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(Received for publication, June 10, 1994; and in revised form, December 2, 1994) From the
The glucocorticoid and transforming growth factor-
The combined effects of systemic steroid and protein hormones as
well as locally acting growth factors control the normal development,
differentiation, and proliferation of the mammary
gland(1, 2, 3, 4, 5) .
Depending on their bioavailability, these extracellular signaling
molecules can interdependently, and in a temporally appropriate manner,
regulate the function of the three main differentiated cell types of
the mammary gland (i.e. stromal, myoepithelial, and epithelial
cells). The systemic humeral signals acting on mammary tissue include
the ovarian steroids and lactogenic hormones, such as prolactin and
glucocorticoids(6, 7, 8) . In addition, each
type of mammary cell can produce and/or respond to several classes of
growth factors (1, 2, 3, 4, 5) . Two
important mammary-derived mitogens are transforming growth factor- Recent evidence suggests that TGF- It is tempting to consider that TGF- A key
issue is whether particular differentiated properties usually
associated with nontransformed mammary cells are also under reciprocal
control by glucocorticoids and TGF-
CAT activity in the cell extracts containing 30-50 µg of
lysate protein was measured by the nonchromatographic assay of Neumann
and co-workers(38) . The enzyme assay was carried out in a
final reaction volume of 250 µl in the presence of 1 µCi of
[
Figure 1:
Dexamethasone stimulates
transepithelial electrical resistance and suppresses DNA synthesis of
Con8 mammary tumor cells. Upper panel, Con8 cells were
cultured on permeable supports in the presence (+DEX) or
absence (-DEX) of 1 µM dexamethasone. At
the indicated times, the monolayer transepithelial electrical
resistance was monitored, and the ohms
Conceivably, dexamethasone may be
stimulating monolayer tight junction-like properties by causing an
overgrowth of Con8 mammary tumors on the filters. However, consistent
with our previous observations using a plastic
substratum(25, 26, 28) , dexamethasone
significantly suppressed [
Figure 2:
Dexamethasone dose-response of
transepithelial electrical resistance, DNA synthesis, and receptor
function. Upper panel, Con8 mammary tumor cells were cultured
on permeable supports for 2 days in the presence of the indicated
concentrations of dexamethasone. The monolayer transepithelial
electrical resistance was determined, and the ohms
The
dexamethasone stimulation in TER also dose-dependently correlated with
glucocorticoid receptor transcriptional activation of the chimeric
GRE-CAT reporter gene as well as with an inhibition of DNA synthesis (Fig. 2, upper versus lower panels). Steroids which are
either neutral with respect to their glucocorticoid activity (such as
Figure 3:
Effects of dexamethasone and TGF-
To determine whether TGF-
Figure 4:
TGF-
Figure 5:
TGF-
Figure 6:
TGF-
Figure 7:
Constitutive expression of transforming
growth factor-
Figure 8:
Effects of glucocorticoids and
constitutive expression of transforming growth factor-
To confirm that constitutive
expression of TGF-
Figure 9:
Effects of glucocorticoids and
transforming growth factor-
Figure 10:
Effects of glucocorticoids and
transforming growth factor-
Our results with Con8 mammary tumor cells have demonstrated
that glucocorticoids can coordinately suppress cell proliferation and
stimulate tight junction formation and thereby confer to this
transformed cell type normal-like growth and differentiation
characteristics. Exposure of glucocorticoid growth-suppressed cells to
the mammary mitogen TGF- The
glucocorticoid stimulation of transepithelial electrical resistance is
a receptor-dependent process which occurs under conditions in which the
mammary tumor cells are growth-suppressed, whereas TGF- The
TGF- Components of intercellular junctional complexes
have recently been implicated in growth regulation, such as the genes
which encode certain tight junction-associated proteins and adhesion
molecules(50) . One such gene product is the tight
junction-associated ZO-1 protein which is homologous to a class of
tumor suppressor genes. The amino-terminal half of ZO-1 displays
significant sequence homology to the product of the lethal discs large (dlg) gene of Drosophila(50, 51) .
The dlg gene product is localized in the undercoat of the
septate junction in Drosophila which is considered to be
analogous to the tight junction of vertebrate epithelial cells.
Mutations in dlg result in a loss of apical-basolateral
epithelial cell polarity and in neoplastic growth which implicates this
gene as a tumor suppressor gene(50) . It is conceivable that
junctional plaque proteins play a role in suppression of the malignant
phenotype by orchestrating the interactions of junctional adhesion
receptors and cytoplasmic signal transducers which are involved in the
negative regulation of cell growth. Consistent with this idea, we have
shown that under conditions in which TGF- The maintenance of tight junction function is a
normal-like differentiated property which prevents the mixing of
molecules from the apical and basolateral membranes and which precludes
paracellular
permeability(34, 53, 54, 55) . Tight
junction permeability of mammary epithelia is regulated during the
onset of lactation in which milk components are strictly secreted into
the lumen of the ducts via apical-directed secretory
pathways(31) . Lactogenic steroid and protein hormones and a
variety of growth factors are known to be involved in regulating the
temporal and tissue-specific development of the lactogenic state. Our in vitro work with nontransformed mammary epithelial cells (35, 36) suggests that glucocorticoids are likely to
be the lactogenic hormones responsible for regulation of tight junction
permeability. 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 controlled genes, or by interfering with the action of other
transcription factors, such as the Jun TGF-
Volume 270,
Number 12,
Issue of March 24, 1995 pp. 6505-6514
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Abrogates Glucocorticoid-stimulated Tight Junction
Formation and Growth Suppression in Rat Mammary Epithelial Tumor Cells (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TGF-) regulation of growth and cell-cell contact was investigated
in the Con8 mammary epithelial tumor cell line derived from a
7,12-dimethylbenz()anthracene-induced rat mammary adenocarcinoma.
