From the Department of Molecular and Cell Biology and the Cancer Research Laboratory, University of California at Berkeley, Berkeley, California 94720-3200
Received for publication, December 23, 2002
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ABSTRACT |
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In Con8 mammary epithelial tumor cells, we
have documented previously that the synthetic glucocorticoid
dexamethasone induces the reorganization of the tight junction and
adherens junction (apical junction) and stimulates the monolayer
transepithelial electrical resistance (TER), which is a reliable
in vitro measurement of tight junction sealing. Western
blots demonstrated that dexamethasone treatment down-regulated the
level of the RhoA small GTPase prior to the stimulation of the
monolayer TER. To test the role of RhoA in the steroid regulation of
apical junction dynamics functionally, RhoA levels were altered in Con8
cells by transfection of either constitutively active (RhoA.V14) or
dominant negative (RhoA.DN19) forms of RhoA. Ectopic expression of
constitutively active RhoA disrupted the dexamethasone-stimulated
localization of zonula occludens-1 and The apical junctional complex, which consists of the tight
junction, the adherens junction, and desmosomes, is located at the
points of contact between cells and controls intercellular adhesion and
the permeability properties involved in epithelial cell-cell
interactions (1-5). The tight junction, or zonula occludens (ZO),1 is the most apical
structure of the junctional complex and forms a continuous
"gasket-like" seal around the lateral circumference of adjacent
cells which controls the selective diffusion of solutes through a
paracellular pathway. Tight junctions also establish and maintain
cellular polarity by restricting the lateral diffusion of lipids and
membrane proteins between the compositionally and functionally distinct
apical and basolateral membrane domains (6-13).
Tight junction assembly is generally thought to require the initial
engagement of cell-cell contacts at the adherens junction, which lies
immediately basal to the tight junction and is responsible for
intercellular adhesion between neighboring cells (14). Adherens junction formation is a process that is mediated by the
calcium-dependent intercellular adhesion between E-cadherin
molecules and the formation of an intracellular protein complex that
includes E-cadherin, Both the tight junction and the adherens junction have been proposed to
associate with the perijunctional actin cytoskeleton through
multiprotein complexes (14, 28, 29) and form an integrated functional
unit. The architecture of the apical junctional complex, the
cytoskeleton, and sealing of tight junctions at the sites of cell-cell
contact can be regulated in a dynamic manner depending on the tissue
origin, physiological state, and availability of specific sets of
extracellular cues (3, 30-32). These observations implicate the
potential involvement of distinct signal transduction cascades that
functionally target the tight junction and/or adherens junctions in
epithelial cells.
We have discovered that glucocorticoids, one class of steroid hormones,
induce tight junction formation and cell polarity through a multistep
cascade in cultured mammary epithelial cells (33-37). An early
glucocorticoid-regulated step in the overall cascade is the rapid
stimulation in expression of the helix-loop-helix transcriptional
regulatory molecule Id-1, which is required for the steroid control of
cell tight junction dynamics (38). In a later set of events,
glucocorticoids induce the recruitment of the tight junction proteins
(ZO-1 and occludin), adherens junction proteins (E-cadherin and
Given the complex nature of the steroid-regulated cascade leading to
tight junction formation, additional classes of regulatory proteins may
functionally link the cytoskeleton to the control of tight junction
dynamics in mammary epithelial cells. One such potential category of
cell signaling molecules is the Rho family of small GTPases which
includes RhoA, Rac, and Cdc42 (39-45). In several cell systems, RhoA
has been shown to be involved in cytoskeletal reorganization and has
been implicated in regulating the spatial organization of adherens
and/or tight junction proteins (41, 42, 46, 47). However, an
unambiguous role for RhoA in controlling cell-cell interactions has not
been defined because of tissue/cell type-specific differences in
RhoA-activating cellular cascades and in the experimental manipulation
of tight junction formation. A unique feature of the Con8 mammary
epithelial tumor cells is the ability to control acutely and reversibly
the organization and function of the apical junctional complex with
glucocorticoid treatment without having to first destroy and then
rebuild tight junctions. In this study, we demonstrate that
glucocorticoids down-regulate RhoA levels and that this regulatory
process is required for the steroid-induced remodeling of the apical
junction architecture that leads to functional tight junctions.
