(Received for publication, February 18, 1997, and in revised form, March 26, 1997)
From the Lankenau Medical Research Center, Wynnewood,
Pennsylvania 19096-3411 and the ¶ Laboratory of Cellular
Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health,
Bethesda, Maryland 20892-4255
Modulation of protein kinase C (PKC) by
12-O-tetradecanoylphorbol-13-acetate (TPA) disrupts the
cell-cell junctions of the epithelial cell line LLC-PK1. To
examine the role of specific PKC isoforms in this process we have
created modified LLC-PK1 subclones that express wild-type
and dominant negative versions of PKC- under control of the
tetracycline-responsive expression system. Overexpression of wild-type
PKC-
rendered the cells more sensitive to the effects of TPA on
transepithelial permeability as measured by loss of transepithelial
resistance across the cell sheet. Conversely, expression of a dominant
negative PKC-
rendered the cells more resistant to the effects of
TPA as measured both by loss of transepithelial resistance as well as
cell scattering. The properties of both subclones could be modulated by
the addition of tetracycline, which suppressed the effect of the
exogenous genes. These results indicate that the
isoform of PKC is
at least one of the isoforms that regulate tight junctions and other cell-cell junctions of LLC-PK1 epithelia.
Epithelial cells cover almost all internal and external body surfaces. All types of epithelia are polarized with apical and basolateral surfaces that have different membrane proteins and thereby different functions. The cells thus compartmentalize the tissues of which they are a part. The cells exist in sheets in which individual cells are interconnected by various types of cell-cell junctions, the regulation of which is at play during development, wound healing, and pathological processes such as cancer or chronic inflammation. Therefore, the control of cell-cell junctions and their disruption and reformation is of interest in several fields.
The LLC-PK1 cell line (1) offers an advantageous model system for studying this process. The cell line grows subconfluently as islands of adherent cells. Upon reaching confluence, it forms a differentiated epithelial monolayer with functionally intact tight junctions. Treatment of subconfluent cultures with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)1 causes a breakdown of the cell junctions resulting in a more scattered growth pattern. Acute exposure of LLC-PK1 confluent cell sheets to TPA causes a rapid decrease in the transepithelial resistance (TER) to less than 15% of its initial value (2) because of its effect on one type of cell junction known as tight junctions.
We have been interested in the molecular events in this process and
have concentrated much of our efforts on protein kinase C (PKC), the
molecular target of TPA. PKC has been shown to be one of the process's
key components. Treatment of LLC-PK1 cells with the PKC
inhibitor GF109203X inhibits the effects of TPA (3). PKC mediates
calcium-induced tight junction assembly (4), and its inhibition
prevents the proper distribution of tight junction-associated proteins
such as ZO-1 and cingulin (5). One of the issues in PKC-mediated cell
junction regulation is which specific isoform(s) is at play. PKC is not
a single protein, as the name implies, but a family of proteins with at
least 11 different members (6). Although each member or isoform is
encoded by a separate gene, they all consist of conserved regulatory
and catalytic regions. The isoforms can be divided into four classes
based on regulatory properties. The first class, the conventional PKCs
,
, and
, is regulated by calcium. The second, nonconventional
or novel class, is made up of PKCs
,
,
, and
, which are
not regulated by calcium. The third class, the atypical PKCs
and
(
/
), is not regulated by calcium or TPA. A recently discovered
isoform, PKC-µ, represents a fourth class, which is topologically
related to the PKC family but possesses a kinase domain more related to calcium/calmodulin-dependent kinases. The complexity of the
PKC family and the differential expression of the isoforms suggest that
the members serve distinct roles in signal transduction processes. To
examine this issue, as it relates to PKC-mediated cell junction regulation in LLC-PK1 epithelial cells, we have begun to
modulate the activities of individual isoforms by exogenously
expressing wild type and dominant negative versions of the genes in an
inducible expression system. LLC-PK1 cells express the
,
,
, and
isoforms of PKC, and, in this study, we report that
alteration of the activity of the
isoform of PKC modulates the
integrity of cell-cell junctions as measured by sensitivity to TPA.
Expression vectors
comprising the tetracycline-responsive expression system described by
Gossen and Bujard (7) were used to express both wild-type and dominant
negative versions of PKC-. cDNA encoding the entire reading
frame of wild-type PKC-
was excised from its original vector (8) by
digestion with EcoRI and recloned into the EcoRI
site of the tetracycline-responsive vector pUHD10-3 creating
pUHD10-3
.
