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(Received for publication, February 15, 1995; and in revised form, July 12, 1995) From the
The synthetic glucocorticoid, dexamethasone, induces the
``normal-like'' differentiated property of tight junction
formation and suppresses growth of the Con8 mammary epithelial tumor
cell line, derived from a 7,12-dimethylbenz(
Differentiation and proliferation of mammary gland tissue is
stringently regulated by a dynamic balance of environmental cues that
include systemic steroid and protein hormones, such as the lactogenic
factors prolactin and glucocorticoids as well as estrogen and progestin
ovarian steroids, locally acting growth factors, and extracellular
matrix
components(1, 2, 3, 4, 5) .
A distinguishing feature of transformed mammary epithelial cells is the
dysregulation of cellular responsiveness to particular sets of
extracellular signals and loss of normal interdependence between these
signals, which can result in uncontrolled proliferation and loss of
differentiated properties(6, 7) . One mechanism by
which transformed mammary epithelial cells lose their ability to be
controlled by the microenvironment is by the inappropriate expression
of growth factors and/or function of their cognate
receptors(1, 2, 5, 6) . For example,
two particularly important mammary-derived growth factors regulated at
different stages of normal mammary gland growth and development are
transforming growth factor- The regulation of tight junction permeability in
normal mammary epithelia prevents the paracellular leakage of
macromolecules, such as growth factors, from the apical and basolateral
membranes during the onset of lactation(8) . In addition to
this ``gate'' function, tight junctions maintain cellular
polarity by physically defining the border between the apical and
basolateral plasma membrane compartments (9, 10, 11) . Tight junction structure is
highly dynamic in that permeability, assembly and/or disassembly can be
controlled by a variety of cellular and metabolic
regulators(9, 10, 12) , which include
calcium(13) , phorbol esters(14) , and members of the
insulin-like growth factor-I family(15) . Our recent studies
have documented that glucocorticoids stimulate the in vitro formation of tight junctions in nontransformed mouse mammary
epithelial cell line of ductal
origin(16, 17, 18) , which directly
implicates glucocorticoids as one of the in vivo physiological
modulators of tight junctions in the mammary gland. Given the
biological importance of structurally distinguishing the lumenal side
from the blood or basolateral side of normal mammary epithelia, it is
tempting to consider that tight junctions mediate the polarized
availability of growth factor receptors and/or their signaling pathways
in mammary cells. To address whether EGF receptor signaling can be
regulated by tight junction formation in mammary epithelial tumor
cells, we have been utilizing the Con8 rat mammary tumor cell line
which is derived from a 7,12-dimethylbenz(
Tight junctions define the distinct boundary between the
apical and basolateral plasma membrane
surfaces(9, 10) , and the in vitro formation
of tight junctions in glucocorticoid-treated Con8 mammary tumor cells (25) provides the opportunity to examine the polarized
responsiveness of mammary tumor cells to environmental signals. A key
issue is to determine whether mammogenic growth factor receptor
responsiveness is polarized, since polarity of the normal epithelia is
critical to the function of the mammary
gland(2, 8, 26) . To test this notion,
confluent monolayers of Con8 mammary tumor cells were cultured on
permeable supports and tight junctions formed by treatment with 1
µM dexamethasone. The trans-well permeable support system
allows the apical or basolateral surfaces of the cells to be
selectively manipulated once tight junctions are formed since cell
surface acting agents, such as TGF-
Figure 1:
Polarized disruption of glucocorticoid
stimulated transepithelial electrical resistance by transforming growth
factor-
To determine whether the observed polarized
disruption of tight junction integrity may simply be due to differences
in EGF receptor ligand responsiveness, 42-h dexamethasone-stimulated
cells were incubated for an additional 72 h with various concentrations
of TGF- To further investigate the dynamic steroid and growth factor control
of tight junction structure, we tested whether the localization or
expression of ZO-1, an intracellular peripheral membrane protein highly
associated with tight junctions in epithelial cells (10, 27) may be a target of glucocorticoid or
TGF-
Figure 2:
Glucocorticoid induced localization of
ZO-1 and the polarized disruption of ZO-1 distribution by transforming
growth factor-
Figure 3:
Western blot analysis of ZO-1 expression
