Overexpression of protein kinase C-
increases tight
junction permeability in LLC-PK1
epithelia
James M.
Mullin1,
Jennifer A.
Kampherstein1,
Kathleen V.
Laughlin1,
Cheryl E. K.
Clarkin1,
R. Daniel
Miller1,
Zoltan
Szallasi2,
Bechara
Kachar3,
Alejandro Peralta
Soler1, and
Dan
Rosson1
1 Lankenau Medical Research
Center, Wynnewood, Pennsylvania 19096-3411;
2 Department of Pharmacology,
Uniformed Services University of the Health Sciences, Bethesda
20814; and 3 Laboratory of Cell
Biology, National Institute on Deafness and Other Communication
Disorders, National Institutes of Health, Bethesda, Maryland 20850
 |
ABSTRACT |
The Ca2+-independent
-isoform of protein kinase C (PKC-
) was overexpressed in
LLC-PK1 epithelia and placed under
control of a tetracycline-responsive expression system. In the absence
of tetracycline, the exogenous PKC-
is expressed. Western
immunoblots show that the overexpressed PKC-
is found in the
cytosolic, membrane-associated, and Triton-insoluble fractions.
Overexpression of PKC-
produced subconfluent and confluent
epithelial morphologies similar to that observed on exposure of
wild-type cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Transepithelial
electrical resistance
(RT) in cell
sheets overexpressing PKC-
was only 20% of that in cell sheets
incubated in the presence of tetracycline, in which the amount of
PKC-
and RT
were similar to those in LLC-PK1 parental cell sheets. Overexpression of PKC-
also elicited a significant increase in transepithelial flux of
D-[14C]mannitol
and a radiolabeled 2 × 106-molecular-weight dextran,
suggesting with the
RT decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC-
overexpression results in a
multilayered cell sheet, the tight junctions of which are almost uniformly permeable to ruthenium red. Freeze-fracture electron microscopy indicates that overexpression of PKC-
results in a more
disorganized arrangement of tight junctional strands. As with
LLC-PK1 cell sheets treated with
12-O-tetradecanoylphorbol-13-acetate, the reduced
RT, increased
D-mannitol flux, and tight
junctional leakiness to ruthenium red that are seen with PKC-
overexpression suggest the involvement of PKC-
in regulation of
tight junctional permeability.
phorbol ester; paracellular; transepithelial; resistance; cytoskeleton; mannitol; freeze fracture; dextran
 |
INTRODUCTION |
EPITHELIAL CANCERS FAR OUTNUMBER any other types of
cancer, and by themselves constitute one of the most prominent causes of death in the US population (67). Cancers from organs
such as colon, lung, breast, bladder, prostate, and uterus typically originate from epithelial cells. One of the most fundamental functions of epithelia is their ability to act collectively as a barrier. The
second fundamental characteristic of epithelia is their polarity. Two
structurally and functionally unique cell membranes face the two fluid
compartments, which the epithelium separates. The tight junction, or
zonula occludens, which encircles each epithelial cell, has a role in
maintaining epithelial polarity and in selectively sealing the
paracellular pathway and, thereby, maintaining the barrier, as
discussed in recent reviews (2, 13). Any effect on the structure and,
thereby, the permeability of the tight junction will have profound
implications on the physiological functions of the tissue that the
epithelia comprise. Evidence for physiological regulation of tight
junctional permeability is becoming increasingly common. Parathyroid
hormone-induced paracellular permeability increase of
Ca2+ and
Mg2+ in the loop of Henle (70),
increased paracellular water permeability in the collecting duct in
response to dehydration (22), and altered paracellular permeability of
the renal proximal tubule in response to glucose elevation (27) are
three examples.
Evidence for altered tight junctional structure in transformed
epithelia has been available for many years (46). For example, inflammatory bowel disease linked with increased incidence of colon
cancer has been suggested to be associated with a genetically based
alteration of tight junctional permeability, producing increased paracellular mannitol flux (29). In addition, mice treated with chemical carcinogens have been observed to have decreased
transepithelial impedance across their colonic mucosae (17). Also,
freeze-fracture electron microscopy of epithelia from transitional
carcinoma of the urinary bladder has shown a pattern of a decreased
number of tight junctional strands (60). All these findings suggest a
compromised barrier function of the epithelium.
As described in recent review articles, epithelial tight junctions are
under the control of a wide variety of agents and signal transduction
pathways (2, 63). Of particular interest in the regulation of tight
junctional permeability is the specific signal transduction component,
protein kinase C (PKC) (5, 20, 21, 28, 43, 45, 56, 64). The discovery
of PKC was intertwined with the field of tumor promoter carcinogenesis,
because the phorbol ester class of tumor promoters was recognized early as potent activators of PKC (8, 19). Phorbol esters act through PKC in
their function as tumor promoters in the two-stage model of
carcinogenesis originally developed by Boutwell (11). This model
postulates that tumor development would require a "first-stage," heritable change in DNA caused by a one-time application of a primary
carcinogen, such as dimethylbenzanthracene or dimethylhydrazine. If
this is followed by a long-term, uninterrupted exposure to tumor
promoters, such as phorbol esters, the eventual yields of tumors are
greatly enhanced.