In Con8 cell monolayers cultured on permeable filter supports, the
synthetic glucocorticoid, dexamethasone, coordinately suppressed
[
H]thymidine incorporation, stimulated monolayer
transepithelial electrical resistance (TER), and decreased the
paracellular leakage of [H]inulin or
[
C]mannitol across the monolayer. These
processes dose dependently correlated with glucocorticoid receptor
occupancy and function. Constitutive production of TGF- in
transfected cells or exogenous treatment with TGF- prevented the
glucocorticoid growth suppression response and disrupted tight junction
formation without affecting glucocorticoid responsiveness. Treatment
with hydroxyurea or araC demonstrated that de novo DNA
synthesis is not a requirement for the growth factor disruption of
tight junctions. Immunofluorescence analysis revealed that the ZO-1
tight junction protein is localized exclusively at the cell periphery
in dexamethasone-treated cells and that TGF- caused ZO-1 to
relocalize from the cell periphery back to a cytoplasmic compartment.
Taken together, our results demonstrate that glucocorticoids can
coordinately regulate growth inhibition and cell-cell contact of
mammary tumor cells and that TGF-, can override both effects of
glucocorticoids. These results have uncovered a novel functional
``cross-talk'' between glucocorticoids and TGF- which
potentially regulates the proliferation and differentiation of mammary
epithelial cells.
(TGF-) (
)and epidermal growth factor (EGF) which both
act through the tyrosine kinase EGF receptor (9, 10) .
TGF- and EGF immunolocalize to morphologically distinct areas of
the mammary gland(11) , and the expression of these growth
factors is regulated at different stages of mammary gland growth and
development(1, 12, 13) . Both in vitro and in vivo studies have shown that TGF- can have a
selective effect on the proliferation of normal mammary epithelial
cells(1, 5, 12, 14, 15, 16, 17) .
For example, exogenous administration of TGF- stimulates ductal
and terminal end bud proliferation in the virgin mouse mammary
gland(11) , enhances lobuloalveolar development in
mitogenically primed mice(1, 14) , and stimulates
angiogenesis(15) .
is involved in the early stages of breast tumorigenesis (1, 2, 5, 18) and acts as a potent
autocrine growth regulator of mammary tumor
cells(5, 12, 14, 19) . For example,
constitutive expression of TGF- in a nontumorigenic mouse mammary
epithelial cell line induces anchorage-independent growth in
vitro(20) , while introduction of TGF- cDNA into the
germ line of transgenic mice induces the appearance of
hormone-dependent mammary adenocarcinomas(21) . Many human and
rodent mammary tumors inappropriately express high levels of TGF- in vivo(22) and in vitro(16) which
has been proposed to account, in part, for the cellular escape from
hormonal growth control of some transformed mammary epithelial cells.
Consistent with the proliferative advantage of mammary tumor cells that
produce this mitogen, TGF- mRNA or protein expression has been
found in 40-70% of primary metastatic human breast
tumors(18, 23, 24) . Moreover, the expression
and secretion of TGF- can be coordinately regulated with the
proliferative state of certain mammary tumors. In hormone-responsive
human mammary tumor cell lines, ovarian steroids stimulate in vitro proliferation with a concomitant increase in the production of
TGF-(1, 5, 12, 14) , whereas
treatment of breast cancer patients with the anti-estrogen tamoxifen
often results in a significant decrease in the levels of TGF- in
the tumor(25) .
mediates the dysregulation of mammary tumor cell responsiveness to
environmental cues by modulation or inhibition of the ability of
systemic factors, such as steroid hormones, to control mammary cell
differentiation and growth. To test this notion, we are utilizing Con8
mammary tumor cells, which are an epithelial cell line derived from a
7,12-dimethylbenz()anthracene-induced rat mammary
adenocarcinoma(4, 26, 27) . These rat mammary
tumor cells constitutively express cell surface glycoprotein antigens
related to the human PAS-O human milk fat globule protein demonstrating
that these cells retain differentiated characteristics of their mammary
epithelial origin(26) . We have shown that TGF- and
glucocorticoid hormones have opposing actions on the proliferation of
these mammary tumor cells. Glucocorticoid hormones suppress the
growth(26, 27, 28) , induce c-jun expression(28) , and inhibit production of an
autocrine-acting TGF-(29, 30) , whereas treatment
of steroid growth-suppressed Con8 cells with TGF- restimulates DNA
synthesis (28, 29, 30) and induces expression
of c-myc and cyclin D1 transcripts(28) . in transformed Con8 cells. One
such differentiated property is the regulation of tight junction
permeability, since the onset of lactation in the normal mammary gland
coincides with an increase in the structural development of the tight
junction(31) . Thus, tight junction formation is important in
establishing cell-cell interactions by the formation of
``seals'' between laterally adjacent
cells(32, 33, 34) . We have recently shown in
a nontransformed mouse mammary epithelial cell line of ductal origin
that dexamethasone regulates tight junction
permeability(35, 36) . Our results suggest that a
similar ``normal-like'' differentiated property may be
conferred upon Con8 mammary tumor cells by glucocorticoids. In this
study, we show that glucocorticoids coordinately regulate tight
junction permeability and suppress the growth of Con8 mammary tumor
cells and that both steroid effects are reversed by exposure to
TGF-.
