 (e.g., 25 ± 1%
control at 2.5 h, Fig. 9C), similar to our earlier
reported findings (7). Inhibition of
Isc was prevented by 5 µM Gö-6850 but
not Gö-6976 or 10 µM rottlerin, consistent with the notion that
PKC
is the key isoform involved in inhibition of epithelial
Cl
secretion. Membrane translocation of PKC
, on the
other hand, occurred substantially later with bryostatin-1 than with
PMA (Fig. 10A), and, also in
contrast to PMA, the total level of both cytosolic and membrane PKC
reduced to 39 ± 10% control, suggestive of PKC
downregulation
or degradation. Barrier function was only transiently (and relatively
minimally) affected (Fig. 10B). The basal TER was 94 ± 5% control at 4 h after bryostatin-1 addition.
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DISCUSSION |
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Activation of PKC by phorbol esters exerts complex and
time-dependent effects on transepithelial Cl secretion.
In dog trachea, for example, PMA induces a transient activation of
Isc within minutes of exposure, followed by a
progressively profound inhibition of cAMP-dependent electrogenic ion
transport (2). In native tissue models, it is difficult to
ascertain whether the effect of PMA (and, by extension, PKC) is exerted directly at the level of the Cl
secretory epithelial cell
or on altered neurohormonal regulatory input. However, PKC has been
shown to acutely regulate several ion transporters and channels
involved in Cl
secretion. PKC has been shown to activate
CFTR Cl
channels at low intracellular Ca2+
concentrations (15), perhaps by facilitating protein
kinase A-dependent phosphorylation of CFTR (12, 48).
Liedtke and coworkers (16-18) showed that
Na+-K+-2Cl
cotransporter (NKCC1)
in airway epithelia is acutely activated by PKC
. The use of cultured
epithelial cell lines as models of electrogenic Cl
secretion has facilitated detailed mechanistic analysis of the effects
of PKC on specific elements of the secretory apparatus. In certain
intestinal lines (e.g., HT29cl.19A cells) (1, 42, 43), but
not in others (T84 cells) (2, 20), PMA induces a
transient, albeit small, increase in Isc.
However, the dominant effect of PMA appears to be inhibitory. Warhurst
et al. (45) noted that phorbol ester markedly inhibited
the T84 cell Cl
secretory response to prostaglandin
E2 by a mechanism that involved receptor desensitization
(45), although a variety of later studies indicated a
profound inhibition of Isc responses to all
cAMP-mediated stimuli, including permeant analog (20, 22,
30). Initially, the mechanism of inhibition of Cl
secretion was thought to involve downregulation of CFTR gene expression
(39). However, later studies indicated that the inhibitory effect of PMA on Cl
secretion in fact preceded effects at
the apical membrane by at least several hours (20,
30). Rather, inhibition of transepithelial Cl
secretion by PMA correlated most closely with
inhibition of several basolateral membrane transport sites including
K+ channels (1, 30) and NKCC1 (8,
20). However, the basis for PMA inhibition of multiple
independent transport pathways at the basolateral membrane by PMA was
not established. As one possible unifying mechanism, we postulated that
PMA promoted the endocytic retrieval (and thereby reduced surface
expression) of multiple transport pathways at the basolateral membrane
(35).
In the current study, we have provided several lines of evidence
linking activation of PKC to the progressive inhibition of
cAMP-dependent Cl
secretion by PMA in the T84 cell model.
First, inhibition of Isc by PMA was prevented by
the cPKC and nPKC isoform inhibitor Gö-6850 but not the
PKC
-selective inhibitor Gö-6976 or the PKC
-specific
inhibitor rottlerin. The specificity of these inhibitors was confirmed
by in vitro kinase assay. Second, early inhibition of
forskolin-stimulated Isc temporally correlates
with immediate activation of PKC
as shown by in vitro kinase assay
as well as early translocation of PKC
, which occurred within 15 min
after PMA addition. Moreover, bryostatin-1 and CCh both inhibited
Isc and translocated PKC
, and in the case of
CCh, PKC
is the only PKC isoform noted to be translocated and
activated during this time period. Finally, immunolocalization studies
showed that in response to PMA, PKC
takes on a distribution
associated with the basolateral membrane.
