(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- 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.
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- (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.
Confocal images were obtained from a Zeiss Axioplan
epifluorescence microscope using a Zeiss 40 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.
Figure 2:
Transforming growth factor- 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.
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)4Mg(OH)2.5H
0, 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.
Figure 1:
Transforming growth factor-
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 ohms
cm
were calculated as described in the
text. TER measurements were performed in triplicate, and the results
are the averages of three separate
experiments.
Consistent with its mode of action on mammary gland
differentiation (35) , the inhibitory effects of TGF- 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.
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-
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.
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-. 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).
Figure 4:
Pretreatment with dexamethasone for 48 h
prevents transforming growth factor- 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 ohms
cm
were calculated as
described in the text. The TER measurements were performed in
triplicate, and the results are the averages of three separate
experiments.
Indirect immunofluorescence microscopy
revealed that the addition of TGF- 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.
Figure 5:
Pretreatment with dexamethasone for 48 h
prevents transforming growth factor- 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.
Figure 6:
Effects of hormone treatment on
glucocorticoid receptor and transforming growth factor- 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.
Figure 7:
Transforming growth factor-
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.
To determine
whether the disruption of glucocorticoid-induced tight junction
formation by TGF- 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.
Figure 8:
Transforming growth factor- 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 ohms
cm
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.
Our results using a nontransformed mammary epithelial cell
line represent the first evidence that glucocorticoids, an important
systemic lactogenic steroid(49) , and TGF-, 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) .
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- 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.
Dexamethasone coordinately induced tight junction
formation and suppressed DNA synthesis, whereas the TGF-
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.
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-. 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.
TGF- 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.
It is tempting to consider that the selective antagonism
between glucocorticoid and TGF- 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.