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(Received for publication, July 6, 1995; and in revised form, September 28, 1995) From the
The synthetic glucocorticoid, dexamethasone, stimulated the
transepithelial electrical resistance and suppressed the DNA synthesis
of 31EG4 nontransformed mouse mammary epithelial cells. The addition of
transforming growth factor-
The precise regulation of cell-cell interactions is an essential
feature of the development and function of the mammary gland. Three
differentiated cell types, stromal, myoepithelial, and epithelial
cells, exist in the mammary epithelium and/or mesenchyme, and
reciprocal communication between these cell types occurs as part of
mammary morphogenesis during postnatal development and puberty, as well
as during pregnancy and lactation(1) . Radioactive tracer
studies and freeze fracture electron microscopy revealed that an
increase in tight junction structural organization and a decrease in
permeability of mammary epithelium correlates with the differentiation
state of the mammary gland during the onset and establishment of
lactation(2, 3, 4) . Environmental cues such
as systemic ovarian steroids and lactogenic hormones, locally acting
growth factors, and the extracellular matrix control the normal
differentiation and proliferation of mammary cells (5, 6, 7) and collectively have been proposed
to regulate the dynamics of cell-cell interactions. Part of the
complexity of understanding the hormonal pathways that control mammary
cell-cell interactions is the likelihood that combinations of steroids,
protein hormones, and growth factors contribute to this process in an
additive, synergistic, or antagonistic fashion. Also, cellular targets
of hormonal pathways that potentially regulate mammary cell-cell
interactions are generally uncharacterized, although our in vitro evidence has shown that glucocorticoids regulate tight junctions
in transformed and nontransformed mammary epithelial
cells(8, 9, 10) . The tight junction or
zonula occludens is the most apical member of a series of intercellular
junctions known as the junctional complex with the adherens junction
immediately below it and desmosomes further basal. Tight junctions form
a continuous seal around the lateral membrane of adjacent cells and
have a highly dynamic structure whose permeability, assembly, and/or
disassembly can be altered by a variety of cellular and metabolic
regulators(11, 12, 13) . At intercellular
contact points, where the membranes of adjacent cells come into close
proximity, tight junctions serve as a selective barrier to the
paracellular diffusion of solutes on the basis of size and charge
across epithelial and endothelial cell
monolayer(14, 15) . Tight junctions also contribute to
the maintenance of cellular polarity by physically defining the border
between the apical and basolateral plasma membrane
surfaces(16) . To date, one transmembrane protein,
occludin(17) , and five cytoplasmic-residing peripheral
membrane proteins, ZO-1(18) , ZO-2(19) ,
cingulin(20) , the 7H6 antigen(21) , and Rab13 (22) have been identified as being localized at tight
junctions. Actin, which forms the characteristic perijunctional ring at
the underlying adherens junction, has also been shown to associate with
the tight junction, suggesting a direct interaction between the
cytoskeleton and tight
junctions(13, 23, 24, 25, 26) .
The 220-kDa phosphoprotein ZO-1(27) , which has been shown to
specifically bind to the related cytoplasmic protein,
ZO-2(19) , and to the cytoskeletal protein,
spectrin(28) , is the only known cytoplasmic link to the
transmembrane protein occludin(29) . Moreover, ZO-1 is a member
of a family of membrane-associated proteins containing potential SH3
and guanylate kinase domains, the first of which is the
lethal(1)discs-large-1 (dlg) tumor suppressor gene located at
the septate junction in Drosophila(30) . This homology
implicates ZO-1 as a potential component of signal transduction
cascades through the plasma membrane(31) . Conceivably, the
availability and combinatorial actions of systemic steroids and locally
acting growth factors, which regulate particular stages of mammary
gland growth and differentiation, concomitantly control tight junction
dynamics. In vivo studies suggest that one or more of the
lactogenic hormones, such as prolactin, insulin, and/or
glucocorticoids(32, 33) , may play an important role
in establishing and regulating appropriate intercellular contacts. Our
recent in vitro studies using cultured 31EG4 mouse mammary
epithelial cells demonstrated that glucocorticoids but not the other
lactogenic hormones stimulated an increase in monolayer transepithelial
electrical resistance and a decrease in the paracellular permeability (10) , implicating this steroid as a key regulator of mammary
tight junctions. The glucocorticoid-dependent stimulation of 31EG4
tight junctions occurs by a receptor-dependent process requiring normal
levels of extracellular calcium (10) and functioning
serine/threonine phosphosphorylation-dephosphorylation
cascades(9) . However, the role of other classes of
extracellular signals on tight junction dynamics is unknown, and
perhaps other mammogenic factors may concurrently modulate tight
junction function and/or structure. One candidate mammogenic factor is
transforming growth factor-
Confocal images were obtained from a Zeiss Axioplan
epifluorescence microscope using a Zeiss 40
Figure 2:
Transforming growth factor-
For luciferase assays, 20 µl
of lysate supernatant for each sample was mixed with 100 µl of
Promega's reconstituted luciferase assay reagent (20 mM tricine, 1.07 mM
(MgCO
Figure 1:
Transforming growth factor-
Consistent with its mode of action on mammary gland
differentiation (35) , the inhibitory effects of TGF-
To precisely compare the distribution of
ZO-1 with that of actin, optical sections were taken in the x-y plane of ZO-1 and pseudocolors were assigned to
ZO-1 as green and actin as red. The images shown in Fig. 2illustrate sections at the apical plane of the tight
junction (top panels) and the plane 2 µm lower (bottom
panels). Cells that were not treated with steroid or growth factor (Fig. 2, a and b) displayed ZO-1 and actin
colocalization as a fine ring at the cell periphery at the most apical