A dominant negative version of the gene was created by in
vitro mutagenesis replacing the conserved lysine in the ATP
binding domain in position 368 with an alanine. The
XcmI-BsiWI fragment spanning the Lys Ala
mutation was then excised from its expression vector to replace the
analogous fragment in pUHD10-3
vector creating pUHD10-3
DN.
LLC-PK1 cells
were maintained in minimum essential medium supplemented with 10%
fetal bovine serum (HyClone Laboratories). When tetracycline was
present, its concentration was 1 µg/ml. For transfection, cells were
harvested by trypsinization, washed, and 5 × 106
cells were resuspended in 0.8 ml of phosphate-buffered saline in an
electroporation cuvette (0.4 cm). 20 µg of pUHD15-1, which encodes
the bacterial transactivator tTA, along with 5 µg of a plasmid
conferring resistance to hygromycin, were added to the suspension. This
was subjected to one pulse (300 V; 250 microfarads) in a Bio-Rad
electroporator. Cells were then plated onto three 10-cm tissue culture
plates, incubated for 1 day, then adjusted to 400 µg/ml hygromycin.
2-4 weeks later, drug-resistant clones were isolated, expanded, and
analyzed by transient transfection for tetracycline-responsive
expression of a luciferase reporter gene cloned into the pUHD10-3
expression vector. One of the resulting subclones,
LLC-PK1tTA, was selected for subsequent constructions, which were to stably transfect the PKC-
expression vectors described above. This was performed by the same electroporation procedure utilizing 5 µg of pSVzeo (Invitrogen) and 1 mg/ml zeocin for
selection. Expression of wild-type and dominant negative PKC-
was
assessed by Western analysis.
Cells were harvested by trypsinization followed by centrifugation. Cell pellets were then lysed in Tris-Cl buffer containing 0.1% sodium dodecyl sulfate and proteinase inhibitors. DNA was sheared to reduce viscosity, and the protein concentration was determined with Bio-Rad protein assay dye reagent. 50 µg of total protein was mixed with an equal volume of 2 × sample buffer (0.125 M Tris-Cl, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 10% 2-mercaptoethanol, 0.002% bromphenol blue). Samples were heated in a boiling water bath for 5 min and then loaded onto a denaturing 6% polyacrylamide electrophoresis gel.
After separation, proteins were electrophoretically transferred to a
nitrocellulose membrane. The filters were first treated with
Tris-buffered saline (50 mM Tris-Cl, pH 7.5, 0.15 M NaCl) with 0.05% Tween 20 (TBST) containing 3% nonfat
milk. This was followed by hybridization in 5 ml of TBST with 1%
bovine serum albumin and 1 µg of anti-PKC- antibody (Upstate
Biotechnology, Inc.) for 5 h at room temperature. The blots were
subsequently washed in TBST and then incubated with anti-mouse
peroxidase-conjugated antibody (Amersham) for 1 h. After washing
in TBST, the blots were developed using the enhanced chemiluminescence
Western blotting system (Amersham).
Electrical equipment and protocols for
electrical measurement have been described before (9). Briefly, 1 × 106 cells were seeded onto Falcon 3102 filter rings (4.2 cm2). On day 4, after which electrical resistance across
the cell sheet had stabilized, cells were refed with fresh media,
incubated for 1-2 h, and then their initial TER was measured. Cell
sheets were then refed with media containing 1.0 × 108 M TPA, and electrical resistance across
the cell sheet was determined through the course of resistance
decreases. Measurements were done in triplicate, and all results are
representative of at least three experiments.
For assessment of cell
scattering 3 × 103 cells were seeded in 24-well
plates in 1 ml of medium containing 108 M
TPA. Growth patterns were followed by light microscopy and photographed
3 days after seeding.
Since we have noted previously that TPA treatment of
LLC-PK1 cells is associated with a translocation of PKC-
from the cytosolic fraction of the cells to the membrane fraction (9),
we decided to modulate the activity of PKC-
by exogenous expression
of the gene. Because randomly isolated sublines can vary from the
parental in any given property, there is consequent difficulty in
ascribing any phenotypic change to the expression of an exogenous gene. We chose to avoid the problems of clonal variation by utilizing the
tetracycline-responsive expression system of Gossen and Bujard (7) to
overexpress PKC-
. In the first step, we cotransfected into
LLC-PK1 cells a plasmid expressing tTA and a plasmid
conferring hygromycin resistance. After screening drug-resistant clones
for tetracycline-repressible expression of tTA, we selected a subline, LLC-PK1tTA, for additional experiments. Next, pUHD10-3
PKC-
was transfected into LLC-PK1tTA using pSVzeo
(Invitrogen) as a coselectable marker. 20 drug-resistant clones
were analyzed for expression of PKC-
, and one was found to have
elevated levels. Fig. 1 shows Western analysis of
extracts of cells cultured with and without tetracycline, expressing
exogenous PKC-
. Parental cells exhibited a single protein of
approximately 75 kDa. LLC-PK1
cells grown in the absence
of tetracycline exhibited PKC-
levels that were 10-20 times higher.