Materials--
Dulbecco's modified Eagle's medium/Ham's F-12
(50:50) and calf serum were supplied by BioWhittaker (Walkersville,
MD). Phosphate-buffered saline, trypsin-EDTA, and dexamethasone were
obtained from Sigma. G418 was purchased from Invitrogen. Permeable
tissue culture supports/filter inserts were manufactured by Nunc and
distributed by Applied Scientific (San Francisco, CA). Polyclonal
rabbit anti-ZO-1, monoclonal mouse anti- Cell Culture and Measurement of Transepithelial Electrical
Resistance--
Con8 rat mammary epithelial tumor cells have been
described previously (37, 49). To generate control, dominant active, and dominant negative Rho cell lines, the neomycin-resistance gene
containing plasmids pcEXV-Myc-RhoA.V14, pcEXV-Myc-RhoA.DN19, and the
pcEXV empty vector were transfected into Con8 cells. After selection
with the neomycin analog G418 for 2 weeks, 50 clones were selected,
expanded, and tested for induction of Myc-tagged RhoA. All cell lines
were routinely grown to 100% confluence on Nunc permeable supports in
Dulbecco's modified Eagle's medium/Ham's F-12 supplemented with 10%
calf-serum and penicillin/streptomycin and maintained at 37 °C in a
humid atmosphere of air/CO2 (95:5). For culturing of cells
transfected with either the RhoA.V14 or RhoA.N19 expression vector,
G418 was added to the culture medium to a final concentration of 600 µg/ml. The cell culture medium was changed every day during the
experimental treatment. For the glucocorticoid treatment of cells, the
synthetic glucocorticoid agonist dexamethasone (Sigma) was added to the
normal growth medium at a final concentration of 1 µM (prepared as a 1,000 µM stock in
ethanol). Formation of tight junction was monitored by daily measurements of transepithelial electrical resistance (TER) using an
EVOM Epithelial Voltohmmeter (World Precision Instruments, Sarasota,
FL), as we have described previously (33-35, 37, 49). Calculations for
ohms·cm2 were determined by subtracting the resistance
measurement of a blank filter and multiplying by the area of the monolayer.
Immunofluorescence and Confocal Microscopy--
Con8- and
RhoA-transfected rat mammary cells were grown on Nunc filters and
incubated with or without 1 µM dexamethasone for several
days. Cells were treated every 24 h. For fluorescent analysis, the
cell layers were washed three times with Dulbecco's phosphate-buffered saline (BioWhittaker) and fixed with 1.75% formaldehyde in
phosphate-buffered saline for 15 min at room temperature. After three
additional washes with phosphate-buffered saline, the plasma membrane
was permeabilized with 0.5% Triton X-100 extraction buffer (0.5%
Triton X-100, 100 mM Tris-HCl, pH 7.5, 120 mM
NaCl, 20 mM Hepes, and 5 mM EDTA) for 15 min at
room temperature. After blocking in 5% non-fat dry milk, filters were
stained for 1 h with anti-ZO-1 antibodies followed by a 1-h
incubation with fluorescein isothiocyanate-conjugated secondary
antibodies. For costaining, the cells were incubated with either
Western Blots--
For Western blot analyses, cells were rinsed
with ice-cold phosphate-buffered saline (BioWhittaker) and extracted in
lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl,
20 mM MgCl2, 1% Nonidet P-40) containing
protease inhibitors. Samples were normalized for protein content with
the Bio-Rad Bradford protein assay. Cell lysates were fractionated on a
12% SDS-polyacrylamide gel. Proteins were transferred
electrophoretically from the gel to a nitrocellulose membrane (Micron
Separations, Inc., Westboro, MA). Blots were blocked in 5% non-fat dry
milk in washing buffer (0.1 M Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for at least 30 min at room temperature and then incubated with anti-RhoA or anti- Glucocorticoid Down-regulation of RhoA Protein Levels and
Stimulation of Monolayer Transepithelial Electrical
Resistance--
Our previous studies (33-37) demonstrated that in
Con8 mammary epithelial tumor cells, glucocorticoids induce tight
junction formation through a multistep process that results in a final intercellular membrane sealing event. As a first step to determine whether the small GTPase RhoA plays a regulatory role in this cascade,
we examined the effects of glucocorticoids on the level of RhoA
protein. After reaching confluence, Con8 cells were serum starved for
24 h, treated with or without 1 µM synthetic
glucocorticoid dexamethasone for up to 4 days, and RhoA protein levels
were determined by Western blot analysis using monoclonal
anti-RhoA-specific antibodies. As shown in Fig.
1A, dexamethasone caused a
significant down-regulation of RhoA protein compared with the untreated
control cells. This down-regulation was typically detectable by 8 h of steroid treatment and reached statistically significant levels by
48 h, which continued throughout the time course. Western blots
probed with anti-actin antibodies (Fig. 1A, upper
blot) and anti-
Parallel sets of time course experiments examined the monolayer TER in
confluent Con8 cells cultured on Nunc filter supports in the presence
or in the absence of dexamethasone. The percent steroid-induced
increase in TER was determined at each time point as the ratio of TER
observed in steroid treated versus nontreated cells.