To further understand the relationship among PKC activity, tight
junctional permeability, and epithelial cancer, it will be important to
address the following questions: 1)
Which junctional proteins are being targeted for phosphorylation with
subsequent alteration of tight junctional permeability?
2) Which members of the PKC family
may be responsible for this alteration? Our laboratory has previously
shown that the level of PKC activity and the amount of
membrane-associated PKC-
correlate with tight junctional
permeability (50, 51, 53, 54). Specifically, we showed that PKC-
expression, particularly in the membrane-associated fraction,
correlates with tight junctional leakiness and that overexpression of
PKC-
in conjunction with phorbol ester exposure results in complete
loss of barrier function (55, 59). In this report we present evidence
that the
-isoform of PKC is also capable of inducing tight
junctional leakiness, but without the requirement of phorbol esters.
 |
MATERIALS AND METHODS |
Construction of expression vectors.
The tetracycline-responsive expression system described by Gossen and
Bujard (24) was used to overexpress the wild-type form of PKC-
. cDNA
encoding the entire reading frame of mouse PKC-
was cloned into the
tetracycline-responsive vector pUHD10-3 creating pUHD10-3
wt. This
PKC-
construct has been used previously to overexpress PKC-
in
rat basophilic leukemia cells (26, 66). In addition to the entire
PKC-
reading frame, the construct also contains a COOH-terminal
epitope tag derived from mouse PKC-
.
Cell cultures and transfections.
LLC-PK1 cells, originally derived
from pig kidney cortex (30), and their derivatives were cultured in
-modified minimum essential medium (JRH) supplemented with 10%
fetal bovine serum (HyClone Laboratories), as previously described
(55). LLC-PK1tTA cells expressing
the tetracycline-responsive transactivator tTA (59) were transfected
with pUHD10-3
wt using one pulse in a Bio-Rad Gene Pulsor set at 300 V and 500 µF. pSVzeo (Invitrogen) was included to confer resistance
to the antibiotic Zeocin. Drug selection was at 1 mg/ml
Zeocin, and subclones were screened by Western analysis. One clone
expressing exogenous PKC,
LLC-PK1
wt (P
5), was selected
for further analysis. When tetracycline was present in the medium, its
concentration was 1 µg/ml.
PKC immunoblotting.
Differentiated cell sheets cultured on
75-cm2 tissue culture flasks
(Falcon), as described above, were scraped into 2 ml of buffer A (20 mM
Tris · HCl, pH 7.5, 0.25 M sucrose, 10 mM EGTA, 2 mM
EDTA, 20 µg/ml leupeptin, 10 µg/ml aprotinin, and 50 µM
phenylmethylsulfonyl fluoride) at 4°C, sonicated, and separated as
described previously into cytosolic (C) and membrane-associated (Triton
X-100-soluble, M) fractions (55). As a check on the completeness of the
Triton X-100 extraction, a second membrane extraction was performed by addition of another 150 µl of buffer
A at 4°C with 1% Triton X-100 to the pellet.
Samples were placed on a rotator for 1 h at 4°C, then centrifuged
for 1 h at 39,000 rpm. This supernatant (M2) was removed to a
separate tube. The pellet (F3) was solubilized in lysis buffer as
described above and represents the Triton-insoluble fraction.
Samples taken for total PKC-
were washed once in PBS at 4°C,
scraped, and rinsed into 2 ml of lysis buffer (150 mM NaCl, 50 mM
Tris · HCl, 1 mM EGTA, 1 mM EDTA, 1% NP-40,
0.1% sodium deoxycholate, 0.1% SDS, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 50 µM phenylmethylsulfonyl fluoride) at
4°C. The samples were then processed as described previously
(55).
SDS-PAGE was performed using 8% polyacrylamide gels on a Protean II
electrophoresis apparatus (Bio-Rad). Protein transfer to 0.45-µm
nitrocellulose (Micron Separation) was performed overnight at 15 V
using a Bio-Rad Transblot cell. After nonspecific binding was blocked
with 5% nonfat dry milk, the immunoblot was incubated with a primary
rabbit polyclonal anti-PKC-
antibody (Research and Diagnostic
Antibodies) at 1:1,000 for 1 h at room temperature. A horseradish
peroxidase-labeled goat anti-rabbit IgG secondary antibody was then
used in conjunction with the Renaissance Western blot chemiluminescence
kit (DuPont-NEN). The labeled immunoblot was then placed against
reflection autoradiography film (DuPont) and developed in a Kodak M35A
X-OMAT processor. The identity of the major band as PKC-
was
supported by experiments in which a PKC-
-blocking peptide (Research
and Diagnostic Antibodies) was first reacted with the anti-PKC-
antibody that prevented the appearance of the band in the immunoblot
analysis.