Materials
Dulbecco's modified
Eagle's medium/F12 (50:50) and the calf serum were supplied by
BioWhittaker (Walkersville, MD), and the PBS (phosphate-buffered
saline), trypsin-EDTA, and dexamethasone were obtained from Sigma.
[H]Thymidine (5 Ci/mmol) was obtained from
Amersham, and [H]inulin and
[C]mannitol were purchased from ICN Flow
Radiochemicals. Permeable supports manufactured by Nunc were
distributed by Applied Scientific. Human recombinant TGF- was
purchased from Promega. The ZO-1 monoclonal antibodies (R40.76) were a
generous gift of Bruce R. Stevenson (Dept. of Anatomy and Cell Biology,
Univ. of Alberta, Edmonton), and fluorescein
5-isothiocyanate-conjugated goat anti-rat IgG antibodies were supplied
by Cappel. All other reagents were of the highest purity available.Cells and Methods of Culture
Con8 is a single
cell-derived epithelial subclone obtained after collagenase digestion
of the 13762NF transplantable rat mammary
adenocarcinoma(26, 27) . CT93 cells are derived from
Con8 mammary tumor cells by transfection of a TGF- expression
vector(30) . Both cell lines were routinely grown to 100%
confluency on permeable tissue culture supports in Dulbecco's
modified Eagle's medium/F-12 supplemented with 5% calf serum, at
37 °C in a humid atmosphere of air/CO
(95:5). Cell
culture medium was routinely changed every 24 hours. The permeable cell
culture support system by Nunc utilizes a rigid, hydrophilic, inorganic
filter membrane to close one end of a short polystyrene cylinder. These
inserts fit inside wells of tissue culture plates and support cell
growth on the porous membrane allowing epithelial cells to import
nutrients and excrete from their entire surface. 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 either hydroxyurea (1 mM) or araC
(cytosine 
-D-arabinofuranoside; 10 µM) at
the indicated times of hormone treatment.Assay of DNA Synthesis by
[
DNA synthesis
was quantitated by determining the incorporation of
[H]Thymidine IncorporationH]thymidine as described previously (28) . Briefly, quadruplicate samples of Con8 or CT93 cells
grown on permeable supports under the indicated hormonal conditions
were incubated with 1 µCi/ml [H]thymidine (1
Ci/mmol) for 1 h at 37 °C in a humid atmosphere of air/CO (95:5). The cells were washed four times with PBS, twice with
100% methanol, and air-dried for 12 h, and the radioactivity was
quantitated by liquid scintillation counting.Flow Cytometric Analysis of DNA Content
Cell
samples containing 3 million cells were centrifuged at 800 
g for 5 min at room temperature. The supernatant fractions
were removed, and resuspended cell pellets were then hypotonically
lysed in 0.5 ml of ice cold DNA-staining solution (0.5 mg/ml propidium
iodide, 0.1% sodium citrate, 0.1% Triton X-100). Nuclear emitted
fluorescence greater than 585 nm was measured with a Coulter Elite
instrument with laser output adjusted to deliver 15 milliwatts at 488
nm. Approximately 10,000 cells were analyzed from each sample at a rate
of 300-500 cells/s. The percentages of cells within the
G, S, and G/M phases of the cell cycle were
determined by analysis with the computer program Multicycle provided by
Phoenix Flow Systems.Transepithelial Electrical Resistance
Measurements
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 (35, 36) . 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).Paracellular Permeability
The degree of monolayer
``leakiness'' was measured by applying 400 µl of 25
µCi/ml [H]inulin (M
= 5000) or 400 µl of 25 µCi/ml
[C]mannitol (M =
182) to the apical surface of confluent cell monolayers. Six hundred
microliters of cell culture medium without
[H]inulin was applied to the basolateral
reservoir of the wells. Immediately after exposing the cells to the
radioactive tracer, baseline readings were taken by assaying a 4 µl
aliquot of medium from the apical side and 6 µl of medium from the
basolateral side of the wells. In five 1-h intervals, 4 µl from the
apical surface and 6 µl from the basolateral reservoir were tested
for the presence of [H]inulin or
[C]mannitol by scintillation counting.Immunofluorescence Staining for ZO-1
Con8 and CT93
cells were grown to 100% confluency on permeable 24-well permeable
supports and incubated with the indicated combinations of dexamethasone
and TGF-. The monolayers were washed three times with
Dulbecco's PBS (BioWhittaker) with 130 mg/liter
CaCl2HO and 100 mg/liter
MgCl6HO. The cells were fixed with
acetone/methanol (50:50) at -20 °C for 5 min and air-dried.
Fixed cells were either used immediately or stored at -20 °C.
The primary and secondary antibodies for ZO-1 were diluted in TBST (50
mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20)
with 3% nonfat dry milk. 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. The secondary antibody was
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. 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. Phase contrast and immunofluorescent
images were photographed at 1600 ASA with Kodak Ektachrome 400ASA film. Transfection Procedures and CAT Assays
Con8 cells
from a logarithmically growing culture were incubated in
Dulbecco's modified Eagle's medium/F12 medium (1:1)
containing 10% calf serum and transfected by electroporation. Briefly,
cells were collected after dispersion by treatment with trypsin-EDTA,
washed twice with sterile calcium- and magnesium-free
phosphate-buffered saline (PBS), and resuspended in sucrose buffer
containing 270 mM sucrose, 7 mM sodium phosphate
buffer, pH 7.4, 1 mM MgCl. The cells (1-2
10
cells/sample) contained in 250 µl were
dispensed into sterile cuvettes and plasmid DNA (15-25 µg
total including 10-15 µg of GRE-CAT) was added to cells and
mixed, and 5 electric pulses (400 V square wave pulse for 99 µs)
delivered to the sample using a BTX 800 Transfector apparatus (BTX Inc.