levels. Con8 mammary tumor cells were treated with no hormones
(-Dex) or 1 µM dexamethasone
(+Dex) for 72 h. 10 ng/ml human recombinant TGF-
Conceivably, the polarized manner in which
TGF-
Figure 4:
Nonpolarized stimulation of Con8 mammary
tumor cell DNA synthesis by transforming growth factor-
To examine whether the
basolateral-mediated disruption of tight junction formation by
TGF-
A key consequence of the glucocorticoid stimulated formation
of tight junctions in Con8 mammary tumor cells is the formation of a
polarized cell monolayer, since TGF- Reduction of
tight junction permeability is an important differentiated function of
normal mammary gland cells(8) , and our results show that
glucocorticoid-regulated tight junctions can control access of growth
factors to one class of receptor signaling networks (30) which
govern the growth and development of normal and transformed mammary
cells(1, 2, 4) . We propose that the
glucocorticoid stimulation of tight junction formation provides a
functional switch which restricts the biological response to TGF- The ZO-1 tight junction-associated
protein, whose cellular localization is regulated in an antagonistic
manner by glucocorticoids and TGF- Glucocorticoids can
exert their effects on gene expression by specific binding of the
steroid receptor complex to DNA transcriptional enhancer elements,
which are present in promoters of steroid regulated genes, or by
interfering with the action of other transcription factors via
protein-protein interactions(40, 41) . We hypothesize
that specific subsets of glucocorticoid-regulated gene products act as
structural and/or regulatory components which modulate tight junction
permeability, selectively target or functionally regulate cellular
trafficking pathways mediating ZO-1 localization, and/or guide the
cellular decision that determines whether TGF-
Volume 270,
Number 47,
Issue of November 24, 1995 pp. 28223-28227
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)anthracene-induced
rat mammary adenocarcinoma. Characterization of the transepithelial
electrical resistance of Con8 mammary tumor cells cultured on permeable
supports revealed that a novel response to dexamethasone is the
generation of a polarized cell monolayer with respect to epidermal
growth factor receptor responsiveness. Administration of transforming
growth factor- (TGF-) to the basolateral, but not the apical,
plasma membrane compartment disrupted the glucocorticoid-stimulated
tight junction barrier. Confocal immunofluorescence microscopy revealed
that dexamethasone caused the ZO-1 tight junction-associated protein to
localize exclusively to the apical border of laterally adjacent
membranes of the cell periphery, whereas basolateral administration of
TGF- caused the redistribution of ZO-1 back to disorganized
aggregates along the cell periphery. In contrast, TGF- was able to
exert its mitogenic effects equally on both sides of the cell monolayer
independent of its polarized disruption of tight junction formation.
Our results represent the first evidence for a functional polarization
of the epidermal growth factor receptor and strongly implicate the
glucocorticoid-regulated formation of tight junctions in policing the
polarized responsiveness of mammary cells to growth factors.
(TGF-) (
)and epidermal
growth factor (EGF), which both act through the EGF receptor and have
been directly implicated in the pathogenesis and growth of mammary
tumors in rodents and
humans(1, 2, 4, 5) . However,
virtually nothing is known about the role of cell-cell interactions in
controlling access to growth factors and other signals from the
microenvironment.)anthracene-induced rat
mammary adenocarcinoma (19) . The synthetic glucocorticoid,
dexamethasone, strongly suppresses the growth of Con8 mammary tumor
cells(20, 21) , by inducing a G block in
cell cycle progression (22) and inhibiting the production of
autocrine-acting TGF-(23, 24) . We have recently
shown that glucocorticoids stimulate tight junction formation in
confluent monolayers of Con8 cells and that constitutive expression and
secretion of TGF- reversed this process under conditions in which
cellular proliferation is stimulated(25) . This result suggests
that exogenously added TGF- may be capable of disrupting fully
functioning tight junctions in glucocorticoid treated Con8 cell
monolayers, depending on the cell surface availability of EGF
receptors. By examining the responsiveness of dexamethasone-treated
monolayers of mammary tumor cells to TGF-, we uncovered the first
evidence for a polarized responsiveness of EGF receptors and provide
direct support for steroid-stimulated intercellular junctions as
crucial structures that police the ability of mammary tumor cells to
respond to locally acting growth factors.