cell-cell border. Consistent with our previous results(9) ,
cells grown in the presence of dexamethasone were larger and had a
greater linear junction length per cell (Fig. 2, c and d versus a and b). In dexamethasone-treated cells,
ZO-1 and actin colocalizes in the lower plane (Fig. 2, d), but actin signal is significantly decreased in the upper
plane (Fig. 2, c). Therefore, under conditions where
TER is greatly enhanced by dexamethasone, actin appears to be absent
from the most apical region of ZO-1 localization. TGF-
Figure 3:
Western blot analysis of ZO-1 and
E-cadherin protein levels as well as expression of the cytokeratin type
II epithelial cell marker protein. A, 31EG4 cell monolayers
were cultured for 72 h with the indicated combinations of 1 µM Dex and 10 ng/ml TGF-
Figure 4:
Pretreatment with dexamethasone for 48 h
prevents transforming growth factor-
Indirect immunofluorescence microscopy
revealed that the addition of TGF-
Figure 5:
Pretreatment with dexamethasone for 48 h
prevents transforming growth factor-
Figure 6:
Effects of hormone treatment on
glucocorticoid receptor and transforming growth factor-
Figure 7:
Transforming growth factor-
To determine
whether the disruption of glucocorticoid-induced tight junction
formation by TGF-
Figure 8:
Transforming growth factor-
Our results using a nontransformed mammary epithelial cell
line represent the first evidence that glucocorticoids, an important
systemic lactogenic steroid(49) , and TGF- During functional differentiation of the
mammary gland, the key hormonal regulators of mammary cell-cell
interactions must be selective and reversible in their actions and in
some instances, counteract each other depending on the stage of
differentiation. Consistent with these biological properties,
glucocorticoids and TGF- Dexamethasone coordinately induced tight junction
formation and suppressed DNA synthesis, whereas the TGF- It has been proposed that junctional plaque
proteins, such as ZO-1, help orchestrate the interactions of integral
membrane proteins with cytoplasmic signaling components and, as a
result, may serve as regulators of cell proliferation, cell adhesion,
and cell-cell interactions. In this context, ZO-1 is homologous to the
lethal(1)discs-large-1 (dlg) tumor suppressor gene of Drosophila, whose mutation resulted in neoplastic overgrowth
of the imaginal discs as well as a loss of cell polarity and adhesion.
Although a role for ZO-1 in intercellular signaling is unknown, several
studies have attempted to ascertain the nature of its regulation. For
example, ZO-1 was shown to be phosphorylated only on serine residues
under normal conditions in MDCK cells (27) and tyrosine
residues during the formation of tight junctions in the slit diaphragms
of glomerular epithelial cells(56) . We have shown that
glucocorticoid stimulation of TER in 31EG4 cells did not alter the
localization, phosphate content, or phosphopeptide digest pattern of
ZO-1 but did increase protein levels(9) . Recent evidence has
shown that the PKC agonist 1,2-dioctanoylglycerol can promote the
assembly of tight junctions, as evidenced by the translocation of ZO-1
and actin filaments to the cell periphery(57) , and PKC can in vitro phosphorylate ZO-1, which may play a role in the
formation of tight junctions(58) . Previous observations
demonstrating that ZO-1 is detected at the tight junction as well as
adherens junction zone only in cells with leaky tight junctions (59) may explain the high colocalization of actin and ZO-1 in
31EG4 cells treated with TGF- TGF- It is tempting to consider that the selective antagonism
between glucocorticoid and TGF-
Volume 271,
Number 1,
Issue of January 5, 1996 pp. 404-412
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
in Mouse Mammary Epithelial Cells (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 (TGF-) to mammary cells
simultaneously with or up to 24 h after dexamethasone treatment
prevented the steroid induction of transepithelial electrical
resistance and stimulated the incorporation of
[
H]thymidine. However, the TGF- inhibition
of tight junction formation did not require de novo DNA
synthesis. Confocal microscopy revealed that the organized
immunostaining pattern of the tight junction protein, ZO-1, and F-actin
at the cell periphery was disrupted by TGF-, resulting in
disorganized and diffuse staining patterns throughout the cell. Western
blot analysis demonstrated that TGF- did not alter the protein
levels of ZO-1. In contrast to cells not treated or pretreated with
steroid for up to 24 h, TGF- had no effect on cells pretreated
with dexamethasone for 48 h. Transfection of chimeric reporter genes
containing promoters responsive to either glucocorticoid or TGF-
demonstrated that the mutual antagonism of tight junction dynamics by
dexamethasone and TGF- occurs in the presence of intact signaling
pathways. Taken together, our results establish for the first time that
glucocorticoids and TGF- can antagonistically regulate tight
junction formation in a nontransformed mammary cell line.
(TGF-), (
)which is
produced within morphologically distinct areas of the mammary gland (34, 35) and signals through its cognate cell surface
serine/threonine kinase receptors. TGF- is a regulator of mammary
branching morphogenesis and cell growth(36) , yet relatively
little is known about its effects on mammary cell-cell
interactions(7, 37) . In this study, we establish that
glucocorticoids and TGF- mutually antagonize each other's
actions to regulate tight junction dynamics, localization of the ZO-1
tight junction protein, and cell growth in nontransformed mammary
epithelial cells.
Materials
Dulbecco's modified
Eagle's medium/Ham's F-12 (50:50) was supplied by
BioWhittaker (Walkersville, MD). Fetal bovine serum, insulin,
dexamethasone, gentamicin sulfate, and the E-cadherin antibody
(uvomorulin, clone DECCMA-1) were purchased from Sigma. Transforming
growth factor- 1 was purchased from Life Technologies, Inc.
Permeable supports were made by Nunc and distributed by Applied
Scientific (San Francisco, CA), and [H]thymidine
(82.9 Ci/mmol) was obtained from NEN Products (Boston, MA). The ZO-1
antibody R40.76 (27) was a generous gift from Bruce Stevenson
(Department of Anatomy and Cell Biology, University of Alberta).