San Diego, CA). Subsequently, the cells and DNA were allowed to sit for
10 min on ice, diluted with Dulbecco's modified Eagle's
medium/F12 medium (1:1), plated in 100-mm Corning plastic culture
dishes, and cultured at 37 °C. Twelve hours after transfection, the
medium was aspirated, and the cells were washed twice in PBS and then
reincubated with fresh medium. After another 12 h, cell cultures were
treated for 48 h with the indicated concentrations of TGF- and/or
dexamethasone, and cells were harvested for CAT assays. Harvested cells
were washed twice in PBS and resuspended in 0.1 M Tris-HCl, pH
7.8, and cell extracts were prepared by four cycles of freeze-thawing,
alternating between ethanol-dry ice bath and 37 °C water bath, 5
min each cycle. The cell lysates were heated at 68 °C for 15 min
and centrifuged at 12,000 g for 10 min, and
supernatants were recovered. The protein contents in the supernatant
fractions were determined with the Bradford protein assay(37) . H]acetyl coenzyme A (specific activity 200
mCi/mmol, DuPont NEN), 25 µl of 1 M Tris-HCl, pH 7.8, and
chloramphenicol (50 µl of a 5 mM aqueous solution). The
reaction mixture was gently overlaid with 4 ml of a water-immiscible
scintillation fluor (Econofluor, DuPont NEN) and incubated at 37 °C
for 4 h. The CAT activity was monitored by direct measurement of
radioactivity by liquid scintillation counting. Measurements of CAT
activity were in the linear range of the assay as determined by a
standard curve using bacterial CAT enzyme. The enzyme activity was
expressed as a function of protein present in corresponding cell
lysates (H-acetylated chloramphenicol produced, cpm/µg
of protein/4 h). Pure bacterial CAT enzyme (0.01 unit; Pharmacia,
Uppsala, Sweden) was utilized as a positive control for the CAT
enzymatic assays, while mock-transfected cells were used to establish
basal level activity. Each experiment was performed in triplicate and
was repeated two or more times.
Glucocorticoids Stimulate Tight Junction Function and
Suppress DNA Synthesis of Con8 Rat Mammary Tumor Cells
We have
previously shown that the synthetic glucocorticoid, dexamethasone,
stimulates tight junction formation in nontransformed mouse mammary
epithelial cells in vitro by monitoring an increase in
monolayer transepithelial electrical resistance (TER) and by the
inhibition of apical to basolateral movement of small radiolabeled
tracers(35, 36) . An intriguing issue is whether
dexamethasone can induce a similar normal-like differentiated property
in growth-suppressible rodent mammary tumor cells. Therefore, confluent
monolayers of Con8 mammary tumor cells were grown on permeable
supports, treated in the presence or absence of 1 µM dexamethasone, and assayed for the formation of tight junctions by
measuring the monolayer TER over a 3-day time course.
[H]Thymidine incorporation was used to determine
rates of DNA synthesis. As shown in Fig. 1(upper
panel), dexamethasone induced a striking increase in monolayer
electrical resistance over a 60-h time course, while, in untreated
cells, the monolayer electrical resistance remained constant and at low
levels. Under these conditions, the apical to basal paracellular
permeability of [H]inulin was inhibited 32-fold
in dexamethasone-treated cells compared to untreated cells (see below)
demonstrating that the increase in electrical resistance was due to the
formation of a tight monolayer.
cm were
calculated as described in the text. 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 (+DEX) or absence (-DEX) of 1 µM dexamethasone. The rate of
DNA synthesis was monitored at the indicated times during a 60-h time
course by determining the incorporation of
[H]thymidine after a 1-h pulse label as described
in the text.
H]thymidine
incorporation of Con8 mammary tumor cells grown on permeable supports
within 36 h of steroid treatment (Fig. 1, lower panel).
The suppression of [H]thymidine incorporation
using cells cultured on permeable supports is within the time frame
observed for glucocorticoids to induce an early G block in
cell cycle progression of cells cultured on plastic
substratum(28) . Thus, in the absence of glucocorticoids, Con8
mammary tumor cells rapidly proliferate and fail to produce a tight
monolayer, whereas stimulation of tight junction formation was
coincident with the dexamethasone inhibition of cell growth.Glucocorticoid Stimulation of Monolayer Transepithelial
Electrical Resistance and Inhibition of Paracellular Leakage Correlates
with Receptor Occupancy and Function
In order to assess the
functional relationship of glucocorticoid receptor occupancy and
function to the steroid-dependent regulation of tight junction
formation, the TER and apical to basolateral
[C]mannitol leakage of Con8 cell monolayers were
monitored after treatment of Con8 cells with different concentrations
of dexamethasone. To determine the dose response of receptor function,
dexamethasone inhibition of DNA synthesis (via
[H]thymidine incorporation) was used to monitor
the growth suppression response. A parallel set of mammary tumor cells
was transfected with a GRE-CAT chimeric reporter gene containing three
functional glucocorticoid response elements and assayed for
dexamethasone-stimulated chloramphenicol acetyltransferase (CAT)
activity. As shown in Fig. 2(upper panel),
dexamethasone dose-dependently stimulated the monolayer TER and
inhibited paracellular leakage with half-maximal responses of
approximately 6 nM dexamethasone which is similar to the
half-maximal binding of this steroid to Con8 cell glucocorticoid
receptors(26) . Importantly, the half-maximal inhibition of
apical to basolateral paracellular leakage of
[C]mannitol (M =
182) approximated the half-maximal concentration of dexamethasone
required to stimulate monolayer TER (Fig. 2, upper
panel). Thus, the dexamethasone-dependent increase in electrical
resistance dose-dependently corresponded with a reduction in
paracellular permeability confirming that the dexamethasone-induced
increase in TER was indicative of a decrease in tight junction
permeability or ``gate'' function(33) .