Consistent with our previous studies (33-36), after an approximate 24-h time lag, dexamethasone-treated cell monolayers displayed a
significant increase in monolayer resistance, and the absolute TER
plateaued at about 1,000 ohms·cm2 by 4 days in steroid
(Fig. 1B). The results from several RhoA Western blots were
also averaged and plotted as the percentage of RhoA protein detected in
dexamethasone-treated cells compared with untreated control cells. As
also shown in Fig. 1B, the dexamethasone down-regulation of
RhoA protein correlates with the stimulation of TER and becomes
statistically significant (p = 0.05) at 48 h.
These results suggest a potential causative relationship between the
glucocorticoid regulation of RhoA protein levels and tight junction formation.
Establishment of Mammary Tumor Cell Lines That Express
Constitutively Active or Dominant Negative RhoA and Remain
Glucocorticoid-responsive--
To assess functionally the requirement
of the glucocorticoid down-regulation of RhoA for tight junction
formation and/or sealing, stable Con8-derived cell lines expressing
either the constitutively active or dominant negative forms of RhoA
were established. The pcEXV-Myc-RhoA.V14 expression plasmid
encodes a constitutively active RhoA protein, which maintains the
GTP-bound form of the protein, and an N-terminal Myc epitope tag. The
pcEXV-Myc-RhoA.N19 expression plasmid also contains an N-terminal Myc
epitope tag and encodes a dominant negative RhoA that binds more
tightly to Rho-GEFs than the wild type GTPase, and thus preventing
activation of endogenous RhoA, without binding to effector proteins
(50). After selection of transfected Con8-derived cells with the
neomycin analog G418 for 2 weeks, individual cell clones were selected, expanded, and tested for their expression of Myc-tagged RhoA mutant proteins using a monoclonal Myc antibody (Fig.
2). Cells expressing the empty vector
behaved identically to the wild type, untransfected Con8 cells,
therefore we chose to use wild type cells as controls for the remainder
of the experiments. The Con8-DN25 (expressing the dominant negative
RhoA) and the Con8-V6 (expressing the constitutively active RhoA) cells
were utilized for further characterization of the role of RhoA in the
glucocorticoid-induced tight junction formation.
To determine whether the Con8-derived cell lines expressing either the
dominant negative or constitutively active RhoA remain responsive to
glucocorticoids, Con8-V6 and Con8-DN25 cells, along with control Con8
cells, were grown on tissue culture dishes and treated either with or
without 1 µM dexamethasone for 5 days. This
relatively long time point was utilized because it represents the
glucocorticoid treatment duration needed to observe the maximal stimulation in TER in the parental Con8 cells (see Fig. 1). Total cell
extracts were fractionated by SDS-polyacrylamide gels, and Western
blots were probed with antibodies specific for RhoA to detect total
RhoA protein. Western blots were also probed with antibodies for the
Myc tag on the RhoA constructs to detect the exogenous RhoA proteins,
as well as antibodies to the Sgk, which we originally identified as a
primary glucocorticoid-responsive gene in Con8 mammary tumor cells
(51). As a control for protein loading, the Western blots were probed
with antibodies against
As expected, Con8 cells show a strong down-regulation of endogenous
RhoA protein upon dexamethasone treatment (Fig.
3, RhoA). Con8-V6 and
Con8-DN25 cells produce a high level of RhoA immunoreactive protein in
the presence of dexamethasone because of the expression of exogenous
RhoA sequences. In these transfected cells, we also observed a
significant reduction in the down-regulation of total RhoA in
dexamethasone-treated cells compared with untreated cells (Fig. 3,
RhoA). Probing the Western blots with monoclonal anti-Myc antibodies revealed that the level of exogenous RhoA protein, either
the constitutive active or dominant negative form, is partially down-regulated upon dexamethasone treatment (Fig. 3,
Myc-RhoA). This down-regulation of exogenous RhoA protein
suggests that one of the effects of dexamethasone may be to alter RhoA
protein stability. Importantly, the impact of constitutively active and
dominant negative RhoA signaling is maintained in the transfected cells because of the relatively high level of RhoA protein which continues to
be produced in dexamethasone-treated cells (Fig. 3,
Myc-RhoA).