Fluorescein isothiocyanate-conjugated anti-rat IgG antibodies were
supplied by Cappel Laboratories (Malvern, PA). Rhodamine-labeled
phalloidin was purchased from Molecular Probes Inc. (Eugene, OR).
GRE-CAT was a generous gift of Keith R. Yamamoto (Department of
Biochemistry and Biophysics, UCSF), p3TP-Lux was a generous gift of
Joan Massagué (Cell Biology and Genetics Program,
Memorial Sloan-Kettering Cancer Center), and pUHC131-1 was a
generous gift of Astar Winoto (Department of Molecular and Cell
Biology, University of California, Berkeley).Cell Culture and Transepithelial Electrical
Resistance Measurements
31EG4 mammary cells were cultured
on permeable filter supports in Dulbecco's modified Eagle's
medium/Ham's F-12 supplemented with 2% fetal bovine serum, 50
µg/ml gentamicin sulfate, and 5 µg/ml insulin with daily
changes of medium as described previously(9, 10) . In
appropriate experiments, dexamethasone was added to a final
concentration of 1 µM, and TGF- was added to a final
concentration of 10 ng/ml. In order to inhibit DNA synthesis, cell
cultures were exposed to 1 mM hydroxyurea. TER was measured on
filter grown cells using the EVOM Epithelial Voltohmmeter (World
Precision Instruments) as described
previously(8, 9, 10) . The EVOM provides an
alternating square wave current of ± 20 µA through the
monolayer to measure the tightness of the tight junction. Daily
resistance measurements were taken at room temperature with the
electrode after alcohol sterilization. Calculations for
ohms
cm
were made by subtracting a blank filter (159
) and multiplying by the area of the monolayer (0.49
cm
).Assay of DNA Synthesis by
[
DNA synthesis was quantitated by determining
the incorporation of [H]Thymidine
IncorporationH]thymidine. Triplicate
samples of cells grown on permeable filter supports under the indicated
hormonal conditions were incubated with 6 µCi/ml of
[H]thymidine (82.9 Ci/mmol) for 2 h at 37 °C
in a humid atmosphere of air/CO (95:5). The cells were
washed three times with cold 10% trichloroacetic acid, and the filters
were placed directly into scintillation vials with 300 µl of 0.3 N NaOH to lyse the cells. Radioactivity was quantified on a
Beckman LS 1801 liquid scintillation counter.Immunofluorescence Microscopy
31EG4
mammary cells were plated at 100% confluency on 24-well filters and
incubated with the indicated combinations and times of exposure to
dexamethasone and/or TGF- for 48 or 72 h. The cell monolayers were
washed three times with PBS and then fixed with acetone/methanol
(50:50) at -20 °C for 5 min. Cells were allowed to air dry
and then washed with TBST-3% NFDM (50 mM Tris-HCl, pH 8.0, 150
mM NaCl, 0.05% Tween 20, and 3% nonfat dry milk). All
subsequent incubations with antibodies and washes were performed with
this buffer. Cells were incubated with ZO-1 monoclonal antibody R40.76
(1:400 dilution) at room temperature for 1 h and then washed three
times. Fluorescein isothiocyanate-conjugated goat IgG anti-rat IgG was
added at a 1:100 dilution for 1 h at room temperature and then washed
three times. The filters were punched out and mounted on glass slides
in 50% glycerol, 50 mM Tris, pH 8-9, and 0.4% n-propyl gallate. Immunofluorescent images were photographed
at 1600 ASA with Kodak Ektachrome 400 ASA film.Confocal Immunofluorescence
Microscopy
For confocal microscopy, filter-grown monolayers
were incubated with combinations of dexamethasone and TGF- for 72
h. After three washes, the monolayers were fixed with 1.75%
formaldehyde in PBS for 15 min at room temperature. The cells were
washed three more times and permeabilized with 0.5% Triton X-100 in PBS
for 10 min. Following three washes, cells were then blocked with
TBST-3% NFDM. ZO-1 staining was performed as described above. After the
ZO-1 immunostaining, cells were stained for F-actin by incubating
monolayers with 165 nM rhodamine phalloidin in PBS for 0.5 h.
After three washes, filters were mounted on glass slides as described
above.
Plan-Neofluar
multi-immersion objective (0.9 numerical aperture) and analyzed with a
Bio-Rad MRC 600 system. A split screen of the double label images was
obtained with a dual filter set for fluorescein and Texas red. A series
of optical sections was collected for each specimen at ascending z
levels beginning at the basal surface in 1-µm increments. Due to
the increased thickness of the cell monolayer, the number of optical
sections collected in the presence of TGF- was approximately twice
the amount of that collected from monolayers cultured in the absence of
TGF-. In Fig. 2, only the most apical regions of ZO-1 are
exhibited in order to investigate whether actin can colocalize with
ZO-1 at the apical level. After contrast and zoom enhancements, images
were assigned pseudocolors representing the original color of the label
used for immunofluorescence and printed with a Kodak Colorease PS
printer.
alters
the cellular morphology and distribution of ZO-1 and F-actin. 31EG4
mammary epithelial cells were cultured on permeable supports with the
indicated combinations of 1 µM Dex and 10 ng/ml TGF-
for 72 h. The cells were fixed and simultaneously analyzed for ZO-1 and
F-actin colocalization by confocal microscopy using ZO-1 monoclonal
primary antibodies (green) and rhodamine phalloidin staining (red) as described in the text. The plane of ZO-1 is shown as
two panels of optical sections 2 µm apart. The most apical images
are shown at the top of the figure (a, c, e, and g), and the lower plane is shown at the bottom
of the figure (b, d, f, and h). The bar represents 25 µm.