Materials
Dulbecco's modified
Eagle's medium/Ham's F-12 medium (50/50) and calf serum
were supplied by Whittaker (Walkersville, MD) and phosphate-buffered
saline (PBS), trypsin-EDTA, araC, and dexamethasone were obtained from
Sigma. [
H]Thymidine (5 Ci/mmol) was obtained from
Amersham Corp. Permeable supports manufactured by Nunc were distributed
by Applied Scientific (San Francisco, CA). Human recombinant TGF-
was purchased from Becton Dickinson (San Jose, CA). The ZO-1 monoclonal
antibodies (R40.76) were a generous gift of Bruce R. Stevenson
(Department of Anatomy and Cell Biology, University of Alberta,
Edmonton) and fluorescein 5-isothiocyanate-conjugated goat anti-Rat IgG
antibodies were supplied by Cappel (Malvern, PA). All other reagents
were of highest available purity.Cells, Method of Culture on Permeable Supports, and
Measurement of Transepithelial Electrical Resistance
Con8 is a
single cell-derived epithelial subclone obtained after collagenase
digestion of the 13762NF transplantable rat mammary
adenocarcinoma(20, 21) . This cell line was routinely
grown to 100% confluency on Nunc permeable tissue culture supports in
Dulbecco's modified Eagle's medium/Ham's F-12 medium
supplemented with 10% calf serum, at 37 °C in a humid atmosphere of
air/CO
(95:5). Cell culture medium was routinely changed
every 24 h. In appropriate experiments, dexamethasone was added to a
final concentration of 1 µM, and human recombinant
TGF- was added to a final concentration of 10 ng/ml. In order to
inhibit DNA synthesis, cells cultures were exposed to 10 µM araC. The transepithelial electrical resistance was measured on
support-grown cells at room temperature, using the EVOM Epithelial
Voltohmmeter (World Precision Instruments, Sarasota, FL) as described
previously (16, 17, 25) . Resistance
measurements were taken aseptically every 8-24 h. Calculations
for ohms
cm were determined by subtracting the
resistance measurement of a blank filter and multiplying by the area of
the monolayer (0.49 cm for the 10-mm filters).Assay of DNA Synthesis by
[
To quantitate
relative rates of DNA synthesis, triplicate samples of Con8 cells were
grown to confluency on permeable supports and treated with indicated
combinations of dexamethasone, TGF-H]Thymidine Incorporation, and/or araC. The media were
replaced with fresh media containing 1-4 µCi/ml of
[H]thymidine (5 Ci/mmol), and the cells were
incubated for 1 h. The cells were washed four times with PBS and twice
with 100% methanol and air-dried for 12 h. The filter inserts were
placed directly into scintillation vials, and radioactivity was
quantitated by liquid scintillation counting.Confocal Immunofluorescence Staining for ZO-1
Con8
cells were grown to 100% confluency on permeable 24-well filters and
incubated with the indicated combinations of dexamethasone and
TGF-. TGF- was added to either the apical or basolateral side
of the monolayer. The monolayers were washed three times with
Dulbecco's PBS (BioWhittaker, Walkersville, MD), and the cells
were fixed with 1.75% formaldehyde in PBS for 15 min at room
temperature. After three washes with PBS, the monolayers were
permeabilized with TX-100 extraction buffer (0.5% Triton X-100, 10
mM Tris-HCl, pH 7.5, 120 mM NaCl, 25 mM KCl,
2 mM EDTA, 2 mM EGTA, 0.1 mM dithiothreitol,
0.5 mM phenylmethylsulfonyl fluoride). The primary and
secondary antibodies were diluted in TBST (50 mM Tris-HCl, pH
8.0, 150 mM NaCl, 0.05% Tween-20) with 3% nonfat dry milk. The
cells were washed three more times with PBS and then blocked with 3%
nonfat dry milk in PBS. ZO-1-specific antibody R40.76 was used at a
dilution of 1:400. Cells were incubated at room temperature for 1 h and
then washed three times with TBST with 3% nonfat dry milk. The
secondary antibody was a fluorescein 5-isothiocyanate-conjugated goat
IgG fraction to rat IgG and used at a dilution of 1:100. Cells were
incubated with the antibody for 1 h in the dark at room temperature.