Immunofluorescent detection of PKC-
and
PKC-
.
After transepithelial resistance
(RT) was read
across cell sheets cultured on Falcon 3102 filter rings, cell sheets
were rinsed in PBS, then fixed in 3.2% formaldehyde and permeabilized
in Triton X-100, as described previously (55). After additional rinses, cell sheets were exposed to 10% normal goat serum, then incubated overnight with a rabbit polyclonal antibody to PKC-
(Research and
Diagnostics Antibodies) or with a mouse monoclonal antibody to PKC-
(Upstate Biotechnology). After rinses in PBS, cell sheets were
incubated with a 1:100 CY3 goat anti-rabbit or goat anti-mouse secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA) for 1 h in the dark. After a final rinse in PBS and distilled water, cell
sheets were mounted in glycerol and viewed with epifluorescence illumination using rhodamine filters.
Freeze-fracture electron microscopy.
Cell monolayers growing on the Falcon 3102 filter inserts were fixed in
2% glutaraldehyde in PBS for 2 h, washed thoroughly in PBS, scraped
from the surface of the filter, and progressively equilibrated with
30% glycerol in PBS for cryoprotection. After 2-3 h in the
glycerol solution the samples were placed on top of a gold disk and
rapidly frozen by immersion in liquid Freon cooled by liquid nitrogen.
After they were frozen, the samples were freeze fractured at
110°C in a Balzers 301 apparatus, shadowed with
platinum-carbon, and viewed in a Zeiss 902 electron microscope. Micrographs were printed with reverse contrast so that the platinum deposits are white.
Transmission electron microscopy and ruthenium red staining.
Cell sheets were exposed on their apical surface to 0.2% ruthenium
red, as described previously (55). After they were washed, cell sheets
were postfixed with osmium tetroxide containing ruthenium red. All
materials used for electron microscopy were purchased from Electron
Microscopy Sciences (Fort Washington, PA).
RT measurements.
As described previously (55), 4 days after cells were seeded into
Millicell PCF filter rings (Millipore), cell sheets were refed fresh
culture medium and incubated for an additional 1 h, then initial
RT measurements
were performed. Cell sheets were again refed fresh medium plus the
phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA),
and changes in RT
were followed for an additional 2 h.
Transepithelial flux of
D-[14C]mannitol
and [14C]dextran (2 ×106 mol wt).
As described previously (55), after initial
RT measurements,
cells were refed culture medium containing 0.1 mM
D-[14C]mannitol
(0.6 µCi/ml) or 14C-methylated
dextran (1.5 µCi/ml) in the basolateral compartment. At 30-min
intervals, 25-µl samples were taken from the apical fluid compartment
for liquid scintillation counting. This process continued for 2.5 h.
Flux rates were expressed as counts per minute per hour per square
centimeter or micromoles per hour per square centimeter of cell sheet
surface area. Radiolabeled mannitol and dextran are products of NEN
(Boston, MA) and Sigma Chemical (St. Louis, MO), respectively.
 |
RESULTS |
As described in a previous publication (59), we sought in these studies
to approach the problem of clonal variation (in the generation of
transfectant sublines involving single cell dilution) by utilizing an
inducible transfectant expression system. In the
tetracycline-responsive expression system of Gossen and Bujard (24),
overexpression of wild-type PKC-
is achieved only in the absence of
tetracycline, whereas its presence reduces expression to the level in
the parental cell line. Initially, the
LLC-PK1 cells were transfected
with a plasmid expressing the tetracycline transactivating protein
(tTA) along with a second plasmid conferring resistance to hygromycin.
Drug-resistant clones were then screened for tetracycline-repressible
expression of tTA. One such clone, LLC-PK1tTA, was selected for
further experimentation.
LLC-PK1tTA, used in a previous
study on exogenous expression of PKC-
(59), was then itself
transfected with a plasmid, pUHD10-3
wt, with Zeocin as a
coselectable marker. Drug-resistant clones were selected and analyzed
for expression of PKC-
. One such clone,
LLC-PK1
wt (P
5), was selected
for further study on the basis of its marked elevation of PKC-
expression.
As shown in the Western immunoblot of Fig.
1, the P
5 subline
exhibited a very high level of PKC-
expression in the absence of
tetracycline. The LLC-PK1tTA
parental cell line and the P
5 transfectant in the presence of
tetracycline showed barely detectable levels of expression of
endogenous PKC-
. The transfected PKC-
comigrated with the PKC-
from a known PKC-
-positive rat brain lysate (Transduction
Laboratories), each having an apparent molecular weight of ~75,000.
It was necessary to maintain P
5 in culture medium with tetracycline
for at least 14 days to reduce transfected PKC-
levels to the point
of indetectability.