As also shown in Fig. 3, dexamethasone strongly induced Sgk in both
Con8-V6 and Con8-DN25 cells to approximately the same extent,
demonstrating that both transfected cell lines retained their overall
responsiveness to glucocorticoids. Similar results were obtained when
the cells were treated with glucocorticoids for short durations,
including 1 h (data not shown). For unknown reasons, the induction
of Sgk protein upon dexamethasone treatment appears higher in Con8-V6
and Con8-DN25 cells compared with Con8 cells.
The Down-regulation of RhoA Is Required for the
Glucocorticoid-induced Organization of the Junctional Complex--
The
Con8-V6 and Con8-DN25 cells were used to characterize functionally the
role of RhoA protein in the glucocorticoid-induced formation of tight
junctions. We documented previously that tight junction sealing is
preceded by a distinct steroid-regulated membrane organization step
that controls remodeling of both the adherens and tight junctions (33).
The glucocorticoid-induced organization of the junctional complex was
examined in Con8-V6, Con8-DN25, and control Con8 cells by
immunofluorescence visualization of the ZO-1 tight junction protein and
the
In a complementary manner, in Con8-DN25 cells both Effects of Expressing Constitutively Active RhoA or Dominant
Negative RhoA on Dexamethasone-induced Tight Junction Sealing--
To
examine the potential effects of RhoA on the regulated sealing of tight
junctions which occurs after the remodeling of the apical junction,
Con8-V6, Con8-DN25 cells, and Con8 cells were grown to confluence on
Nunc filter supports, and the monolayer TER was monitored in cells
treated with and without 1 µM dexamethasone for 5 days.
Dexamethasone treatment significantly increased TER in control Con8
cells and Con8-DN25 cells, compared with untreated Con8 cells
(p = 0.05; Fig. 5). In
the absence of steroids, the TER was ~200 ohms·cm2,
whereas in glucocorticoid-treated cells the TER reached ~950 ohms·cm2 (Fig. 5, black bars). The TER
obtained for glucocorticoid-treated Con8 cells is used as the 100%
level, with which all of the TER measurements were compared. In
contrast to the untransfected Con8 cells, in Con8-V6 cells, which
express constitutively active RhoA, dexamethasone failed to induce an
increase in the monolayer TER, which remained at the basal level of
~200 ohms·cm2 in the presence or in the absence of
steroid (Fig. 5, Con8-V6 bars). The TER in cells expressing
the dominant negative Con8-DN25 was partially inducible, with the TER
reaching ~500 ohms·cm2 in steroid-treated cells
compared with the 360 ohms·cm2, TER observed in untreated
cells (Fig. 5, gray bars). The basal level TER for the
Con8-DN25 cells was not significantly different from either the Con8 or
Con8-V6 cells; however, the dexamethasone-induced TER was
statistically significant (p = 0.05) compared with the basal TER. Thus, tight junction sealing remains
glucocorticoid-inducible in cells expressing the dominant negative form
of RhoA, although the magnitude of this response was less than that
observed in the control Con8 cells. Interestingly, in the absence of
glucocorticoids, Con8-DN25 cells displayed an organized junctional
complex (see Fig. 4) without tight junction sealing. This result
suggests that the inhibitory effects of RhoA protein likely disrupt
membrane organization at the apical junction, whereas other
glucocorticoid-responsive signaling pathways are responsible for the
induction of tight junction sealing.
Effects of RhoA on the Glucocorticoid-regulated Organization of
F-actin at the Apical Junction and on Actin Stress Fiber
Formation--
We observed previously that glucocorticoids stimulate
perijunctional F-actin ring formation at adherens junctions of Con8 mammary tumor cells (33). Several studies have shown that F-actin is
physically associated with the adherens junction by its tethering to
After a 5-day treatment of cells cultured on filter supports in the
presence or absence of dexamethasone, the localization pattern of
F-actin was examined by confocal laser immunofluorescence microscopy.