Western Blot Analysis for ZO-1 and E-Cadherin Protein
Production
Cell lysates were subjected to 6%
SDS-polyacrylamide gel electrophoresis and then transferred to
nitrocellulose membranes (Micron Separations Inc., Westboro, MA). The
membranes were blocked overnight at 4 °C with TBST-5% NFDM and
incubated with a mixture of primary rat monoclonal antibodies directed
against ZO-1, R40.76 (1:1000), and primary polyclonal E-cadherin
antibodies (1:1600) in TBST-1% NFDM overnight at 4 °C; secondary
antibody directed against rat IgG conjugated to HRPO (Cappel) was
diluted 1:10,000 in TBST-1% NFDM and incubated for 1 h. The signal was
detected by enhanced chemiluminescence on Hyperfilm-ECL from Amersham
Corp. Parallel cell samples were electrophoretically fractionated, and
equivalent protein loading was demonstrated by Coomassie Blue staining
of the protein gel.Transfection Procedures
31EG4 mammary
cells from a logarithmically growing culture were transfected by
electroporation. Briefly, cells were harvested with trypsin-EDTA,
washed twice with sterile Ca- and
Mg
-free PBS, and resuspended in sucrose buffer
containing 270 mM sucrose, 7 mM sodium phosphate
buffer, pH 7.4, and 1 mM MgCl
. 250 µl of cell
suspension and plasmid DNA (15-25 µg total including
10-15 µg of either GRE-CAT or p3TP-lux) were mixed, and 5
electric pulses (400 V square wave pulse for 99 µs) were delivered
to the sample using a BTX 800 Transfector apparatus (BTX Inc., San
Diego, CA). The samples were incubated for 10 min on ice and cultured
at 37 °C. 16 h after transfection, the cells were treated for the
indicated times with TGF- and/or dexamethasone in fresh media and
harvested for CAT or luciferase assays. Cells harvested for CAT assays
were washed three times in PBS, resuspended in 0.1 M Tris-HCl,
pH 7.8, and prepared by four cycles of freeze-thawing. The cell lysates
were heated at 68 °C for 15 min and centrifuged at 12,000 g for 10 min, and the supernatants were recovered. Cells for
the luciferase assay were washed three times with PBS and lysed with 1
ml of reporter lysis buffer (Promega) according to the
manufacturer's instructions.CAT and Luciferase Assays
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
[H]acetyl co-enzyme A (specific activity, 200
mCi/mmol; DuPont), 25 µl of 1 M Tris-HCl, pH 7.8, and 50
µl of 5 mM aqueous solution of chloramphenicol. 4 ml of a
water-immiscible scintillation fluor (Econofluor, DuPont NEN) was
added, and the samples were 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
[H]acetylated chloramphenicol produced
(cpm/µg protein/4 h). Pure bacterial CAT enzyme (0.01 units;
Pharmacia Biotech Inc.) was utilized as a positive control for the CAT
enzymatic assays, whereas mock transfected cells were used to establish
basal level activity. Each experiment was performed in triplicate and
was repeated three or more times.)4Mg(OH)2.5H0, 2.67 mM MgSO
, 0.1 mM EDTA, 33.3 mM
dithiothreitol, 270 µM coenzyme A, 470 µM luciferin, and 530 µM ATP, pH 7.8) at room
temperature. The light produced was measured in Beckman LS 6000IC
scintillation counter with the coincidence circuit turned off (under
the Single Photon Monitor option) or in a Monolight 2010 luminometer
(Analytical Luminescence Laboratory, San Diego, CA). Cells transfected
with pUHC131-1(38) , a plasmid that constitutively
expresses the luciferase gene, were utilized as positive control,
whereas mock transfected cells were used to establish background level
of activity in the assay.
TGF-
We have previously shown that the
synthetic glucocorticoid, dexamethasone, stimulates formation of tight
junctions in cultured 31EG4 cells by measuring an increase in TER and a
decrease in apical to basolateral movement of small radiolabeled
tracers(10) . To test the effects of TGF- Reversibly Inhibits Glucocorticoid
Stimulation in Transepithelial Electrical Resistance of 31EG4 Mammary
Epithelial Cells on
glucocorticoid stimulated tight junction function, confluent monolayers
of mammary cells were grown on permeable supports and treated with or
without 1 µM dexamethasone (Dex) in the presence or the
absence of 10 ng/ml TGF-. After an approximately 24-h time lag,
dexamethasone induced a significant increase in monolayer TER, whereas
in untreated cells, the basal TER remained low (Fig. 1). When
added simultaneously with dexamethasone, TGF- completely repressed
the ability of glucocorticoids to stimulate TER (Fig. 1). The
apical to basal leakage of [
C]mannitol was
significantly reduced in dexamethasone-treated cells compared with
cells treated with steroid and TGF- (data not shown), confirming
that the failure to increase TER in the presence of TGF-
correlated with a decrease in paracellular permeability through the
monolayer.
reversibly inhibits the dexamethasone stimulation in transepithelial
electrical resistance. 31EG4 mammary epithelial cells were cultured on
permeable supports with the indicated combinations of 1 µM Dex and 10 ng/ml TGF- for 120 h. In one set of cultures,
TGF- was withdrawn from the medium of cells treated with
dexamethasone and TGF- for 48 h (large arrow) by
incubating the cells with medium supplemented with dexamethasone alone
for an additional 72 h (Dex/TGF- Withdrawal). Throughout
the 120-h time course, the TER was determined at the indicated times,
and the ohmscm were calculated as described in the
text. TER measurements were performed in triplicate, and the results
are the averages of three separate
experiments.