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Fig. 1.
Western immunoblot of total protein kinase C (PKC)- in
LLC-PK1tTA parental cells
expressing tetracycline-responsive transactivator (tTA) and P 5 cell
sheets with and without tetracycline. Differentiated cell sheets
cultured in 75-cm2 tissue culture
flasks were rinsed, placed in an SDS lysis buffer, sonicated, and
processed for PKC- SDS-PAGE Western immunoblot. Each lane received
50 µg of total protein. A commercially available positive control (+)
from rat brain lysate (Transduction Laboratories) was run, and band
observed comigrated with 75-kDa band shown here. Exclusion of primary
antibody resulted in disappearance of all bands. These bands also
disappeared when blocking peptide used to generate primary antibody was
added before incubation with primary antibody. Lanes
A and B,
LLC-PK1tTA with and without
tetracycline, respectively; lanes C
and D,
LLC-PK1P 5 with and without
tetracycline, respectively.
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LLC-PK1 epithelia typically grow
in the subconfluent state as tightly associated islandlike formations.
Exposure to TPA or other PKC activators (Fig.
2, A and
B) causes the cells to partially disaggregate and develop a scalloped cell border (51, 53). In the
absence of tetracycline, P
5 manifests the morphology of a
subconfluent LLC-PK1 culture
already treated with TPA (Fig. 2C).
P
5 cultures treated for 2 h with TPA then exhibit an exaggerated morphological response as individual cells begin to partially round up
and become refractile (Fig. 2D).
P
5 cultures in the presence of tetracycline (Fig.
2E) appear morphologically similar to parental cultures (Fig. 2F).

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Fig. 2.
Phase-contrast micrographs showing effect of protein kinase C-
overexpression on morphology and response to acute phorbol ester
exposure of subconfluent LLC-PK1
cell sheets. A and
B: parental
LLC-PK1-tTA cell line in absence
and presence of 10 7 M
12-O-tetradecanoylphorbol-13-acetate (TPA, 2 h),
respectively. C and
D: P 5 transfectant (without
tetracycline) in absence and presence of TPA (2 h).
E and
F: P 5 transfectant (with
tetracycline) in absence and presence of TPA (2 h). Parental cells
(A and
B) organize in characteristic
epithelial islandlike formations, which then partially disaggregate in
presence of phorbol ester as individual cell membrane edges begin to
ruffle and scallop. P 5 transfectant in absence of tetracycline
(C and
D) is overexpressing PKC- , and
its cells never associate in islandlike formations. In presence of TPA
its cells scallop and ruffle but also eventually round up and detach
from culture dish. P 5 transfectant in presence of tetracycline
(E and
F) is virtually indistinguishable
from control.
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For confluent LLC-PK1 cultures on
a solid plastic substratum, the hallmark morphological characteristic
is the appearance of fluid-filled domes or hemicysts randomly across
the cell sheet (Fig.
3A).
These structures indicate the presence of vectorial salt and water
transport, as well as the presence of intact tight junctional seals.
Exposure of confluent LLC-PK1 cell
sheets to TPA for 2 h results in the near-complete disappearance of
domes (Fig. 3B), a phenomenon that
was previously shown to be due to the increased leakiness of tight
junctions (51, 53). Overexpression of PKC-
in the P
5 cell line
produces cell sheets with generally fewer and smaller domes or, in some
cases, no domes at all, even in the absence of TPA (Fig.
3C). Exposure of P
5 cell sheets
to TPA for 2 h causes partial rounding and detaching of cells, a response never seen in parental cells regardless of the duration of
exposure to TPA (Fig. 3D). The
morphology of P
5 cell sheets in the presence of tetracycline is
nearly identical to that of parental cell lines in control conditions
or in the presence of TPA (Fig. 3, E
and F).

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Fig. 3.
Phase-contrast micrographs showing effect of PKC- overexpression on
morphology and response to phorbol ester exposure of confluent
LLC-PK1 cell sheets.
A and
B: parental
LLC-PK1tTA cell line in absence
and presence of 10 7 M TPA
(2 h), respectively. C and
D: P 5 transfectant (without
tetracycline) in absence and presence of TPA.
E and
F: P 5 transfectant (with
tetracycline) in absence and presence of TPA. Parental cell sheets
(A) show 3-dimensional fluid-filled
domes characteristic of polarized, differentiated
LLC-PK1 cell sheets. Domes totally
disappear after 2 h in 10 7
M TPA as cells assume an arabesque-like morphology but never actually
round up or detach (B). P 5
transfectant in absence of tetracycline
(C) typically manifests fewer or, in
some areas, no domes at confluence. After 2 h in TPA medium, its cells
begin to round and detach (D).
Behavior is similar in P 5 transfectant in presence of tetracycline
and in parental cell line in absence
(E) and presence
(F) of TPA.