The roles of PKC in cell function, particularly in epithelial cells,
remain poorly understood. A role in cytoskeletal organization was
suggested on the basis of findings that F-actin can serve as an
isozyme-selective RACK (receptor for activated C kinase) for PKC
(29) and that PKC
is a MARCKS (myristoylated
alanine-rich C kinase substrate) kinase (3). In cardiac
myocytes, PKC
appears to play a major role in ischemic
preconditioning and functions within the context of p42/p44
mitogen-activated kinase pathway in response to diverse cellular growth
factors and forms of cell stress (27, 28). PKC
has been
shown to associate with caveolae in cardiac myocytes (33),
suggesting that it may play a role in membrane traffic and in
coordinating integrated signaling responses within these specialized
membrane microdomains. Weller et al. (47) recently showed
that activation of PKC
in colon cancer cells may be a trigger for
proliferative responses to PMA, and, indeed, overexpression of PKC
promotes tumorigenicity (26). In T84 cells, Chow et al.
(4) showed that PKC
is activated in response to
epidermal growth factor (EGF) and may participate in the negative
regulation of Ca2+-dependent Cl
secretion by
EGF and CCh.
In addition to vectorial transport, epithelial cells also possess the property of barrier function. PKC appears to play an important role in junction formation after epithelial disassembly, such as in the Ca2+ "switch" model, although the precise mechanism whereby PKC regulates this process remains to be established (37). PKC-dependent junction assembly is initiated at the level of E-cadherin and the zonula adherens, rather than at the tight junctions, and thus this process may not necessarily be mediated by the same PKC isoenzyme(s) that influence paracellular permeability (which is largely determined at the level of the zonula occludens). PKC-dependent junctional hyperpermeability in confluent T84 monolayers is known to be induced simply by an elevation of cell Ca2+ (38), although whether the Ca2+ dependence of junctional permeability reflects a specific role for a cPKC is uncertain.
The present experiments closely link extended activation of PKC to
impaired barrier function in model T84 epithelia. First, we observed
that TER remains relatively constant after PMA treatment (and PKC
translocation) until a time that follows the later translocation of
PKC
to the membrane fraction. Second, the PMA-associated fall in TER
was prevented by Gö-6976, a PKC inhibitor that is highly selective for Ca2+-dependent cPKC isoforms. Third, in
response to PMA, PKC
translocated from the basal cytoplasm to the
apical zone of T84 monolayers, in the vicinity of the junctional
complexes and perijunctional actomyosin ring known to affect junctional
integrity. Comparison of the effect of PMA with that of other PKC
agonists provides indirect support for the hypothesis that PKC
is
the key isoform involved in junctional regulation. For example, CCh,
unlike PMA, had no effect on TER and did not alter PKC
subcellular
distribution at any time point.
Bryostatin-1, compared with PMA, induced a delayed membrane
translocation of PKC that was associated with a smaller and
transient fall in TER. The return of TER toward control levels with
extended bryostatin-1 treatment may reflect accelerated degradation
(downregulation) of PKC
. Indeed, bryostatin-1 has been shown to
induce proteosome-mediated degradation of PKC
through enhanced
ubiquitinization (14). Bryostatin-1, which shares with PMA
an affinity for the DAG binding site of PKC, is known to induce a
subset of the cellular responses evoked by PMA and, interestingly, to
antagonize many of the responses it does not share with PMA
(11). Thus our earlier finding that bryostatin-1 is able
to partially antagonize the effect of PMA on TER (7) is
likely to reflect the ability of bryostatin-1 to downregulate PKC
.