The cell monolayers were then washed three times with TBST containing
3% nonfat dry milk. The supports were punched out and mounted on glass
slides in 50% glycerol, 50 mM Tris, pH 8-9, and 0.4% n-propyl gallate. Confocal images were obtained from a Zeiss
Axioplan epifluorescence microscope using a Zeiss 40X Plan-Neofluar
multi-immersion objective (0.9 numerical aperture) and processed with a
MRC 600 system. A series of digitized optical sections were collected
starting from the basal side of the monolayer and stepping toward the
apical membrane in 1.0-µm steps. The sections representing the
plane of ZO-1 were printed on a Kodak Colorease PS printer.Western Blot Analysis for ZO-1 Protein
Production
For Western blot analysis, whole cell samples in
lysis buffer were normalized for protein content with the Bio-Rad
Bradford protein assay. Cell lysates were subjected to 6%
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose
(nitro ME, Micron Separations Inc, Westboro, MA), Western blots
sequentially incubated with rat anti-ZO-1 monoclonal antibodies
(R40.76), and horseradish peroxidase-conjugated goat anti-rat IgG
secondary antibody, and the signal was detected by enhanced
chemiluminescence as described previously(16) . Parallel cell
samples were electrophoretically fractionated, and equivalent protein
loading was demonstrated by Coomassie Blue staining of the protein
gels.
, can be added to either the
basolateral (via the ``outer compartment'') or apical (via
the ``inner compartment'') plasma membrane of the cell
monolayer. Consistent with our previous results (25) ,
dexamethasone induced a significant increase in transepithelial
electrical resistance (TER) of the cell monolayer over a 42-h time
course, which remained essentially unchanged in the absence of steroid (Fig. 1, upper panel; +DEX versus -DEX). To test for the polarized distribution
and/or function of EGF receptors, TGF- was added to either the
apical or basolateral surfaces of dexamethasone-treated mammary tumor
cells, and the effects on monolayer TER were monitored over 42 h. When
TGF- was added to the apical side of a Con8 mammary tumor cell
monolayer treated with dexamethasone for 24 h, TER continued to
increase at a rate slightly greater than that of cells treated only
with dexamethasone (Fig. 1, upper panel;
+DEX/TGF Ap versus +DEX) which
indicates that the tight junctional machinery was intact. In striking
contrast, when TGF- was added to the basolateral surface of the
cells, TER was reduced to basal levels within 18 h of growth factor
treatment (Fig. 1, upper panel; +DEX/TGF Bl versus -DEX). Similarly, when both the apical
and basolateral surfaces of the monolayer were exposed to TGF- (Fig. 1, upper panel; +DEX/TGF Ap/Bl), the monolayer electrical resistance was quickly restored
to basal levels. Basolateral, but not apical, addition of TGF-
reversed the dexamethasone-inhibited paracellular leakage of small
radiolabeled tracers, such as [H]inulin (data not
shown), further confirming that TGF- disrupts the
``gate'' function and increases monolayer permeability in a
polarized manner.