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Because it has been reported that PKC-
has a regulatory role in the
cell cycle (68) and its overexpression decreased growth rate (47), the
effect of PKC-
overexpression on the log-phase growth rate of
LLC-PK1 cells was examined. As
shown in Fig. 4, the P
5 transfectant did
have a slightly slower growth rate in the absence of tetracycline
(PKC-
overexpressing) than in the presence of tetracycline. In
addition, although possibly unrelated, the confluent density of P
5
was significantly higher in the absence than in the presence of
tetracycline: 2.3 × 105 vs.
1.76 × 105
cells/cm2. This
likely arises in part from the multilayering of the
LLC-PK1 cells overexpressing
PKC-
.

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Fig. 4.
Growth curve of P 5 cells in presence and absence of tetracycline
(tet). Cells from a previously confluent culture were trypsinized and
seeded at a density of 1 × 106 cells per
75-cm2 culture flask per 25 ml of
culture medium. Each day for 3 days, cells were again trypsinized and
counted individually in hemocytometers. Values are means ± SE of 3 flasks.
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When cell sheets were cultured on permeable filters, the effect on
tight junctional permeability of overexpression of PKC-
and exposure
to TPA could be evaluated by measuring
RT. The confluent LLC-PK1 cell sheet is
characterized by an
RT of ~200
· cm2. This
value was unaffected by the presence of tetracycline (Table 1). A 2-h exposure to TPA caused a >90%
decrease in RT.
Tight junctions in the P
5 subline were already leaky, as evidenced by an RT that was
only 17% of that of the parental
LLC-PK1tTA cell sheets under
control conditions. Exposure to TPA caused the RT of P
5 cell
sheets to decrease even further. In P
5 cell sheets cultured in the
presence of tetracycline,
RT values were
86% of the values in parental cell sheets, and the response of the
P
5 cell sheets to TPA was similar to the response of the parental cell lines.
To corroborate that the above differences and changes in
RT are indeed due
to changes in paracellular permeability (as opposed to changes in
transcellular conductance), the effects of PKC-
overexpression and
TPA exposure on the transepithelial flux of mannitol were also
measured. Because mannitol is limited to the paracellular route in its
flux across the cell sheet and cannot be metabolized by
LLC-PK1 cells (49), it is a
suitable marker to measure tight junctional permeability. Previous work
has shown that a 2-h exposure to a phorbol ester, such as TPA or
phorbol 12,13-dibutyrate, can increase transepithelial (paracellular) mannitol flux across these cell sheets by as much as 20-fold (53). Overexpression of PKC-
had a similarly dramatic effect (10.6-fold) on mannitol flux (Table 2), even in the
absence of TPA. Inhibition of PKC-
expression by tetracycline
reduced the difference to only a 27% increase in mannitol flux.
Demonstrating junctional leakiness to mannitol (182 mol wt) does not
address whether the tight junctions are leaky to large macromolecules.
However, the flux of 2 × 106-molecular-weight
14C-methylated dextran across
P
5 cell sheets was substantially greater in the absence than in the
presence of tetracycline (Fig. 5). This
signifies that overexpression of PKC-
confers paracellular leakiness
to a wide range of macromolecules.

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Fig. 5.
Transepithelial flux of
14C-methylated dextran across
parental and P 5 cell sheets in presence and absence of tetracycline.
After measurement of transepithelial electrical resistance
(RT), 1.5 µCi/ml of 14C-methylated dextran
(2 × 106 mol wt) was added
to basolateral fluid compartment. At 1-h intervals, 25-µl samples
were withdrawn from apical fluid and analyzed by liquid scintillation
counting. Samples were also analyzed by gel filtration chromatography
(Sephadex G50) to determine fraction of counts per minute (cpm) that
was undegraded dextran. Values are means ± SE of 3 cell sheets,
with cpm corrected for percentage of undegraded dextran.
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An additional means of ascertaining tight junctional leakiness in the
PKC-
-overexpressing P
5 cells is to observe the penetration of an
apically administered, electron-dense probe into the intercellular space. As shown in Fig. 6 and Table
3, the electron-dense dye ruthenium red
is able to permeate across all tight junctional bands of the P
5 cell
sheet. We previously observed that phorbol ester exposure of
LLC-PK1 cell sheets results in the
ability of ruthenium red to penetrate across all tight junctions (55). A P
5 cell sheet maintained in the presence of tetracycline is similar to the parental cell line, in that ruthenium red is unable to
penetrate across 95% of the tight junctional bands. In addition to its
effect on tight junctional permeability, overexpression of PKC-
in
P
5 cell sheets also resulted in multilayering of cells.
Multilayering has likewise previously been observed for chronic
exposure of LLC-PK1 cell sheets to
phorbol esters (54, 55). However, the multilayering in
LLC-PK1 cell sheets chronically treated with TPA was heterogeneous, whereas in the absence of tetracycline the multilayering occurred uniformly throughout the P
5
cell sheet.