The same subline grown in the presence of tetracycline showed levels
that were almost equal to levels in the parental LLC-PK1
tTA line. Phenotypically, the cells resemble the parental line, growing
as islands of coherent cells. Upon reaching confluence, both the
transfectant and the parental line remain a single layer and
differentiate into a polar epithelial-like cell sheet with apical and
basolateral surfaces. Both lines form dome-like or cystic structures as
a result of the vectorial transepithelial transport and the tight
junctions of the cell sheet. However, as seen in Fig. 2,
not only do domes collapse when treated with TPA, but the transfected
cells rapidly round up and begin to detach from the dish. When
expression of the exogenous PKC-
was down-regulated by the addition
of tetracycline, the normal dome collapse occurred on TPA treatment but
not the cell rounding and detaching. The disruption of tight junctions
was assessed quantitatively in measurements of TER decreases on TPA
treatment. As seen in Fig. 3, cells overexpressing PKC-
showed a more rapid response to all concentrations of TPA tested compared with wild type LLC-PK1. However, when these
cells were grown in the presence of tetracycline, TER decreases matched that of the parental line. The presence of tetracycline had no effect
on TER decreases in wild-type LLC-PK1 (data not shown).
To generate a subline of LLC-PK1 cells expressing an
inducible dominant negative version of PKC-, we transfected
pUHD10-3
DN into LLC-PK1tTA by the same
procedure as described above. Fig. 4 shows the Western
analysis of four clones that were isolated. Each showed an exogenous
protein running 4-5 kDa below the endogenous PKC-
. In all four
clones, the exogenous protein was down-regulated significantly by
tetracycline. PKC is first synthesized as an inactive unphosphorylated
polypeptide. It is first phosphorylated by an unknown PKC kinase and
then undergoes two autophosphorylations to generate the active species
(10). The kinase-dead dominant negative protein is obligatorily unable
to autophosphorylate and therefore displayed a faster electrophoretic
migration compared with the endogenous protein.
Physiologically, the effect of PKC-DN was the opposite
of its wild-type counterpart. LLC-PK1
DN
cells grown in the absence of tetracycline showed a much slower
response to 10
8 M TPA than did parental
counterparts. As seen in Fig. 5, at 1 h of
treatment, TERs of LLC-PK1
DN were 80% of
their initial value, whereas the parental cells had decreased to 25%.
The degree of tetracycline responsiveness of the system is also seen in
electrical measurements with LLC-PK1
DN cells
grown in the presence of tetracycline which showed responses to TPA
nearly identical to those of parental LLC-PK1tTA.
We next assessed the effects of PKC-DN expression on
growth patterns of the line. LLC-PK1, like most epithelial
lines, grow as islands of coherent cells. Treatment of subconfluent
cultures with TPA disrupts the cell junctions that produce this compact architecture. The result is a more fibroblastic growth pattern with
individual cells migrating away from the island and a more scattered
appearance. Fig. 6 shows the results of TPA treatment of
LLC-PK1
DN cells grown in the presence and
absence of tetracycline. With expression of PKC-
DN
turned off, treatment of cells with TPA induced a scattered growth
pattern identical to that of wild type cells treated with TPA
(panel B). However, with the expression of
PKC-
DN turned on, TPA-induced cell scattering was
completely inhibited; that is, growth patterns were identical to either
wild type LLC-PK1 or LLC-PK1
DN
in the absence of TPA.