Apical staining visualized the localization of F-actin at the adherens
and tight junctions (Apical), whereas actin stress fibers
were detected at the basolateral surface (Basal). As shown in Fig. 6 (top panels,
Basal), staining for F-actin showed that actin stress fiber
formation was relatively unaffected by dexamethasone in Con8 cells
under conditions in which the steroid induces F-actin ring formation
(Fig. 6, top panels, Apical). In contrast to the control cells, in Con8-V6 cells expressing constitutively active RhoA,
the formation of F-actin rings was less strongly induced by steroid
treatment, and actin staining could be found throughout the cytoplasm
(Fig. 6, middle panels, Apical). The actin stress fibers remained unchanged in Con8-V6 cells in the presence or absence
of dexamethasone (Fig. 6, left panels, Basal),
suggesting that the general cellular adhesion properties remained
unaffected. In Con8-DN25 cells expressing dominant negative RhoA, we
observed an induced perijunctional F-actin ring formation independent
of dexamethasone treatment (Fig. 6, bottom panels,
Apical). These results are consistent with the organized
localization of RhoA Does Not Affect the Dexamethasone-induced Formation of Mammary
Tumor Cell Monolayers--
We observed previously that dexamethasone
treatment of multilayered Con8 cells induces the cells to form a
monolayer under conditions in which tight junctions are formed (35). To
determine whether the observed effects of RhoA on apical junction
remodeling and F-actin ring formation may be a fortuitous consequence
of a disruption in cell monolayer formation, the XZ planes of
actin-stained cell samples were analyzed by confocal laser microscopy.
As also shown in Fig. 6 (Z-planes), 5 days treatment with
dexamethasone induced cell monolayer formation in the control Con8
cells, Con8-V6 cells, and Con8-DN25 cells. Thus, the disrupted
formation of tight junctions and F-actin rings observed in cells
expressing constitutively active RhoA and the precocious membrane and
cytoskeletal remodeling observed in cells expressing dominant negative
RhoA cannot be explained by any secondary effects on monolayer formation.
A key characteristic of the Con8 mammary tumor
epithelial cells used in our study is that physiologically relevant
hormonal cues stimulate the formation of tight junctions and a
polarized cell monolayer (33-35, 38). After an early transcriptional
event (38), glucocorticoids induce the organization of the apical junctional complex which results in the recruitment of tight junction proteins, adherens junction proteins, and several intracellular signaling proteins to the cell periphery at the sites of cell-cell contact (33). This junctional reorganization, which is reversed by
growth factor exposure (34-36), is followed by a distinct
Ras-dependent step that results in formation of highly
sealed tight junctions (33). In the current study, we now establish
that the steroid down-regulation of the RhoA small GTPase is required
for junctional reorganization. Ectopic expression of constitutively
active RhoA protein interferes with glucocorticoid-induced localization
of both the ZO-1 tight junction protein and the -catenin to sites of
cell-cell contact, inhibited tight junction sealing, and prevented the
complete formation of the F-actin ring structure at the apical side of
the cell monolayer. In a complementary manner, dominant negative RhoA
caused a precocious organization of the tight junction, adherens
junction, and the F-actin rings in the absence of steroid, whereas the
monolayer TER remained glucocorticoid-responsive. Taken together, our
results demonstrate that the glucocorticoid down-regulation of RhoA is a required step in the steroid signaling pathway which controls the
organization of the apical junctional complex and the actin cytoskeleton in mammary epithelial cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin,
-catenin, plakoglobin, and the
actin cytoskeleton (8, 14, 15). These events trigger a series of
molecular processes leading to the recruitment of tight junction
components to the points of cell-cell contact and formation of a tight
paracellular seal of the epithelium (13). The tight junction complex
includes several classes of transmembrane proteins, including occludin (8), the claudin protein family (16-19), intracellular peripheral membrane proteins, and potential cell signaling molecules (3, 5). The
cytoplasmic tail of occludin interacts with and tethers a complex of
related peripheral membrane proteins ZO-1 (20) and its two related
family members ZO-2 (21) and ZO-3 (22), which are members of the MAGUK
(membrane-associated guanylate kinase) protein family. The MAGUK
protein family has been proposed to recruit a variety of proteins to
the membrane at the tight junction to form a tight extracellular seal
(3, 5). Several potential cell signaling molecules, such as atypical
protein kinase C (23), the heterotrimeric G protein
subunit (24,
25), as well as the Ras target AF-6/Afadin, which binds to ZO-1 (26, 27), have been localized to the tight junction.
-catenin), as well as the Ras and phosphatidylinositol 3-kinase cell
signaling proteins to the cell periphery at the sites of cell-cell
contact (38). This junctional reorganization process is followed by a
distinct Ras- and phosphatidylinositol 3-kinase-dependent
step that results in formation of highly sealed tight junctions in a
polarized cell monolayer (38). Glucocorticoids down-regulate the level
of fascin protein (37), an actin-bundling protein that binds to the
adherens junction protein
-catenin. Fascin acts as inhibitor of
tight junction formation, and we have recently shown that transforming
growth factor-
disrupts junctional organization by preventing the
steroid down-regulation of fascin (35).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin antibodies,
monoclonal anti-
-occludin antibodies, monoclonal anti-Myc
antibodies (clone 9E10), monoclonal anti-actin antibodies, as well as
anti-actin Texas Red-conjugated rhodamine phalloidin were purchased
from Zymed Laboratories, Inc. (South San Francisco, CA). The anti-serum
and glucocorticoid-inducible kinase (Sgk) polyclonal antibodies
have been described previously (48). Fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG antibodies were supplied
by Cappel Laboratories (Malvern, PA). Monoclonal anti-RhoA antibodies
were obtained from Santa Cruz Biotechnology. The plasmids expressing
dominant active and dominant negative RhoA (pcEXV-Myc-RhoA.V14 and
pcEXV-Myc-RhoAD.N19, respectively) and empty vector (pcEXV) were a kind
gift from Dr. Marc Symons (Onyx Pharmaceuticals, Richmond, CA).