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Fig. 6.
Effect of PKC- overexpression on permeability of
LLC-PK1 tight junctions to
ruthenium red. Electron micrographs of P 5 transfectant cells in
presence (A) or absence
(B) of tetracycline were exposed to
electron-dense dye ruthenium red, on apical surface only, for
evaluation of tight junctional permeability. A: monolayered
cells showing no penetration of ruthenium red through tight junctions
into lateral intercellular space (arrows); B:
multilayered cells with darkly stained intercellular membranes
(arrow), indicating tight junctional leakiness to apical ruthenium red.
Scale bars, 2 µm.
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Table 3.
Quantitation of electron-microscopic evaluation of tight junctional
leakiness using ruthenium red on cell sheets of P 5
transfectants with or without tetracycline
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Although the PKC-
-overexpressing cell sheets are transepithelially
leaky (as assessed electrically, by transport of radiolabeled D-mannitol and dextran, and by
penetration of apical ruthenium red into intercellular spaces), the
cells do in fact possess tight junctions, as observed by routine
transmission electron microscopy. PKC-
overexpression, however, does
cause a structural change in the tight junctions, as shown by
freeze-fracture electron microscopy. A comparison of Figs.
7 and 8
illustrates this fact, showing that with overexpression of PKC-
the
normal parallel arrays of junctional strands seen in control cells are
altered to a more disorganized network of strands with many
discontinuities. This provides a structural correlation to the
functional permeability changes described earlier (31).

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Fig. 7.
Images from freeze-fracture replicas showing cross-fracture
(A) and en face membrane fracture
views (B and
C) of tight junctions from confluent
P 5 transfectant cells in presence of tetracycline. Tight junctions
(arrows) consist of a network of 2-5 long parallel strands and
short interconnecting strands forming a continuous band separating
apical membrane (am) from lateral membrane (lm) and sealing luminal
compartment (lu). mv, Microvilli. Scale bar, 0.2 µm.
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Fig. 8.
Freeze-fracture views of tight junctions from confluent P 5
transfected cells cultured in absence of tetracycline and
overexpressing PKC- . About one-third of cell contacts have tight
junctions with morphology very similar to wild-type cells, where long
parallel strands are connected by short strands to form a continuous
belt structure (arrows in A and
B) that separates apical membrane
(am) from lateral membrane (lm) closing luminal compartment (lu).
However, in majority of cell contacts, tight junctional strands are not
organized in parallel but form a loose network with discontinuities
(C and arrowheads in D).
Loose tight junctional strands that are clearly not forming a barrier
structure are also found dispersed along lateral plasma membrane
(E and
F).
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After 6 h of TPA exposure, we did not observe pronounced disappearance
of PKC-
from the cytosolic fraction of P
5 (without tetracycline),
nor did PKC-
exhibit significant translocation to a
membrane-associated fraction (Fig. 9).
Cytosolic downregulation and translocation to membrane-associated
fractions occurred dramatically for PKC-
(50). The amount of PKC-
in the Triton-insoluble fraction (F3), however, decreased noticeably
after TPA exposure.

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Fig. 9.
Western immunoblot of PKC- (without tetracycline) in cytosolic,
membrane-associated, and Triton-insoluble fractions of P 5 cell
sheets exposed to 10 7 M
TPA. Differentiated cell sheets cultured in
75-cm2 tissue culture flasks were
refed control medium + 10 7
M TPA and reincubated at 37°C for 6 h. Cell sheets were rinsed,
placed in buffer A, sonicated, and
processed for PKC- SDS-PAGE Western immunoblot. Each lane received
50 µg of total protein. Cytosolic (C) fraction is supernatant of
buffer A centrifugation.
Membrane-associated fraction (M1) is supernatant of a subsequent
centrifugation in 1% Triton X-100. This step was then repeated once
(M2) to more completely extract fraction. Pellet of second 1% Triton
X-100 centrifugation (F3) was solubilized in 0.1% SDS lysis buffer.
Exclusion of primary antibody resulted in disappearance of all bands.
|
|
Immunofluorescent localization of PKC-
in the parental and P
5
transfectant subline in the presence of tetracycline shows similar
patterns of diffuse PKC-
staining. In the absence of tetracycline
and consequent overexpression of PKC-
(Fig.
10, A and B), one can see not only a
dramatically increased level of expression amidst a multilayering cell
sheet but also a uniform pattern of expression across all cells of the
cell sheet.

View larger version (153K):
[in this window]
[in a new window]
|
Fig. 10.
Immunofluorescent localization of - and -isoforms of PKC in
PKC- -overexpressing transfectant
LLC-PK1 subline.
A and
B: PKC- expression in P 5 cells
in presence and absence of tetracycline, respectively.