. Upper panel, Con8 mammary tumor cells were
cultured on permeable supports in the presence (+DEX) of
1 µM dexamethasone and a control culture was incubated in
the absence of steroid (-DEX). After 24 h in
dexamethasone, 10 ng/ml of human recombinant TGF- was added to
either the apical side of the monolayer (+DEX/TGF Ap), the basolateral side of the monolayer (+DEX/TGF Bl), or to both sides of the monolayer (+DEX/TGF Ap/Bl). Throughout the 42-h time course, the monolayer
transepithelial electrical resistance was determined at the indicated
times, and the ohmscm were calculated. Each assay was
performed in triplicate, and the results are an average from three
separate experiments. Lower panel, Con8 cells were cultured on
permeable supports in the presence of 1 µM dexamethasone
for 42 h and then incubated for 72 h with the indicated concentrations
of TGF- added to either the apical or basolateral sides of the
monolayer. The transepithelial electrical resistance at each
concentration of growth factor was determined, and the difference in
ohmscm was calculated relative to
dexamethasone-treated cells not treated with TGF- (0
point). Each assay was performed in triplicate, and the results
are an average from three separate
experiments.
(0-20 ng/ml) added to either the apical or
basolateral sides of the monolayer. Cells treated with dexamethasone
without TGF- displayed a 6-fold stimulation in TER (defined as 0 point in lower panel in Fig. 1). Basolateral
addition of TGF- dose-dependently reduced the TER to basal levels,
whereas apical addition of TGF- further increased the TER (Fig. 1, lower panel). Although most dramatic at the
higher doses of TGF-, the polarized effects are observed at the
lower concentrations of TGF- corresponding to the physiological
levels of growth factor. The similarities in each dose response suggest
that the EGF receptor affinities are similar on both sides of the
monolayer and supports the hypothesis that TGF- activates
different components of the EGF-receptor signal transduction pathways
linked to the apical versus the basolateral compartments. signaling in Con8 mammary tumor cells. The images shown in Fig. 2illustrate sections taken in the apical plane of ZO-1 at
the top through the basolateral plane at the bottom.
The distance between each optical section is 2 µm. In the absence
of steroid (Fig. 2, Panels a-c), ZO-1 staining
displayed a spotty, discontinuous pattern, which appeared as apparent
aggregates along the cell periphery, especially at the junctions
between several cells. In addition, ZO-1 at the middle and basolateral
plane (Fig. 2, Panels b and c) in certain
cells of the untreated monolayer were visualized as intense, linear
series of accumulations at the cell border. When cells were treated
with dexamethasone (Fig. 2, Panels d-f), ZO-1
localization exhibited a very discrete and continuous band at the
lateral membrane of adjacent cells in all three planes. Consistent with
the effects on TER, addition of TGF- to the apical side of a
glucocorticoid-induced polarized monolayer (Fig. 2, Panels
g-i) had no effect on ZO-1 localization which was maintained
as a distinct and continuous band surrounding the peripheries of the
cells. In contrast, when cells were treated with TGF- at the
basolateral surface of the monolayer (Fig. 2, Panels
j-l), ZO-1 distribution reverted back to a disorganized and
discontinuous pattern of staining, analogous to that seen in control
cells without drug treatment. Although these cells retained the
peripheral ZO-1 localization, the more intense staining was at the
junctions shared by numerous cells. A similar disorganized ZO-1
immunostaining was also observed after the addition of TGF- to
both the apical and basolateral sides of the monolayer (data not
shown). This growth factor alteration of ZO-1 localization is
indicative of the disruption in the apical/basolateral boundary
mediated by the loss of tight junctions. Under conditions in which
glucocorticoids or TGF- modulates the pattern of ZO-1
immunostaining, Western blot analysis revealed that there were no
detectable differences in ZO-1 protein levels (Fig. 3). Thus,
the coordinate and opposing regulation of the cellular distribution of
ZO-1 correlates with the regulation of monolayer transepithelial
electrical resistance of mammary tumor cells by glucocorticoids and
basolateral addition of TGF-. These results demonstrate for the
first time a polarity of EGF receptor responsiveness in epithelial
cells and strongly implicates the glucocorticoid induction of tight
junction formation in mammary tumor cells as playing a role in
governing this process.