cm were calculated as described in the text. The apical to
basolateral leakage of [C]mannitol (M = 182) was monitored as described in the
text. The results are an average of triplicate samples. Lower
panel, Con8 cells were cultured on permeable supports and analyzed
for DNA synthesis by incorporation of
[H]thymidine. A parallel set of cultures were
transfected with the GRE-CAT chimeric reporter gene by electroporation
and assayed for dexamethasone-stimulated CAT activity as described in
the text. The results are an average of triplicate
samples.
-estradiol or testosterone) or which show glucocorticoid
antagonist activity (RU38486, progesterone) failed to induce Con8
monolayer TER (data not shown). Thus, the dexamethasone stimulation of
Con8 monolayer TER dose-dependently correlated with glucocorticoid
receptor occupancy and function which suggests that tight junction
permeability is regulated in Con8 cells by a glucocorticoid
receptor-mediated process, rather than as a result of an aberrant
interaction between the steroid and the plasma membrane. Furthermore,
consistent with our previous results with nontransformed mammary cells (35) , the glucocorticoid-induced formation of tight junctions
in Con8 mammary tumor cells required de novo protein synthesis
and normal levels of extracellular calcium (data not shown).TGF-
Dexamethasone disrupts a
TGF- Stimulates the Cell Cycle of Glucocorticoid
Growth-suppressed Con8 Cells and Inhibits Glucocorticoid-induced
Transepithelial Electrical Resistance autocrine loop in Con8 mammary tumor cells(29) ,
while exogenously added TGF- restimulates the growth of
glucocorticoid suppressed cells(28, 29, 30) .
The growth effects of TGF- was utilized to address the question of
whether the glucocorticoid-induced differentiated property of tight
junction formation is related to or independent of the proliferative
state of mammary tumor cells. To directly assess the cell cycle effects
of TGF-, nuclei isolated from Con8 cells treated with combinations
of dexamethasone and TGF- for 48 h were analyzed for their nuclear
DNA content. Flow cytometry profiles of nuclear DNA content after
propidium iodide fluorescent staining revealed that TGF- reversed
the dexamethasone-mediated block in cell cycle progression. As shown in Fig. 3, dexamethasone treatment altered the DNA content of the
mammary tumor cell population from an asynchronous population of
growing cells in all phases of the cell cycle to one in which
approximately 85% of dexamethasone-treated mammary tumor cells
exhibited a 2n DNA content, which is indicative of the G block in cell cycle progression(28) . Exposure of Con8
cells to TGF- prevented dexamethasone from inducing a cell cycle
arrest since the FACS profile in the presence of both steroid and
growth factor was that of a population of proliferating cells; 61% of
growth factor treated cells have a 2n DNA content equivalent to the
G phase of the cell cycle, 29% of the cells have a S phase
DNA content, and 10% have a 4n G/M DNA content (Fig. 3).
on
cell cycle phase distribution of Con8 mammary tumor cells. Con8 cells
were treated with the indicated combinations of 1 µM dexamethasone (DEX) and 10 ng/ml human recombinant
TGF-, cell extracts were stained with propidium iodide, and
nuclei were analyzed for DNA content by flow cytometry with a Coulter
Elite Laser. Approximately 10,000 cells were analyzed from each sample.
The percentages of cells within the G, S, and
G/M phases of the cell cycle were determined with the
Multicycle computer program as described under ``Experimental
Procedures.''
reverses the
glucocorticoid-stimulated formation of tight junctions under the
conditions that promote cell cycle progression, confluent mammary tumor
cell monolayers grown on permeable supports were treated with 1
µM dexamethasone for 24 h to induce tight junction
formation and then incubated in the presence or absence of growth
factor. As shown in Fig. 4, the monolayer transepithelial
electrical resistance was reduced to basal levels within 18 h of
TGF- treatment. We have previously shown that this time course is
sufficient for TGF- to stimulate the cells to move through G and S phases(28) . Thus, under conditions in which
TGF- restimulates proliferation of glucocorticoid suppressed
cells, the function of pre-formed tight junctions is coordinately
disrupted.
overrides the
dexamethasone-stimulated monolayer transepithelial electrical
resistance. Con8 mammary tumor cells were cultured on permeable
supports in the presence (+DEX) of 1 µM dexamethasone, and one control culture was incubated in the
absence of steroid (-DEX). After 24 h in dexamethasone,
cells were treated in the presence (+DEX/+TGF)
or absence (+DEX) of 10 ng/ml of human recombinant
TGF-. Throughout the 42-h time course, the monolayer
transepithelial electrical resistance was determined at the indicated
times, and the ohmscm were calculated. The results
are an average from triplicate samples.
De Novo DNA Synthesis Is Not Required for TGF-
Given the mitogenic effects of
TGF- to
Disrupt Tight Junction Formation on Con8 mammary tumor cells, it was important to test whether
the disruption of tight junction formation by this growth factor is an
indirect consequence of the onset of cell proliferation or a more
direct target of TGF--mediated receptor signaling. To test this
notion, the effects of TGF- on monolayer TER was monitored in the
presence or absence of either of two DNA synthesis inhibitors,
hydroxyurea or cytosine -D-arabinofuranoside (araC).