C and
D: PKC- expression in presence and
absence of tetracycline. Note high expression of PKC- in
multilayered cells in absence of tetracycline
(B) compared with low level of
expression in monolayered cells in presence of tetracycline
(A). Expression of PKC- is the
same with (C) and without
(D) tetracycline. Scale bar, 25 µm.
|
|
Overexpression of one PKC isoform has been shown to affect levels of
other isoforms (69). Coupled with the fact that PKC-
expression was
correlated with tight junctional permeability of LLC-PK1 cell sheets (50), the
level of PKC-
was also measured in the transfectant subline.
Immunofluorescence detection of PKC-
does not show any dramatic
difference of PKC-
content in the P
5 subline with or without
tetracycline (Fig. 10, C and
D). This lack of any difference
concerning PKC-
expression in the P
5 subline with or without
tetracycline was then confirmed by Western immunoblots (data not
shown). Overexpression of PKC-
did not affect PKC-
expression in
this transfectant subline. These results suggest that the observed
changes in tight junctional permeability in P
5 cells are a
consequence of PKC-
overexpression and not an indirect effect
mediated by PKC-
.
 |
DISCUSSION |
The PKC-
isoform was overexpressed in the
LLC-PK1 epithelial cell
line, which by itself was sufficient to increase tight junctional
permeability. Previously, we showed that overexpression of the PKC-
isoform affected transepithelial permeability only in the presence of
phorbol esters (59). These two studies suggest that
1) PKC-
and PKC-
can exert a
regulatory role in transepithelial paracellular permeability and
2) the unusual intracellular
localization of PKC-
in the P
5 subline may obviate the need for
exogenous PKC activators, such as phorbol esters, to achieve subsequent transepithelial leakiness. Not only do the radiotracer flux and electron-dense dye studies indicate that the transepithelial leakiness is due to paracellular leakiness, but they also further define the
RT data to show
that the increased paracellular leakiness is due to greater tight
junctional permeability and not a change in the lateral intercellular
space (4, 36, 65).
In tight junctional research, some data suggest that the paracellular
pathway is not simply "open" or "closed." Instead, a wide
variety of intermediate stages may exist, depending on the varying
physiological needs and roles of each specific epithelial tissue. For
example, the need to move glucose paracellularly across the
gastrointestinal tract after a meal (44) is very likely to involve a
tight junctional mode different from that during the chemotaxis of
neutrophils through the paracellular pathway in response to infection
(57). Both of these states would also differ from the relatively
closed mode, wherein the junctions can even discriminate
between the permeability afforded to
Na+ and that afforded to
Cl
(23). Physiological need
for a variety of tight junctional "states" suggests that many
signal transduction elements exist to create and dissipate these
states. Our studies provide evidence that PKC-
and PKC-
are two
such elements that may normally regulate tight junctions. Other PKC
isoforms are likely to regulate transepithelial permeability as well
(20, 61).
Our studies also indicate that PKC-
and PKC-
act independently to
regulate tight junctional permeability. Furthermore, the fact that
exposure of cells to phorbol esters is required to make tight junctions
leaky in PKC-
-overexpressing cells, whereas PKC-
-overexpressing cells do not share this requirement to achieve a similar physiological change, suggests that these two members of the PKC family use different
mechanisms to regulate tight junctional states. This reflects back on
the known differences in the molecular structure of PKC-
and
PKC-
, differences that are likely to affect substrate specificity,
intracellular localization, and the mechanisms by which the kinase
activity of each of these molecules is regulated.
Like all PKC isoforms, PKC-
is a single polypeptide chain with
separate regulatory and catalytic domains. Although catalytic domains
(ATP and substrate binding sites) are relatively conserved among all
three major classes of PKC (Ca2+
dependent, Ca2+ independent, and
atypical), regulatory domains vary from one class to the next as well
as among individual isoforms of a given class. Thus, although
phospholipid and diacylglycerol (phorbol ester) binding sites are
present in the regulatory domains of PKC-
and PKC-
, this domain
of PKC-
lacks a Ca2+-binding
site and has V1 and V2 variable regions unique from those in PKC-
.
It is then noteworthy that PKC-
and PKC-
display fourfold differences in dissociation constants for the phorbol ester phorbol 12,13-dibutyrate, with PKC-
having the higher affinity (34).
PKC-
and PKC-
are frequently found in different cellular
compartments in most cell types. PKC-
tends to localize in the cytosolic compartment, whereas PKC-
tends to be found in
membrane-associated compartments (10, 14, 35). In the P
5
transfectant, even in the absence of TPA, PKC-
already is
distributed in membrane-associated (Triton-soluble) and
cytoskeletal-associated (Triton-insoluble) fractions, whereas PKC-
is normally almost totally found in the cytosolic fraction of
LLC-PK1 cells (50). This might
therefore place PKC-
in a significantly different lipid environment
from that in which PKC-
exists in these cells. This may in turn lead to a difference in the normal occupancy of phospholipid and/or diacylglycerol binding sites of these two isozymes, which then determines their relative degree of activation.