. Con8 mammary tumor cells were treated with no
hormones (-Dex, Panels a-c), 1 µM
dexamethasone (+Dex, Panels d-f),
dexamethasone for 24 h followed by 10 ng/ml human recombinant TGF-
applied on the apical surface of the monolayer for 18 h
(+Dex/+TGF Ap, Panels
g-i) or dexamethasone for 24 h follow by 10 ng/ml TGF-
added on the basolateral surface of the monolayer for 18 h
(+Dex/+TGF Bl, Panels
j-l). The cells were fixed and analyzed for ZO-1
localization by confocal microscopy. The upper panels display
the apical sections, and the bottom panels display the
basolateral sections. The bar represents 25
µm.
was
applied to 24-h dexamethasone-treated cells for an additional 48 h on
the apical surface of the monolayer, +Dex+TGF (Ap), basolateral surface, +Dex+TGF (Bl), or simultaneously to the apical and basolateral surface,
+Dex+TGF (Ap+Bl). Cell lysates
were normalized for total protein (confirmed by Coomassie Blue staining
of a parallel polyacrylamide gel), electrophoretically fractionated,
blotted onto nitrocellulose filters, and analyzed for ZO-1 protein as
described under ``Experimental Procedures.'' The protein
molecular mass standards are myosin (200 kDa) and
phosphorylase b (97.4
kDa).
disrupts the dexamethasone-stimulated formation of tight
junctions may be due to the selective localization of the EGF receptor
protein or due to the polarized function of the receptor-signaling
pathway either at the apical or basolateral side of the cell. To test
this possibility, TGF-'s mitogenic activity on Con8 cells
was examined in monolayers exposed at the apical and/or basolateral
surfaces to this growth factor. Dexamethasone strongly inhibited the
DNA synthesis of Con8 cell monolayers grown on permeable supports (Fig. 4). In contrast to the polarized effects on monolayer TER,
the addition of TGF- to either the apical, basolateral, or both
sides of the monolayer strongly restimulated
[H]thymidine incorporation of dexamethasone
treated mammary tumor cells to approximately the same extent (Fig. 4). This result demonstrates that EGF receptors are
distributed to and are functional at both the apical or basolateral
cell surfaces in glucocorticoid-treated cells; however, the signal
transduction events triggering the disruption of tight junctions appear
to be distinct for the apical versus the basolateral
compartments of the mammary tumor cells.
. Con8
mammary tumor cells were cultured on permeable supports in the presence
of 1 µM dexamethasone (DEX), and one control
culture was incubated in the absence of steroid. After 24 h in
dexamethasone, 10 ng/ml human recombinant TGF- was added to either
the apical side of the monolayer (Ap), the basolateral side of
the monolayer (Bl), or to both sides of the monolayer (Ap/Bl). After an additional 18-h incubation, DNA synthesis
was determined by the incorporation of
[H]thymidine. The results are an average of
quadruplicate samples.
is independent of the proliferative response, the
glucocorticoid and growth factor regulation of
[H]thymidine incorporation and TER of Con8 cell
monolayers were analyzed after treatment with araC, an inhibitor of DNA
synthesis. As shown in Table 1, dexamethasone stimulated the
monolayer TER to the same extent in the presence or absence of 10
µM araC. Under conditions in which
[H]thymidine incorporation remains strongly
inhibited by araC, basolateral exposure to TGF- rapidly reduced
the monolayer TER back to basal levels (Table 1). The monolayer
TER remained at approximately the steroid-induced level after apical
addition of TGF- in the presence or absence of araC. This result
suggests that the basolateral specific signaling pathway leading to the
disruption of cell-cell interactions by TGF- is distinct from and
independent of the proliferative pathway mediated by this growth
factor.