After 48 h of dexamethasone treatment, Con8 cells were treated with
combinations of TGF- and hydroxyurea or araC and assayed for
monolayer TER over the next 48 h. As shown in Fig. 5, TGF-
reduced the monolayer TER in the presence of either araC (upper
left panel) or hydroxyurea (lower left panel), although
this effect was slightly delayed compared to cells treated with
TGF- only. Neither of these DNA synthesis inhibitors prevented
dexamethasone from further stimulating the monolayer TER in the absence
of TGF-. Under the conditions of this experiment, araC (upper
right panel) or hydroxyurea (lower right panel) abolished
the incorporation of [H]thymidine in the presence
or absence of TGF-, demonstrating that these metabolic agents
effectively inhibited DNA synthesis in cells cultured on permeable
supports. These results demonstrate that the TGF- inhibition of
tight junction formation does not require de novo DNA
synthesis and, since these inhibitors act at or near the
G/S border(39, 40) , cell cycle
progression into and past S phase is not needed in order for TGF-
to disrupt the function of intercellular junctional complexes.
disrupts the
glucocorticoid-stimulated formation of tight junctions in the presence
of DNA synthesis inhibitors. Con8 mammary tumor cells were cultured on
permeable supports in the presence of 1 µM dexamethasone (DEX), while one set of control cultures were not treated with
hormone (No Additions). After a 48-h steroid treatment (arrow), dexamethasone-treated cells were incubated with the
indicated combinations of 10 ng/ml TGF- (TGF-) or either 10
µM cytosine -D-arabinofuranoside (top
panels: araC) or 1 mM hydroxyurea (lower panels:
HU). At the indicated times, the monolayer transepithelial
electrical resistance was determined, and the ohmscm were calculated (left panels). At the final time point,
DNA synthesis was monitored by the incorporation of
[H]thymidine as described in the text (right
panels). The results are an average from triplicate
samples.
TGF-
Conceivably, one indirect mechanism by which TGF- Does Not Impair Glucocorticoid Receptor
Function
disrupts tight junction formation is by inhibiting glucocorticoid
receptor function. To test this possibility, Con8 cells were
transfected with the GRE-CAT chimeric reporter gene and assayed for CAT
activity after treatment with combinations of dexamethasone and
TGF- during a 48-h time course. The dexamethasone strongly
stimulates transcriptional activation of the chimeric GRE-CAT reporter
gene after 48 h of continuous treatment in steroid in the presence or
absence of TGF- (Fig. 6, upper four bar graphs).
Since TGF- stimulates cell cycle progression of
dexamethasone-treated cells, the CAT enzymatic specific activity of
dexamethasone/TGF--treated cells is approximately 50% lower than
dexamethasone-treated cells due to the increase in total cell protein
with no corresponding change in CAT plasmid levels. Consistent with
this concept, if TGF- is added during the last 24 h of a 48-h
dexamethasone time course, the dexamethasone-induced CAT specific
activity is intermediate between cells treated with TGF- for 48 h
or without any added TGF- (Fig. 6, bottom bar graph
versus 2nd and 4th bar graphs). Finally, when
dexamethasone is added during the last 24 h of a 48-h time course in
TGF-, the dexamethasone-induced CAT specific activity is
essentially the same as that observed in cells treated with only
dexamethasone for 24 h (Fig. 6, 6th versus 7th bar
graph). Taken together, these results demonstrate that TGF-
does not impair glucocorticoid receptor function under conditions in
which tight junction formation is rapidly disrupted.
does not inhibit glucocorticoid
receptor function. Con8 mammary tumor cells were transfected with the
GRE-CAT chimeric reporter gene by electroporation and then cultured on
permeable supports for 48 h with the indicated combinations and time of
incubation with 1 µM dexamethasone (DEX) and 10
ng/ml TGF- (TGF-). Cell extracts were assayed for
CAT specific activity as described in the text. The results are an
average of two independent sets of triplicate
samples.
Constitutive Expression of TGF-
Compared
to their normal cell counterparts, many mammary tumor cells
constitutively produce autocrine acting growth factors which cause the
cells to proliferate in an uncontrolled manner. Therefore, as a
complementary approach to examining the relationship between the
continuous presence of TGF- Prevents the
Glucocorticoid-stimulated Formation of Tight Junctions and disruption of cell-cell contact,
the glucocorticoid-regulated formation of tight junctions was monitored
in mammary tumor cells which constitutively overexpress TGF-. Con8
cells were transfected with a cytomegalovirus promoter-driven TGF-
expression vector; one subclone of transfected Con8 cells (designated
CT93) was found to constitutively produce high levels of secreted
TGF-(30) . The ability of dexamethasone to induce tight
junction formation and suppress proliferation of Con8 and CT93 cells
was examined in cells cultured on permeable filter supports. Monolayer
transepithelial electrical resistance was measured over a 48-h time
course. Dexamethasone failed to stimulate the TER of mammary tumor cell
monolayers which constitutively express TGF- (Fig. 7). In
contrast, glucocorticoids regulated the monolayer tight junction
permeability of nontransfected Con8 cells (Fig. 7) as well as
vector-transfected controls (data not shown). To determine whether
constitutive expression of TGF- also prevents the
glucocorticoid-mediated inhibition of paracellular transport, the
apical to basolateral movement of [H]inulin (M = 5,000) was monitored in
TGF--transfected and nontransfected Con8 cells. As shown in Fig. 8, dexamethasone induced a 32-fold reduction of
[H]inulin paracellular transport in
nontransfected Con8 cells, while steroid treatment failed to reduce
[H]inulin leakage in CT93 cells which
constitutively express TGF-.