on
steroid-regulated tight junction formation in 31EG4 mammary cells were
completely reversible. As also shown in Fig. 1, TGF-
withdrawal from cells treated for 48 h with both dexamethasone and
TGF- led to a rapid stimulation in TER after a 24 h time lag. The
observed time course of tight junction formation after TGF-
withdrawal was similar to that in cells initially treated with steroid
alone, suggesting that TGF- may be preventing tight junction
function at the earlier steps in the glucocorticoid signal transduction
pathway. The reversible nature of the TGF--mediated inhibition of
tight junction formation demonstrates that TGF- does not
indirectly prevent a stimulation in TER as a result of cytotoxic
effects on the mammary cells.TGF-
To further investigate the mechanism
of hormone-regulated tight junction dynamics, we tested whether
TGF- Disrupts Cellular Distribution of ZO-1 and
Alters Cell Morphology can alter the characteristic tight junction staining and/or
expression of ZO-1. As part of this study, F-actin localization was
also evaluated because the actin-based cytoskeleton is essential for
tight junction assembly and maintenance(24) . 31EG4 cells were
treated with combinations of dexamethasone and TGF- for 72 h and
doubly stained with primary ZO-1 monoclonal antibodies and fluorescein
isothiocyanate-conjugated goat anti-rat IgG as well as with
rhodamine-phalloidin for F-actin. Confocal microscopy was utilized to
investigate the spatial relationship between ZO-1 and actin under
conditions where tight junctions are tightly sealed by dexamethasone
and disrupted by TGF-.
drastically disrupted the overall pattern of ZO-1 and actin staining in
glucocorticoid-treated (Fig. 2, e and f) and
untreated (Fig. 2, g and h) cells, causing a
redistribution from points of cell-cell contacts to a more broad and
cytoplasmic staining. Immunostaining at the upper plane of
TGF--treated cells revealed a complete loss of ZO-1 junctional
staining with both ZO-1 and actin displaying a dispersed colocalization
in this region (Fig. 2, e versus a). At the more
basolateral plane of TGF--treated cells (Fig. 2, f), some ZO-1 is seen to localize at the cell periphery, but
the staining lacks the complete tight rings typical of junctional
associated plaque proteins. When the mammary cells were cultured in the
presence of both dexamethasone and TGF- (Fig. 2, g and h), there is slightly more specific junctional
staining of ZO-1 and actin. In the upper left portion, ZO-1 and actin
staining is detected only in the apical plane (Fig. 2, g), indicating that certain areas of cells are raised higher
than others. ZO-1 and actin staining in the mammary cells treated with
glucocorticoids and TGF- appear to be heterogeneous in that
although most cells have no tight perijunctional staining, some cells
have both specific junctional staining as well as diffuse cytoplasmic
staining and still others have only the characteristic cell peripheral
staining. Similar to cells treated with TGF- alone, actin
colocalizes with ZO-1 even in the most apical plane (Fig. 2, g). Thus, under conditions in which TGF- prevents
dexamethasone from stimulating TER, a remodeling of cell morphology
occurs with a cellular redistribution of junctional associated
proteins.TGF-
To determine whether TGF- Does Not Regulate ZO-1 Protein
Levels treatment alters the
level of ZO-1 protein expression, 31EG4 mammary cells were cultured on
permeable supports and treated in the presence or the absence of the
indicated combinations of dexamethasone and/or TGF- for 72 h.
Western blots of electrophoretically fractionated whole cell extracts
were incubated simultaneously with monoclonal antibodies to ZO-1 and
the adherens junction protein, E-cadherin. As shown in Fig. 3,
TGF- treatment did not affect either the basal levels or the
dexamethasone-induced expression of ZO-1 previously characterized in
these cells(9) . Approximately equivalent gel loading was shown
by similar amounts of E-cadherin in each sample. Importantly, under all
hormonal conditions, the monoclonal antibodies to ZO-1 recognized a
single protein band of approximately 220-kDa proteins. Thus, under
conditions in which the transepithelial electrical resistance fails to
be induced by glucocorticoids, the pattern of ZO-1 distribution in
mammary cells but not its expression is significantly altered by
exposure to TGF-.
. Cell lysates were normalized for total
protein (that was confirmed by Coomassie Blue staining of a parallel
polyacrylamide gel), electrophoretically fractionated, and blotted onto
nitrocellulose filters. The nitrocellulose blot was incubated
simultaneously with ZO-1 and E-cadherin primary antibodies, and protein
signals were detected by chemiluminescence after incubation of
secondary antibodies as described in the text. The protein molecular
mass standards are myosin (200 kDa) and phosphorylase b (97.4 kDa). B, cell extracts from dexamethasone-treated (Dex) and
untreated (No Addition) 31EG4 mammary epithelial cells as well
as NIH 3T3 fibroblasts (NIH-3T3) were electrophoretically
fractionated, and nitrocellulose blots were analyzed for cytokeratin
type II protein as described in the text. The molecular mass markers
are bovine serum albumin (66.2 kDa) and ovalbumin (45
kDa).
Pretreatment with Dexamethasone Precludes TGF-
When added simultaneously with dexamethasone,
TGF- from Disrupting Tight Junction Integrity or Altering ZO-1
Distribution precludes glucocorticoids from inducing the sealing of tight
junctions. To determine whether TGF- can disrupt tight junctions
once they are well sealed due to glucocorticoid treatment, TGF-
was added for 24 h to 24 or 48 h dexamethasone-pretreated cells, and
the monolayers were assayed for changes in TER or ZO-1 localization. As
previously mentioned, dexamethasone rapidly induces the TER of 31EG4
mammary cells after an initial time lag of 24 h. As shown in Fig. 4, TGF- addition at 48 h (Dex/TGF- 48-72) has only a minimal effect on dexamethasone-stimulated
TER. In contrast, similar to cells simultaneously treated with
TGF- and dexamethasone (Dex+TGF-), TGF-
completely inhibited tight junction formation when added at 24 h of
glucocorticoid treatment (Dex/TGF- 24-48). The
addition of TGF- at any time to dexamethasone-treated cells after
the stimulation in TER had no effect on the maintenance of well
established tight junctions.