-catenin antibodies followed by Texas Red-conjugated secondary
antibodies or with Texas Red-conjugated actin-phalloidin. Fluorescent
images were obtained from a Zeiss axiograph, and confocal fluorescent
images were taken with a Leica confocal microscope and processed with
the corresponding Leica software on Microsoft Windows NT.
-occludin antibodies overnight at 4 °C. After three washes for 10 min each in
1% non-fat dry milk in washing buffer, the blots were incubated with
horseradish peroxidase-conjugated anti-mouse antibodies (Bio-Rad) for
1 h. The blots were then washed twice with 1% non-fat dry milk in
washing buffer and once with washing buffer. The blots were developed
with a PerkinElmer Life Sciences chemiluminescence reagent kit. The
level of RhoA protein observed in the Western blots was quantified with
the Scion image program (Scion Corp., Frederick, MD).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-occludin antibodies (Fig. 1A,
lower blot) show that these protein levels remained generally constant during the time course, with some fluctuations in
-occludin protein. These protein levels did not change in response
to dexamethasone treatment, thus providing a control for the
specificity of the effects of glucocorticoids on RhoA protein levels.
In untreated cells, RhoA protein levels at different time points can
vary slightly from experiment to experiment, without a statistically
significant change (p = 0.05) overall during the period
of the time course (data not shown).
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Fig. 1.
Time course of the glucocorticoid
down-regulation of RhoA protein and stimulation of the monolayer
transepithelial electrical resistance. A,
postconfluent Con8 mammary tumor cells were cultured at the indicated
times in the presence (+Dex) or absence ( Dex)
of 1 µM dexamethasone over a 4-day time course. Total
cell lysates were subjected to SDS-PAGE, and Western blots were
analyzed using monoclonal anti-RhoA antibodies. As a control, Western
blots were also probed with anti-actin and anti-
-occludin
antibodies. B, the results of a series of Western blots were
analyzed with the NIH Image program. RhoA protein levels of each time
point were calculated as the percentage of RhoA protein levels observed
in treated cells compared with untreated cells and plotted in
comparison with the percentage increase in TER observed in
dexamethasone-treated Con8 cells grown on filter inserts. Each
data point is the average in percentage of TER, or protein level,
respectively, from four or more independent experiments.
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Fig. 2.
Establishing stable transfected
Con8-derived mammary tumor cell lines expressing constitutively active
RhoA (Rho.V14) or dominant negative RhoA (RhoA.DN19). To generate
control, constitutively active and dominant negative Rho-expressing
cell lines, the neomycin-resistant plasmids pcEXV-Myc-RhoA.V14,
pcEXV-Myc-RhoA.DN19, and pcEXV empty vector were transfected into Con8
cells. After selection with the neomycin analog G418 for 2 weeks, 50 clones were selected, expanded, and tested for expression of Myc-tagged
RhoA by Western blots. The Western blots were probed with anti-Myc
epitope-tagged antibodies to determine the cell lines expressing high
levels of the RhoA proteins. The ectopic expression of either
constitutively active RhoA (RhoA.V14) or the dominant negative RhoA
(RhoA.N19) is shown for four of the cell lines; two cell clones,
Con8-V6 and Con8-DN25, were utilized for further study.
-actin.
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Fig. 3.
The Con8-derived cell lines ectopically
expressing either constitutively active or dominant negative RhoA are
glucocorticoid-responsive. Con8-V6, Con8-DN25, and control Con8
cells were cultured to confluence and either treated (+Dex)
or not ( Dex) with 1 µM dexamethasone for 5 days. Total cell lysates were subjected to SDS-PAGE. Western blots were
probed with anti-RhoA antibodies (RhoA), anti-Myc monoclonal
antibodies (Myc-RhoA), or polyclonal antibodies to Sgk
(SGK), a known primary glucocorticoid-responsive gene in
Con8 cells. As a control, Western blots were probed with anti-
-actin
antibodies.