blocks the glucocorticoid stimulation of monolayer
transepithelial electrical resistance. Con8 mammary tumor cells and
CT93 cells, which constitutively express TGF-, were cultured on
permeable supports in the presence (+DEX) or absence (-DEX) of 1 µM dexamethasone. The
transepithelial electrical resistance was monitored over a 48-h time
course, and the ohmscm were determined as described
in the text.
on
paracellular transport of [H]inulin. Con8 mammary
tumor cells and CT93 cells, which constitutively express TGF-,
were cultured on permeable supports in the presence or absence of 1
µM dexamethasone for 3 days.
[H]Inulin (M = 5000)
was added apically and assayed in the basolateral media after 4 h. The
paracellular transport was calculated as the amount of radiolabeled
[H]inulin detected in the basolateral media,
divided by the total amount of [H]inulin added to
the apical media compartment. The baseline used to determine the -fold
induction is defined by the amount of [H]inulin
which diffused through the support membrane of cell-free
filters.
overrides the glucocorticoid-mediated growth
suppression response, nontransfected Con8 cells and
TGF--transfected CT93 cells were cultured on permeable supports,
and DNA synthesis was examined in cells treated with or without
dexamethasone. Dexamethasone inhibited
[H]thymidine incorporation in nontransfected Con8
cells but not in the CT93 cells (Fig. 9). Thus, constitutive
expression of TGF- prevents glucocorticoids from suppressing the
growth and regulating tight junction permeability. Conceivably, the
failure of glucocorticoids to stimulate TER and reduce paracellular
transport in CT93 cells could be due to clonal variation of transfected
cells and not due to the effects of TGF- per se. Two
lines of evidence argue against this possibility. First, several
independently isolated subclones of transfected Con8 cells which
constitutively express TGF- (30) show the same phenotype
as CT93 cells (data not shown). Secondly, the direct addition of
TGF- to dexamethasone-treated Con8 cells reduced the monolayer TER
back to basal levels within 24 h of growth factor treatment,
concomitantly with a stimulation in [H]thymidine
incorporation (Fig. 9). TGF- addition to transfected CT93
cells did not further reduce the TER or stimulate DNA synthesis (Fig. 9).
on DNA synthesis and transepithelial
electrical resistance of mammary tumor cells. Con8 mammary tumor cells
and CT93 cells, which constitutively express TGF-, were cultured
on permeable supports in the presence or absence of 1 µM dexamethasone for 3 days. Cells which had been treated with 1
µM dexamethasone for 48 h were then exposed to medium
containing dexamethasone and 10 ng/ml human recombinant TGF-. The
cells were then incubated with both factors for 2 days. The
incorporation of [H]thymidine and transepithelial
electrical resistance were monitored as described in the
text.
Effects of Glucocorticoids and TGF-
ZO-1 is an intracellular peripheral membrane
protein that is associated with tight junctions in epithelial
cells(32, 41) . Immunofluorescence studies using
monoclonal ZO-1 specific antibodies demonstrated that after 48 h of
glucocorticoid treatment, during which monolayers of Con8 cells
established significant electrical resistance, ZO-1 protein is
localized to the tight junction at cell peripheries in a sharp
continuous band of immunostaining surrounding each cell (Fig. 10B). In the absence of steroid treatment, a much
higher proportion of ZO-1 immunofluorescence was distributed throughout
the cytoplasm (Fig. 10A). Treatment with (C)
or constitutive expression of (D) TGF- on ZO-1
Distribution disrupted the
distribution of ZO-1 at the cell periphery that is typically observed
in glucocorticoid growth suppressed Con8 mammary tumor cells. In the
presence of TGF-, a high proportion of ZO-1 was detected in the
cytoplasmic compartment as indicated by the diffuse staining throughout
the cell. In addition, the residual ZO-1 immunostaining at the cell
periphery exhibited a spotty and discontinuous pattern (Fig. 10, C and D). Thus, the differential distribution
patterns of the ZO-1 tight junction protein reflect the ability of
TGF- to override the glucocorticoid stimulation of tight junction
formation in mammary tumor cells.
on ZO-1 localization. Con8 mammary
tumor cells were treated with no hormones (panel A: Con8 - DEX), 1 µM dexamethasone (panel B: Con8 + DEX), or dexamethasone and 10 ng/ml human recombinant TGF- (panel C: Con 8 + DEX/+ TGF) for 2 days. CT93
cells, which constitutively express TGF-, were treated with 1
µM dexamethasone (CT93 + DEX) for 2 days.
The cells were fixed and analyzed for ZO-1 localization by indirect
immunofluorescence as described in the text. Cell pictures were
originally photographed at 430
magnification.
rapidly stimulated cell proliferation and
caused the dysregulation of tight junction permeability, resulting in a
loss of monolayer tightness. Moreover, constitutive expression of
TGF- precluded glucocorticoids from mediating either the growth
suppression or tight junction responses. The TGF- disruption of
tight junctions is based on the observed reduction in monolayer
transepithelial electrical resistance, stimulation of paracellular
transport, and redistribution of the ZO-1 tight junction protein.
Malignantly transformed mammary cells can often display a loss of
responsiveness to particular sets of extracellular signals. One
mechanism of dysregulation is the inappropriate production of growth
factors and/or function of their cognate receptors, which alter
proliferative and/or differentiated
properties(1, 2, 3, 4, 5) .