from inhibiting the
stimulation in transepithelial electrical resistance. 31EG4 mammary
epithelial cells were cultured on permeable supports and treated with 1
µM Dex. A control culture was maintained without the added
steroid (No Addition). At 0, 24, or 48 h of dexamethasone
treatment (large arrows), the cells were exposed to 10 ng/ml
TGF-, and the incubations were continued for up to 48 or 72 h. The
monolayer transepithelial electrical resistance was determined at the
indicated times, and the ohmscm were calculated as
described in the text. The TER measurements were performed in
triplicate, and the results are the averages of three separate
experiments.
to steroid-treated cells with
an induced TER had no effect on the cellular distribution of ZO-1
protein. TGF- was added to mammary cells at various times (0, 24,
or 48 h or no addition) during exposure to 1 µM dexamethasone and then incubated for up to 48 or 72 total hours. A
control set of cultures were treated with TGF- for the same time
points in the absence of steroid. Under conditions in which the
monolayer TER is stimulated by dexamethasone for 72 h (no TGF- or
TGF- treatment 48-72 h), ZO-1 protein was localized to the
tight junction as a sharp continuous band of immunostaining at the cell
periphery (Fig. 5). The 48 h dexamethasone-treated and untreated
cells displayed the same peripheral ZO-1 staining as mammary cells
exposed to steroid for 72 h. In contrast, when TGF- was added to
24 h steroid-treated cells (TGF- 24-48 h) or simultaneously
with dexamethasone (0-72 h), TGF- caused a disruption of the
overall ZO-1 staining pattern with most of the immunostaining being
diffused throughout the cell (Fig. 5). A similar TGF-
effect on ZO-1 immunostaining was also observed in the absence of
glucocorticoids (Fig. 5). Taken together, these observations
demonstrate that glucocorticoid or TGF- receptor signaling
pathways can regulate tight junction dynamics of 31EG4 cells.
from disrupting the cellular
distribution of ZO-1. 31EG4 mammary cells were cultured on permeable
supports with (+ Dex) or without (- Dex) 1
µM dexamethasone. Cells were treated with 10 ng/ml
TGF- simultaneously with dexamethasone during an entire 72-h time
course (0-72 hr), for the last 24 h of a 48 h time
course (24-48 hr), or for the last 24 h of a 72-h time
course (48-72 hr). A control set of cell cultures were
not treated with TGF- (none). The cells were fixed and
analyzed for ZO-1 localization by indirect immunofluorescence using a
ZO-1 monoclonal primary antibody as described in the
text.
Mutual Antagonism of Tight Junction Integrity by
Dexamethasone and TGF-
One possible mechanism through which
dexamethasone and TGF- Is Not Due to Inhibition of Receptor
Signaling treatment can mutually exclude the actions
of the other signal is by directly inhibiting receptor function. To
test this possibility, 31EG4 mammary cells were incubated for 72 h with
or without combinations of dexamethasone and TGF- after
transfection with reporter genes containing promoter elements
responsive to either glucocorticoid receptor function (GRE-CAT) or
TGF- receptor signaling (p3TP-Lux). GRE-CAT contains six
glucocorticoid response elements and the minimal promoter of the
alcohol dehydrogenase gene linked to the bacterial CAT reporter gene.
p3TP-Lux, which has been shown to be TGF--responsive(39) ,
contains three consecutive TPA response elements and a portion of the
plasminogen activator inhibitor gene promoter linked to the luciferase
gene. As shown in Fig. 6(top panel), dexamethasone
strongly stimulated GRE-CAT activity both in the presence of TGF-
for 72 h, in which the monolayer TER remains low, and in the presence
of TGF- only during the last 24 h (48-72 h) of a 72-h
dexamethasone treatment, during which the monolayer TER remains high at
the steroid-induced level. Similarly, TGF- stimulates the activity
of p3TP-lux in 31EG4 cells not treated with steroids in cells incubated
simultaneously with dexamethasone and TGF- or in cells treated
with TGF- during the last 24 h of a 72-h dexamethasone treatment (Fig. 6, lower panel). Taken together, these data show
that the glucocorticoid receptor and the TGF- receptor are both
functional in the presence of both hormones and suggest that the mutual
antagonism regulating tight junction dynamics are specific postreceptor
events.
receptor
signaling. 31EG4 mammary cells were transfected with either the
glucocorticoid-responsive GRE-CAT chimeric reporter plasmid or the
TGF--responsive p3TP-Lux reporter plasmid by electroporation and
then cultured for 72 h with the indicated combinations of 1 µM Dex and 10 ng/ml TGF-. In one set of cultures, TGF- was
included in the medium only during the last 24 h of dexamethasone
treatment (48-72 hr). Cell extracts were assayed for
either CAT-specific activity (top panel) or luciferase
specific activity (bottom panel) as described in the text. The
results are the averages of two independent sets of triplicate
samples.
TGF-
Our previous results with both
nontransformed and transformed rodent mammary cells showed that the
glucocorticoid stimulation of tight junction formation is a
differentiated property that is accompanied by and perhaps related to
an inhibition of cell proliferation(9) . This observation
suggests that TGF- Stimulates DNA Synthesis of 31EG4 Mammary
Epithelial Cells might reverse or prevent the growth suppression
effects of glucocorticoids under conditions in which the formation of
tight junctions is inhibited. Depending on the cell type, TGF- can
either suppress or stimulate cell proliferation. TGF- has been
shown generally to inhibit or not effect epithelial cell
growth(40, 41) , although several studies have
documented growth stimulatory effects of TGF- on a small number of
epithelial-derived cells(42, 43) . Conflicting
evidence exists on the growth effects of TGF- on mammary
epithelial cells because in certain mammary epithelial tumors,
TGF- production correlates with tumor progression and
growth(44, 45) , whereas in many mammary tumor cell
lines, TGF- is growth inhibitory(46, 47) . 31EG4
mammary cells are epithelial in nature because they express the
cytokeratin type II epithelial cell marker protein that is not produced
in murine fibroblasts (Fig. 3B). We therefore
investigated TGF- effects on the growth of 31EG4 cells cultured on
permeable supports in the presence of combinations of dexamethasone and
TGF- during 48- or 72-h time courses. As shown in Fig. 7,
dexamethasone significantly inhibited the incorporation of
[H]thymidine after either 48- (left
panel) or 72-h (right panel) treatment. In contrast,
TGF- treatment stimulated [H]thymidine
incorporation of these mammary epithelial cells both in the presence
and the absence of dexamethasone. During a 72-h time course,
simultaneous exposure to TGF- and dexamethasone (0-72)
counteracted the effects of either hormone added alone. Moreover, the
addition of TGF- to dexamethasone-treated cells during the last 24
h of a 72-h time course (48-72), which does not disrupt tight
junction integrity, caused only a minor stimulation of
[H]thymidine incorporation. In general, the
absolute level of [H]thymidine incorporation was
higher in TGF--treated cells under conditions that prevent
dexamethasone from inducing monolayer TER. Thus, TGF- reverses the
glucocorticoid-stimulated formation of tight junctions under the
conditions that promote a stimulation in DNA synthesis.