reduces monolayer electrical
resistance when added to the basolateral membrane compartment but not
when exposed to the apical or lumenal compartment. Growth factor
addition to either compartment of a dexamethasone-treated monolayer
restimulates DNA synthesis, demonstrating for the first time that
TGF-, which binds to the EGF receptor(28) , appears to
trigger different signal transduction cascades or activate polarized
intermediates depending on the compartmental localization of its
cognate receptor (Fig. 4). The proliferative pathway of
TGF- involves activation of the mitogen-activated protein kinase
and protein kinase C signal transduction pathways via the EGF receptor
tyrosine kinase(29) , and we are currently attempting to
uncover the basolateral specific components which selectively transduce
the TGF- disruption of cell-cell interactions.,
and perhaps other factors, selectively secreted into the apical or
lumenal side of the monolayer. Both in vitro and in vivo studies have implicated a role for TGF- in the growth and
development of the normal mammary gland as well as in tumor progression
and proliferation of rodent and human mammary tumor
cells(2, 4, 5, 31) . For example,
the introduction of TGF- cDNA into the germ line of transgenic
mice causes the appearance of hormone-dependent mammary adenocarcinomas (32) . Mammary tumor cells produce TGF- (33) and
the biological implication of basolateral exposure to TGF-, and
subsequent disruption of the apical/basolateral boundary and
intercellular ``gate'' functions controlled by tight
junctions may be the permissive passage of substances between lumenal
and basolateral sides of mammary epithelia, thereby allowing the
epithelium to be responsive to a variety of factors which normally are
confined to one side of the mammary epithelium. Therefore, it is
tempting to consider that dynamic interactions between steroid and EGF
receptor-signaling networks, which have opposing effects on tight
junction formation, ZO-1 protein localization, and cell polarity, may
suppress or mediate the dysfunctional autocrine signaling cascades
within mammary tumor cells. in Con8 mammary tumor cells,
has significant sequence homology to the product of the lethal (l) disc
large-1 (dlg) gene of Drosophila(27, 34) which has been implicated as a
tumor-suppressor gene. Drosophila mutations in dlg result in a loss of apical-basolateral epithelial cell polarity
and in a stimulation of neoplastic growth(35) . Recent evidence
has implicated other junctional components, adhesion molecules, and
integral membrane proteins as potential tumor suppressors (34, 36, 37) or interactions with tumor
suppressor proteins, such as 
-catenin's direct interaction
with APC(38, 39) . Thus, in mammalian cells, the
hormonal control of junctional plaque proteins, such as ZO-1, may play
a key role in suppression of a malignant phenotype by modulating growth
factor receptor signal transduction pathways involved in tumor
proliferation, dedifferentiation, or metastasis. disrupts the tight
junction belt. Steroid hormones have been shown to regulate the
expression of certain cell adhesion molecules, for example C-CAM
(cell-cell adhesion molecule) expression is repressed by androgens in
rat ventral prostatic epithelium (42) and glucocorticoids
reduce integrin transcript levels in PC12
pheochromocytoma cells (43) as well as stimulate expression of
the connexin 26 and connexin 32 gap junction gene transcripts in rat
hepatocytes and hepatoma cells(44) . Thus, an emerging subset
of steroid-regulated genes have been detected which mediate different
types of cell-cell interactions in a variety of tissues. We are
currently attempting to define the glucocorticoid-regulated components
which target tight junction complex proteins and induce functional
polarity to growth factor responsiveness in mammary cells.
), transforming growth factor-; EGF, epidermal growth
factor; TER, transepithelial electrical resistance; PBS,
phosphate-buffered saline; araC, cytosine
-D-arabinofuranoside.