We speculate that bryostatin-1-activated PKC
can induce only minimal
effects on TER before it is depleted and that the early downregulation
of PKC
by bryostatin-1 prevents extended PMA activation of PKC
and thereby attenuates the fall in TER.
A role for PKC in junctional regulation in epithelia has previously
been postulated (32). Notably, Mullin et al.
(24) showed that overexpression of wild-type PKC
in
LLC-PK1 cells renders the cells more sensitive to
PMA-induced junctional disruption, whereas expression of a
dominant-negative PKC
construct renders them resistant. Other PKC
isoforms may also influence junctional structure and permeability under
certain conditions. For example, junctional permeability is increased
by overexpression of PKC
in cultured renal epithelial cells
(24), and in Madin-Darby canine kidney and Caco-2
epithelial cells, the aPKC
is the only isoform that specifically
localizes near the tight junctional complex (6). Because
the PMA-induced decrease in TER was partially inhibited by rottlerin, a
role for PKC
cannot be entirely excluded. However, at the
concentration used, rottlerin also partially inhibited PKC
. It is
unknown, and our studies could not address, whether PMA can alter
PKC
activity, and thus a role for this aPKC also cannot be excluded.
Our localization data indicate that PMA induces an intracellular
redistribution of PKC toward the apical zone of the cell, where it
may potentially interact directly or indirectly with various components
of the tight junction. The target of PKC
that leads to altered
junctional permeability remains to be elucidated but is likely to
involve the cytoskeleton. Hecht et al. (10) showed that
disruption of T84 monolayer integrity by PMA is associated with
disruption of perijunctional F-actin (10). The
permeability characteristics of tight junctions are known to be
modulated by the tension of the perijunctional actin-myosin ring,
which, in turn, is mediated by myosin light chain kinase (MLCK)
(41). PKC is known to itself alter phosphorylation of both
MLC and MLCK, although whether this mechanism can account for the
observed effects of PMA in T84 cells remains speculative. Conflicting
reports have appeared regarding this concept. Turner et al.
(40) showed in a Caco-2 subclone that PMA acutely
increased MLCK phosphorylation and decreased MLC phosphorylation; this
response was associated with an acute increase in TER, presumably due
to relaxation of the perijunctional actin-myosin ring. Other Caco-2
clones, however, have been shown to behave similarly to T84 cells with
a progressive decrease in TER in response to PMA (36).
Enhanced actin-myosin contractile activity through PKC-mediated
regulation of MLCK also has been reported (25), but in one
instance, enhanced junctional permeability due to phorbol ester was
shown to be independent of MLCK (34). Other potential
targets of PKC
must also be considered. Interestingly, phorbol
ester-induced barrier dysfunction in endothelial cells appears to
involve extracellular signal-regulated kinase (ERK1/2) signaling via
Ras (44).
In summary, by using multiple agonists and isozyme selective
inhibitors, we have been able to dissociate PKC actions on transport function and barrier function in T84 model epithelia. Activation of
PKC appears to inhibit electrogenic Cl
secretion,
whereas extended activation of PKC
decreases TER. The present
studies demonstrate that PKC-dependent stimuli can elicit divergent
effects on epithelial cell function through differential activation of
distinct PKC isoenzymes, which in turn act at distinct subcellular
localizations. It thus may prove possible to selectively target
specific PKC isoenzymes for activation, inhibition, or downregulation
in the context of antidiarrheal and anticancer drug development.
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ACKNOWLEDGEMENTS |
---|
This work was presented in part at the American Gastroenterological Association Annual Meeting, May 2000, San Diego, CA.
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FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48010 and DK-51630 (J. B. Mathews).
Address for reprint requests and other correspondence: J. B. Matthews, Department of Surgery, University of Cincinnati College of Medicine, 231 Albert B. 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.
Received 5 October 2000; accepted in final form 13 March 2001.
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