stimulates incorporation of [H]thymidine in 31EG4
mammary epithelial cells under conditions that inhibit tight junction
formation. 31EG4 mammary cells were cultured on permeable supports in
the presence or the absence of 1 µM Dex for either 48- (left panel) or 72-h (right panel) time courses. In
the indicated samples, cells were treated with 10 ng/ml TGF- with
or without dexamethasone for the last 24 h of the 48- (24-48) and 72-h (48-72) time courses.
Mammary cells were also treated with TGF- during the entire 72-h
time course (0-72). The rate of DNA synthesis was
monitored by determining the incorporation of
[H]thymidine after a 2-h pulse label as described
in the text. The results are the averages of triplicate
samples.
requires the stimulation of
[H]thymidine incorporation, the effects of
TGF- on tight junction dynamics was monitored in the presence or
the absence of a DNA synthesis inhibitor, hydroxyurea. After a 24-h
dexamethasone treatment, 31EG4 cells were incubated with combinations
of TGF- and/or hydroxyurea for an additional 24 h and assayed for
monolayer TER and incorporation of [H]thymidine.
The control set of cultures had no additions of hormone or metabolic
inhibitor. As shown in Fig. 8, TGF- inhibited the
dexamethasone-stimulated monolayer TER in the presence or the absence
of hydroxyurea. Dexamethasone induced a significant increase in
monolayer TER in the presence of hydroxyurea, although with a somewhat
reduced response. Under the conditions of this experiment, hydroxyurea
abolished the incorporation of [H]thymidine in
the presence or the absence of TGF-, demonstrating that this
metabolic agent effectively inhibited DNA synthesis in cells cultured
on permeable supports. These results demonstrate that dexamethasone
induction of and TGF- inhibition of tight junction formation do
not require de novo DNA synthesis and, because hydroxyurea
acts at or near the G/S border(48) , cell cycle
progression into and past the 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 a DNA synthesis inhibitor. 31EG4 mammary cells were
cultured on permeable supports in the presence of the indicated
combinations of 1 µM Dex, 10 ng/ml TGF-, and/or 1
µM hydroxyurea for 48 h; one set of control cultures were
not treated with either hormone or hydroxyurea (No Addition). Top panel, the TER was monitored, and the ohmscm were calculated as described in the text. Each assay was
performed in triplicate, and the results are the averages of three
separate experiments. Lower panel, the rate of DNA synthesis
was monitored by determining the incorporation of
[H]thymidine after a 2-h pulse label as described
in the text. The results are the averages of triplicate
samples.
, a locally
produced factor, can antagonistically regulate tight junction dynamics.
This observation implicates these two distinct extracellular signals as
playing important roles in controlling cell-cell interactions in
vivo. The mammary gland undergoes a progression of morphological
and functional changes during pregnancy, lactation, and involution,
including temporal regulation of tight junction permeability. For
instance, colostrum, the milk secreted by the mammary gland a few days
before or after parturition, contains more protein, immunoglobulins,
sodium, and chloride and less potassium and lactose than the milk
secreted during established lactation(2) . These differences
indicate an alteration in paracellular movement that is principally due
to the state of the tight junction. During late pregnancy, freeze
fracture studies have shown that the tight junctional network was
diffuse with relatively scant ridges, depicting a leaky epithelia,
whereas the network at 1 day post-partum and the duration of lactation
were condensed with abundant ridges between the lumen and the
intercellular space, representing a tight
epithelia(3, 50) . Thus, at a stage when
glucocorticoids function in the lactation process, milk components are
strictly secreted into the lumen of the ducts via apically directed
secretory pathways, and highly developed tight junctions are required
to prevent paracellular leakage of blood and milk components from
opposite sides of the mammary epithelium. Tight junction permeability
increases concomitantly with a decline in mammary blood flow and milk
secretion as part of the involution process after
lactation(51, 52) . It is likely that the local
factors responsible for maintaining a relatively unrestricted
paracellular pathway is developmentally regulated depending on the
stage of differentiation of the mammary gland. In this regard,
TGF- 1 transcript levels were detected in all stages (5 week,
mature, pregnant) of the mammary gland development except during
lactation(35) . In addition, TGF- can suppress the
synthesis and secretion of milk caseins from mammary explants in
pregnant mice(53) . antagonistically control tight junction
dynamics in vitro in a temporally regulated manner. In our
studies of mammary cells pretreated with glucocorticoids for 48 h,
TGF- failed to reduce the monolayer TER or alter ZO-1
localization. In contrast, the addition of TGF- simultaneously
with or up to 24 h after glucocorticoid treatment disrupted the
structural organization and function of tight junctions. Activation of
glucocorticoid-responsive or TGF--responsive reporter plasmids
demonstrated that the mutual antagonism displayed by dexamethasone and
TGF- targets signaling pathways that regulate cell-cell
interactions and growth control rather than having direct effects on
receptor function. The time lag required for TER induction suggests
that this process is mediated by steroid-regulated events of about 24
h. Because exposure to TGF- during but not after this lag period
precludes the ability of dexamethasone to induce tight junction
formation, the timing of glucocorticoid-induced gene expression and/or
function is critical for regulating TGF- responsiveness. Given the
known transcriptional mechanism of action of glucocorticoid receptors (54, 55) , it is tempting to consider that expression
and/or activity of the immediate early steroid-regulated gene products
that initiate the tight junction response can be reversed by TGF-
receptor signaling. Conceivably, the delayed dexamethasone-responsive
gene products, which are responsible for the increase in TER and
development of well sealed tight junctions, are relatively unaffected
by exposure to TGF-. Alternatively, components of the TGF-
receptor signaling pathway that directly or indirectly target the tight
junction may not be functional or adequately expressed in
glucocorticoid-treated mammary cells. Identification of the signaling
components that regulate tight junction assembly, disassembly, and
integrity will be important to clarify the precise mechanism of
hormonal control.