We thank Helen Cha, Carolyn Cover, Terry E. Machen,
Anita C. Maiyar, and Ross A. Ramos for their constructive comments
during the course of the work and for their critical comments of this
manuscript. We also express our appreciation to Anna C. Fung for her
superb typing and preparation of this manuscript, Jerry Kapler for his
skillful photography, and Charles Jackson, William J. Meilandt, Marina
Chin, Ritu Patel, Vinh Trinh, and Thai Truong for their technical
support.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 J. Murtagh, E. McArdle, E. Gilligan, L. Thornton, F. Furlong, and F. Martin Organization of mammary epithelial cells into 3D acinar structures requires glucocorticoid and JNK signaling J. Cell Biol., July 5, 2004; 166(1): 133 - 143. [Abstract] [Full Text] [PDF]
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 Y. Zhu, J. Maric, M. Nilsson, M. Brannstrom, P.-O. Janson, and K. Sundfeldt Formation and Barrier Function of Tight Junctions in Human Ovarian Surface Epithelium Biol Reprod, July 1, 2004; 71(1): 53 - 59. [Abstract] [Full Text] [PDF]
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 Y. Guan, N. M. Rubenstein, K. L. Failor, P. L. Woo, and G. L. Firestone Glucocorticoids Control {beta}-Catenin Protein Expression and Localization through Distinct Pathways that Can Be Uncoupled by Disruption of Signaling Events Required for Tight Junction Formation in Rat Mammary Epithelial Tumor Cells Mol. Endocrinol., January 1, 2004; 18(1): 214 - 227. [Abstract] [Full Text] [PDF]
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 N. M. Rubenstein, Y. Guan, P. L. Woo, and G. L. Firestone Glucocorticoid Down-regulation of RhoA Is Required for the Steroid-induced Organization of the Junctional Complex and Tight Junction Formation in Rat Mammary Epithelial Tumor Cells J. Biol. Chem., March 14, 2003; 278(12): 10353 - 10360. [Abstract] [Full Text] [PDF]
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M. Betson, E. Lozano, J. Zhang, and V. M. M. Braga Rac Activation upon Cell-Cell Contact Formation Is Dependent on Signaling from the Epidermal Growth Factor Receptor J. Biol. Chem., September 27, 2002; 277(40): 36962 - 36969. [Abstract] [Full Text] [PDF]
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 J. D. Stockand New ideas about aldosterone signaling in epithelia Am J Physiol Renal Physiol, April 1, 2002; 282(4): F559 - F576. [Abstract] [Full Text] [PDF]
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H. Hasegawa, H. Fujita, H. Katoh, J. Aoki, K. Nakamura, A. Ichikawa, and M. Negishi Opposite Regulation of Transepithelial Electrical Resistance and Paracellular Permeability by Rho in Madin-Darby Canine Kidney Cells J. Biol. Chem., July 23, 1999; 274(30): 20982 - 20988. [Abstract] [Full Text] [PDF]
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P. Buse, S. H. Tran, E. Luther, P. T. Phu, G. W. Aponte, and G. L. Firestone Cell Cycle and Hormonal Control of Nuclear-Cytoplasmic Localization of the Serum- and Glucocorticoid-inducible Protein Kinase, Sgk, in Mammary Tumor Cells. A NOVEL CONVERGENCE POINT OF ANTI-PROLIFERATIVE AND PROLIFERATIVE CELL SIGNALING PATHWAYS J. Biol. Chem., March 12, 1999; 274(11): 7253 - 7263. [Abstract] [Full Text] [PDF]
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V. Wong, D. Ching, P. D. McCrea, and G. L. Firestone Glucocorticoid Down-regulation of Fascin Protein Expression Is Required for the Steroid-induced Formation of Tight Junctions and Cell-Cell Interactions in Rat Mammary Epithelial Tumor Cells J. Biol. Chem., February 26, 1999; 274(9): 5443 - 5453. [Abstract] [Full Text] [PDF]
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 K. Amsler and S. K. Kuwada Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium Am J Physiol Cell Physiol, January 1, 1999; 276(1): C91 - C101. [Abstract] [Full Text] [PDF]
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P. L. Woo, A. Cercek, P.-Y. Desprez, and G. L. Firestone Involvement of the Helix-Loop-Helix Protein Id-1 in the Glucocorticoid Regulation of Tight Junctions in Mammary Epithelial Cells J. Biol. Chem., September 8, 2000; 275(37): 28649 - 28658. [Abstract] [Full Text] [PDF]
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