-catenin adherens junction protein. Cells were grown to
confluence on filter supports, treated with or without
dexamethasone for 5 days, and the localization of ZO-1 and
-catenin was examined by indirect immunofluorescence microscopy
using specific primary antibodies. As expected for Con8 cells (33-35),
dexamethasone induced an overall rearrangement of the apical junction
in which the staining of both ZO-1 and
-catenin changes from a
relatively disorganized pattern with protein localized to the
cytoplasm and/or nucleus to a
honeycomb-like staining pattern (Fig. 4, top panels).
We have shown previously that this staining pattern results from the
redistribution of the tight junction and adherens junction proteins to
contact points along the cell periphery (34). In contrast,
dexamethasone treatment failed to induce completely the junctional
complex organization in Con8-V6 cells, which produce constitutively
active RhoA, showing significant cytoplasmic staining for ZO-1 and
mostly nuclear/cytoplasmic staining for
-catenin (Fig. 4,
middle panels). This result demonstrates that ectopic
expression of active RhoA, which overrides the dexamethasone down-regulation of endogenous RhoA, interferes with proper
glucocorticoid-induced organization of the apical junction, thereby
implicating the requirement to lower RhoA levels to induce tight
junction formation.
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Fig. 4.
Effects of ectopic expression of
constitutively active or dominant negative RhoA on the
glucocorticoid-induced organization of the junctional complex.
Con8-V6 cells and Con8-DN25 cells, along with control Con8 cells, were
grown on filter supports and either treated (+Dex) or not
( Dex) with 1 µM dexamethasone for 5 days.
Cells were fixed, and the localization of ZO-1 and
-catenin proteins
was detected by immunofluorescence microscopy.
-catenin and ZO-1
localized to cell junctions in the absence of dexamethasone (Fig. 4,
bottom panels), showing a distinct honeycomb-like staining pattern. These results suggest that expression of dominant negative RhoA protein causes a precocious membrane organization in the absence
of steroid. After glucocorticoid treatment, Con8-DN25 cells show a
slightly enhanced localization of ZO-1 to the cell periphery, whereas
-catenin staining remained unchanged compared with untreated cells.
Taken together, our results imply that the down-regulation of RhoA
protein is required for the regulated membrane reorganization that
leads to the localization of adherens junction and tight junction
proteins to the apical junctions.
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Fig. 5.
Effects of expressing constitutively active
RhoA or dominant negative RhoA on the dexamethasone stimulation of
TER. Con8-V6 and Con8-DN25 cells along with control Con8 cells
were grown to confluence on filter inserts and cultured in the presence
(+Dex) or absence ( Dex) of 1 µM
dexamethasone for 5 days. The monolayer TER was measured and the
ohms·cm2 calculated as described under "Experimental
Procedures." The average of the TER data for dexamethasone-treated
Con8 cells was set at 100%, and the average TER data for all other
cell lines is represented as a fraction thereof. Significance
(p < 0.005) was established by conducting a two-sample
t test analysis. Data shown represent an average of at least
three independent experiments.
-catenin via
-catenin (52, 53) and to the tight junction through
association with ZO-1 (28), ZO-2, ZO-3, occludin (54), and cingulin
(55). Furthermore, the absence of an intact actin cytoskeleton has been
associated with ZO-1 (56, 57) and occludin disorganization at the tight
junction (57). Although RhoA has been implicated in stress fiber
formation in different cell lines (44, 58-60), RhoA has also been
demonstrated to lose its regulatory control over stress fiber formation
when up-regulated as an indirect consequence of Ras transformation
(61). Given the apparent involvement of RhoA in actin cytoskeleton
reorganization, Con8-V6 and Con8-DN25 cells were utilized to determine
the potential effects of RhoA on the glucocorticoid control of F-actin
ring formation and on stress fiber formation in Con8 mammary tumor cells.
-catenin and ZO-1 in the junctional complex (shown
in Fig. 4). Thus, in mammary tumor cells, the down-regulation of RhoA
appears to be required for the steroid induced F-actin ring formation,
whereas there are no significant effects on actin stress fiber
formation.
View larger version (91K):
[in a new window]
Fig. 6.