Regardless of whether the level of expression of these isoforms in the
membrane-associated compartment is increased by TPA-induced translocation or by transfection, increased tight junctional
permeability results. It may be possible, therefore, that PKC-
(after translocation induced by phorbol esters) and PKC-
(after
transfection) are targeting the same substrate in the
membrane-associated fraction and that phosphorylation of this substrate
leads to increased paracellular permeability. This putative substrate
may be a junctional protein or a protein regulating the insertion of
junctional proteins into the membrane. In any event, the mechanisms of
PKC-
and PKC-
downregulation from the membrane-associated
compartment (38, 39) may, therefore, prove just as physiologically
important as their induced increases (1, 3).
Considering the phosphorylation state of PKC, there is only a single
report of Tyr phosphorylation of PKC-
(41), despite strong evidence
of phosphorylation of PKC-
on Ser/Thr residues (9, 12). However, Tyr
phosphorylation of PKC-
has been shown in numerous studies, where it
appears to play an important stimulatory role in its kinase activity.
Two PKC-
bands, ~6 kDa apart, have been seen in Western
immunoblots of various cell extracts (18, 40), and in one cell type a
second band was attributed to phosphorylation of PKC-
by the protein
product of the v-src oncogene (25). The doublet PKC-
bands seen in P
5 extracts before but not after phorbol ester exposure (Fig. 9) may therefore assume added
significance. Future work in our laboratory will determine whether
these doublet PKC-
bands are in fact due to a difference in PKC-
phosphorylation state and whether their presence and disappearance
correlate with changes in paracellular permeability.
Because of the suspected role of PKC and tumor promoters in
carcinogenesis, the aforementioned compromise of epithelial barrier function in tumorigenesis, and the demonstrated regulation of epithelial barrier function by PKC-
and PKC-
, it may be very significant that the
- and
-isoforms of PKC exhibit levels of expression in human adenocarcinoma markedly different from those in
adjacent normal mucosa (14, 16). Other reports have demonstrated differences in PKC activity and individual PKC isoform content between
tumor tissue and normal tissue (33, 37). Previous studies from our
laboratory have shown a correlation between phorbol ester induction of
tight junctional leakiness and multilayering of epithelia (50). This
same correlation was likewise found to exist in this study with
overexpression of the
-isoform of PKC. Because a strong correlation
exists between PKC activity and tight junctional permeability, a key
question becomes whether tight junctional seals are leaky as a result
of tumor development or whether such leakiness is causally involved in
tumor development. Prolonged activation of one or several PKC isoforms
may be responsible for maintaining a focus of epithelial tight
junctions in an open state, which will allow passage of growth factor
proteins across the epithelium for an extended period (48, 52). The
altered pattern of tight junctional strands shown for the
PKC-
-overexpressing cells (Fig. 8) suggests a structural change that
is significant and not instantaneously reversible.
Prolonged flux of growth factors from luminal fluid compartments into
the intercellular space and interstitium may have the potential to
produce abnormal growth kinetics and architecture and, thereby, be
causally involved in epithelial cancer. The intrinsic polarity of
epithelia places their growth factor receptors normally on their
basolateral cell surface (7, 58, 62). Luminal fluid compartments,
delimited by epithelial tissues, however, frequently contain a very
high concentration of growth stimulatory proteins. The high levels of
epidermal growth factor in urine and in the upper gastrointestinal
lumen are two examples (6, 32, 71). The epithelial barrier and
epithelial polarity together operate to separate ligand from receptor.
If, however, a focus of epithelial cells develops a condition of
chronic tight junctional leakiness and this same group of cells has
been rendered vulnerable to tumor promotion by a previous exposure to
an initiating carcinogen, a sufficient condition may exist for tumor
development. In this case, tumor development would be due specifically
to growth factor access to basolateral growth factor receptor sites
(48, 52).
 |
ACKNOWLEDGEMENTS |
We thank Dr. Peter Blumberg (National Cancer Institute), in whose
laboratory the cDNA PKC-
construct was generated, and Dr. Thomas
O'Brien (Lankenau Medical Research Center), in whose laboratory the
transfections were performed. We also thank Dr. Janet Sawicki for
editorial comments, Dr. Hilary Clarke for careful proofreading, and
Loretta Rossino and Michelle Darby (Editorial Department, Lankenau
Medical Research Center) for preparation of the manuscript.
 |
FOOTNOTES |
This work was supported in part by National Cancer Institute Grants
CA-48121 (J. M. Mullin) and CA-67113 (A. P. Soler).
Present address of C. E. K. Clarkin: Dept. of Biochemistry, University
of Pennsylvania, Philadelphia, PA 19104.
Address for reprint requests: J. M. Mullin, Lankenau Medical Research
Center, 100 Lancaster Ave., Wynnewood, PA 19096-3411
Received 28 October 1997; accepted in final form 4 May 1998.
 |
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