disruption of ZO-1 localization and prevention of steroid-induced TER
were accompanied by a stimulation in [H]thymidine
incorporation. Flow cytometry analysis revealed that TGF- also
induced a shift in cellular DNA content to a profile consistent with a
growing population of cells. ()The ability of TGF- to
preclude the steroid induction of tight junction formation occurred in
the presence of hydroxyurea, a DNA synthesis inhibitor, demonstrating
that this disruptive effect on tight junction integrity is not an
indirect consequence of an increase in DNA synthesis. This observation
suggests that the tight junction machinery is a selective target of
TGF- receptor signaling. However, this evidence does not exclude
the possibility that TGF--mediated stimulation in
[H]thymidine incorporation and cell growth may be
a consequence of the disruption in tight junction structure and ZO-1
redistribution.. Clearly, the modification and
regulation of ZO-1 are complicated processes that are dependent on the
physiological environment, cell type, and integrity of the tight
junction. Conceivably, the TGF--mediated stimulation in
[H]thymidine incorporation and tight junction
disassembly may involve modification and redistribution of the ZO-1
protein. is a potent growth inhibitor of many
epithelial-derived cells but generally stimulates proliferation of many
fibroblast cells(60) . The 31EG4 mammary cells used in our
study are epithelial in nature because they are polarized and express
the epithelial-specific marker gene cytokeratin type II, which is not
produced in mouse fibroblasts. Thus, the untransformed cells are one of
only a few characterized epithelial cell lines in which TGF-
stimulates their growth(42, 43) . Perhaps one reason
for this relatively rare effect of TGF- on epithelial cells is
that the mammary cells are maintained on filters at confluency, which
exposes the entire surface of the cells to nutrients in the
extracellular environment. In addition, several studies have shown a
general correlation between TGF- expression and tumor cell
growth(61, 62) . For example, highly proliferative
mammary tumors contain an increased level of TGF- transcripts
compared with their normal human mammary cell
counterparts(62) . The mechanism by which TGF- promotes
cell growth is not well understood, although the stimulation in
proliferation in a fibroblast cell line is accompanied by an activation
of cyclin E-Cdk2 kinase and down-regulation of the p27/Kip1 cell cycle
inhibitor(63) . Another mechanism by which TGF- may be
stimulating mammary epithelial cell growth is by inducing
transdifferentiation to a nonepithelial, mesenchymal-like phenotype (37) . Whatever the precise mechanism of growth regulation of
mammary epithelial cells, the novel antagonistic regulation of tight
junction dynamics by TGF- and glucocorticoids may have important
implications for understanding hormonal contributions to controlling
differentiation processes of the mammary gland, as well as cell-cell
interactions involved with invasiveness and metastasis of mammary
tumors. signaling observed in vitro parallels that of in vivo cellular events associated with
the control of mammary cell-cell interactions. Transgenic mice
expressing TGF- 1 targeted to the pregnant mammary gland showed
inhibited alveolar development and lactation(64) . We propose
that TGF-, which is developmentally regulated during mammary gland
differentiation, plays a critical role in maintaining a relatively
leaky paracellular pathway, and that during lactation, dexamethasone is
the predominant regulator of tight junction integrity. In an analogous
manner, TGF- has been shown to alter the synthesis of
extracellular matrix components(65, 66, 67) ,
which can potentially affect extracellular signaling and cell function.
For instance, tenascin-C, which inhibits fibronectin-mediated adhesion,
is stimulated by TGF- 1 and down-regulated by
glucocorticoids(68, 69) . The downstream targets of
TGF-- and/or glucocorticoid receptor-activated signaling cascades
in mammary epithelial cells are mostly unknown. Conceivably, ZO-1 or
other tight junction proteins could serve as potential targets for
steroid or growth factor signaling pathways. We are currently
attempting to elucidate the cellular events underlying the mutual
antagonism of tight junction dynamics by glucocorticoids and TGF-
that operate in mammary epithelial cells. Such pathways could
potentially represent an important cross-talk between growth factor and
steroid receptor signal transduction cascades that is necessary to
guide the functional relationships between particular sets of
environmental cues that control mammary cell-cell interactions.
), transforming growth factor- 1;
TER, transepithelial electrical resistance; PBS, phosphate-buffered
saline; CAT, chloramphenicol acetyltransferase; Dex, dexamethasone.)
We thank Patricia Buse, Anita C. Maiyar, and Kay E.
Simon for constructive comments during the course of the work and for
critical comments of this manuscript. We also express our appreciation
to Jerry Kapler for skillful photography, Anna Fung for preparation of
this manuscript, and Richard D. Fetter, William J. Meilandt, Marina
Chin, Ritu Patel, Vinh Trinh, and Thai Truong for technical support.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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