It is therefore tempting to consider that TGF- may exert many of
its tumorigenic effects on mammary epithelial cells by not only
providing a proliferative advantage to transformed cells expressing EGF
receptors, but also by altering the way in which the cells respond to
steroid-induced signals, which are normally responsible for maintaining
critical cell-cell interactions at junctional complexes.
simultaneously stimulates DNA synthesis and reverses the steroid
effects on tight junction permeability. This inverse relationship
between cell proliferation and regulation of cell-cell interactions may
have important implications for understanding mechanisms of
invasiveness and metastasis of mammary tumors. The unrestricted growth
of tumors is dependent upon vascularization of the tumor(42) .
It is conceivable that autocrine or paracrine growth factors with
angiogenic activities may regulate this process, in part, by causing
the dissolution of epithelial cell tight junctions to allow invasion of
new blood vessels. A number of other studies have shown that other
types of cell-cell interactions are altered in transformed cells. For
example, the formation of desmosomes, which are patches of
intercellular contacts(43) , is inversely related to the stage
of lung cancers and their ability to metastasize(44) .
Similarly, it was found that certain connexins, which are gap junction
proteins that regulate the formation of intercellular
channels(45, 46) , are transcriptionally
down-regulated in human mammary tumor cell lines but not in primary
normal or nontransformed cells(47) . It has been proposed that
the connexin-mediated channels help to transmit growth-controlling
signals between cells(47, 48) . Several connexins have
been shown to be selectively produced in nontumorigenic cells and when
overexpressed slow the growth of transformed
cells(48, 49) . Thus, an alteration in cell-cell
communication may protect transformed cells from particular types of
growth inhibition, or alternatively, growth-inhibited cells may be more
capable of forming particular intercellular junctional complexes. In
this regard, we have previously shown that glucocorticoids induce a
G block in cell cycle progression of Con8 mammary tumor
cells(28) , suggesting that this growth suppression is a
prerequisite for the assembly of functional tight junctions. disruption of tight junction formation did not require the
mammary tumor cells to be actively cycling since the growth
factor-mediated reduction in monolayer TER occurs in the presence of
two different inhibitors of DNA synthesis. Both araC and hydroxyurea
block cell cycle progression at the G/S
boundary(39, 40) , whereas, as discussed above,
TGF- overrides the glucocorticoid-mediated block in cell cycle
progression early in the G phase(28) . These
results suggest that the regulation of tight junction functionality may
either be directly linked to the control of the G phase of
the cell cycle up to the S phase boundary or that tight junction
formation may not be a cell cycle-regulated process per se.
Regardless of the precise connection between cell cycle control and
tight junction formation, the disruption of monolayer TER by TGF-
in the presence of DNA synthesis inhibitors implicates the tight
junction machinery as a selective target of EGF receptor signaling and
not just an indirect consequence of cell cycle progression after growth
factor treatment. mediates a dysregulation
of Con8 cell growth and tight junction permeability, ZO-1 is
redistributed from a pericellular location to a more cytoplasmic
compartment. The regions of ZO-1 most homologous to the tumor
suppressor genes are the filamentous domain, an SH3 domain, and a
guanylate kinase domain(50, 51) . These domains
represent interesting potential targets for steroid or growth factor
control of the localization and/or function of this tight
junction-associated protein, since similar structural features are
fundamentally involved in receptor-mediated signal transduction (52) .Fos AP-1 transcription
complex, via protein-protein
interactions(56, 57, 58, 59) . Given
this mechanism of glucocorticoid hormone action, is it likely that
dexamethasone regulates the transcription of key genes encoding protein
components or regulatory factors which modulates tight junction
formation. The timing of glucocorticoid-induced gene expression is
critical in that it may initiate a transcriptional cascade in which
early regulated gene products initiate the growth suppression response,
whereas later-acting response genes may maintain the growth-inhibited
state and regulate tight junction permeability. overrides
glucocorticoid growth suppression and coordinately causes a
dysregulation of tight junction permeability and ZO-1 localization
which suggests a novel role for TGF- in disrupting the
differentiated function of mammary tumor cells. The degree of
permeability of the tight junctions is known to be regulated by
intracellular signals initiated by protein kinase C, phospholipase C,
adenylate cyclase, and GTP-binding proteins, as well as calcium (60, 61, 62, 63, 64) . Any
of these signaling components may be downstream targets of
TGF--mediated cascades initiated by activation of the EGF receptor
tyrosine kinase. We are currently attempting to elucidate the
transcriptional and secondary signaling events underlying TGF-
control that operate in mammary tumor cells and allow this growth
factor to override the effects of glucocorticoids. Conceivably, such
pathways represent an important ``cross-talk'' between growth
factor and steroid receptor signal transduction cascades that are
necessary to guide the functional relationships between particular sets
of environmental cues acting on mammary epithelial cells.
),
transforming growth factor-; EGF, epidermal growth factor; TER,
transepithelial electrical resistance; PBS, phosphate-buffered saline;
araC, cytosine -D-arabinofuranoside; CAT, chloramphenicol
acetyltransferase; GRE, glucocorticoid response element.
We thank Carolyn Cover, Anita C. Maiyar, and Ross
Ramos for their constructive comments during the course of the work and
for their critical reading of this manuscript. We also express our
appreciation to John Underhill and Jerry Kapler for their skillful
photography, Peter Schow for his assistance with the flow cytometry,
and Anna Fung for her preparation of this manuscript as well as Charles
Jackson, William 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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