Protein phosphorylation is the primary mechanism for controlling diverse and complex cellular processes that affect structure, growth, and differentiation. Among the many kinases that are at play in these events is PKC, which often plays a pivotal role in the signal transduction process. Additionally, because PKC is the major receptor for tumor-promoting phorbol esters such as TPA, the long history of tumor promotion research has led to a wealth of information indicating that the modulation of PKC activity is a key step in tumorigenesis as well. The development of an invasive tumor involves a disruption of normal cell-cell junctions that serve to maintain tissue architecture. Additionally, more recent studies have indicated that the mechanism of action of many growth factor receptors involves activation of PKC. This occurs via phospholipase C, which produces diacylglycerol, which binds to and activates the enzyme in the same manner as TPA. The receptor for scatter factor or hepatocyte growth factor, c-Met, is an example of such a receptor, indicating that PKC plays a role in producing a response to this cytokine.
Soon following the initial discovery of PKC, the activity was found to consist of more than one species. With the advent of techniques in molecular biology, the family of isoforms has since grown to include 11 members (6). Differences in tissue distribution, subcellular distribution, and substrate specificity suggest that there is a divergence of function among the isoforms. However, despite the abundance of PKC research, little information is available on the roles of individual isoforms. Since inhibitors of the enzyme are not specific for individual isoforms, we and others have begun to address this issue by the use of exogenous expression studies. This technique, which utilizes both wild-type and dominant negative versions of the gene to modulate the activity of specific individual isoforms, is beginning to indicate which isoforms are involved in particular processes.
We have begun examining the role of the isoform in the disruption
of cell junctions. Calcium plays an established role in the maintenance
of various cell junctions including tight junctions (4, 11), and PCK
is the major calcium-dependent isoform expressed in
LLC-PK1 cells. Its activity is essential for
v-ras transformation of keratinocytes (12), and we have
previously noted that, upon TPA treatment, PKC-
translocates from
the cytosolic to the membrane fraction. This occurs in a
time-dependent manner, which correlates with increased
leakiness of tight junctions. Furthermore, in cells chronically treated
with TPA, which form uneven cell sheets of single and multilayered
areas, PKC-
stays up-regulated in areas in which there is
multilayering, which also is where tight junctions are most leaky (9).
Ellis et al. (13) report that an LLC-PK1 subline
that decreases and then rapidly recovers its TER in response to TPA
also rapidly down-regulates its PKC activity, whereas a subline whose
TER does not recover in the presence of TPA does not down-regulate its
PKC.
Our results show that a direct correlation exists between activity
levels of PKC- and the sensitivity of LLC-PK1 cell
junctions to TPA; that is, up-regulation by expression of exogenous
wild-type PKC-
enhances sensitivity, and down-regulation by
expression of the dominant negative form decreases sensitivity.
Although in vitro studies of PKC show a lack of specificity
toward certain substrates, several literature studies have reported
that the technique of exogenous expression of individual members of the PKC family produces results specific for individual isoforms. For
example, Li et al. (14) expressed six different isoforms of
PKC in 32D cells and reported that only the expression of the
isoform rendered the cells sensitive to TPA-induced differentiation. In
experiments with NIH 3T3 cells, the
isoform inhibited cell growth,
whereas the
isoform rendered the cells tumorigenic (15). Similarly,
Baier-Bitterlich et al. (16) reported that only the
isoform was competent in stimulation of AP-1 activity in T-lymphocytes. This specificity might be explained in part by the fact that isoforms are compartmentalized differently in cells, conferring specificity to
given substrates by spatial accessibility. For example, PKC-
has
been localized to the cytoskeleton of HL-60 cells (17), and PKC-
has
been localized by immmunoelectron microscopy in Madin-Darby canine
kidney epithelia to the region of the zonula occludens and/or zonula
adherens (18). Transfection of endothelia with antisense PKC-
blocked the phorbol ester-induced increase in tight function
permeability normally seen in these cell sheets (19). Similarly,
overexpression of PKC-
yields an endothelial cell sheet with a
dramatically increased effect of phorbol ester on tight junction
permeability (20). We have begun to examine other isoforms of PKC in
terms of the effect they have on regulating cell junctions in
LLC-PK1 and are finding similar results.
Our work represents a significant step toward identifying the relevant isoforms of PKC in regulating cell junctions. More work is necessary to identify the mechanism of this regulation. Many putative targets of PKC have been identified which are associated with cell junctions. Among these are c-Raf (21), vinculin (22), talin (22), MARCKS (23), glycogen synthase kinase-3 (24) and focal adhesion kinase (25). PKC-mediated modulation of any one of these might conceivably play a role in reorganizing cytoskeletal structures during TPA-induced cell migration and disruption of cell junctions.
We thank Drs. Gossen and Bujard for plasmids
making up the tetracycline-responsive expression system and Dr.
Shigeo Ohno for the cDNA for wild type PKC-.