Effects of ectopic expression of
constitutively active or dominant negative RhoA on the
glucocorticoid-regulated localization of F-actin to the apical junction
and on actin stress fiber formation. Con8-V6, Con8-DN25, and
control Con8 cells were grown to confluence on filter inserts and
either treated (+Dex) or not ( Dex) with 1 µM dexamethasone for 5 days. Cells were fixed and
incubated with Texas Red phalloidin-conjugated antibodies. Mounted
cells were analyzed by confocal microscopy. Left panels, XY
sections of the basolateral surface of the cells; middle
panels, XY sections of the apical surface of the cells;
right panels, representative XZ planes of each cell
line.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin adherens junction protein to the cell periphery. The subsequent sealing of the
tight junction, as monitored by the monolayer TER, is also disrupted.
In a complementary manner, expression of a dominant negative RhoA
protein results in the precocious organization of the apical junctional
complex, although sealing of the tight junction still required
glucocorticoids. As diagrammed in Fig. 7,
our results implicate RhoA as an inhibitor of the
glucocorticoid-regulated cascade leading to tight junction formation in
mammary epithelial tumor cells, which needs to be down-regulated prior
to or during the reorganization of the overall apical junction. Our
previous studies demonstrated that Ras, another small GTPase family
member, is required for the steroid-inducible tight junction sealing in rat mammary epithelial cells, suggesting distinct cellular functions for RhoA and Ras in regulating apical junctional complex formation and
function.
View larger version (8K):
[in a new window]
Fig. 7.
Model for the multistep
glucocorticoid-regulated cascade that controls apical junction
remodeling and tight junction sealing. We propose that the
steroid-induced down-regulation of RhoA protein is a critical step in
the glucocorticoid-induced remodeling of the junctional complex
organization that includes both the adherens junction and the tight
junction. The subsequent glucocorticoid-regulated tight junction
sealing occurs in a Ras- and phosphatidylinositol 3-kinase
(PI3K)-dependent fashion. Ectopic expression of
a constitutively active RhoA (RhoA.V14) interferes with apical junction
remodeling, resulting in a loss of tight junction sealing. In a
complementary manner, ectopic expression of dominant negative RhoA
(RhoA.N19) stimulates junctional remodeling in the absence of
steroids.
A precise role for RhoA in tight junction dynamics has not
been defined previously, in part because of apparent cell type differences in RhoA function and regulation. MDCK cells are widely used
to examine cell-cell interactions because these cells constitutively organize their apical junctional complex with sealed tight junctions (42, 62-68). However, in these cells nonphysiological conditions, such
as the removal and readdition of calcium or ATP, are generally used to
study tight junction dynamics. With an ATP depletion protocol, overexpression of a dominant negative RhoA in transiently transfected MDCK cells was shown to induce the accumulation of the ZO-1 and occludin tight junction proteins at the apical cell junctions (43).
Using calcium switch assays, several studies demonstrated that
ectopically expressed RhoA increased the monolayer TER (44, 69),
although paradoxically, in one of these studies overexpression of
constitutively active RhoA caused ZO-1 localization to be highly disorganized despite an increase in barrier function (69).
Interestingly, ectopic expression of either the constitutively active
RhoA or dominant negative RhoA in MDCK cells was shown to perturb tight junction sealing, suggesting that RhoA inhibits tight junction formation but only at an optimal cellular level (42). Furthermore, expression of active RhoA, but not the dominant negative RhoA, disrupted the tight junction strand morphology and localization of
occludin and ZO-1 tight junction proteins (42). Thus, in combination
with our hormonal studies using mammary epithelial cells, the emerging
evidence generally favors an inhibitory role for RhoA in tight junction
formation. Although it is not known how downstream effectors of RhoA
mediate their effects on the apical junctional complex, a family of
Rho-activated serine/threonine protein kinases, p160ROCK (ROCK-I) and
ROK/Rho kinase (ROCK-II), has been shown to be required for tight
junction assembly and function in polarized intestinal epithelial cells
(70) and MDCK cells (44). We are currently examining the role of
individual downstream RhoA effectors in glucocorticoid-induced
formation of tight junctions in mammary epithelial cells.
The tight junction, the adherens junction, and the
cytoskeleton form an integrated functional unit through multiprotein
complexes. For example, the cadherin proteins of the adherens junction
are structurally connected to F-actin polymers through -catenin and
-catenin (53). Also, the actin cytoskeleton interacts with the ZO-1
tight junction protein (31) or potentially indirectly through
ZO-1-binding proteins such as spectrin (71) and cortactin (72).
Furthermore, the actin cytoskeleton interacts with additional proteins
localized to the tight junction, such as ZO-2, ZO-3, occludin (54), and
cingulin (55). Therefore, one mechanism by which cellular RhoA activity
could potentially disrupt the junctional complex is by selectively
altering the cytoskeleton. Consistent with this viewpoint, we observed
that ectopic expression of constitutively active RhoA in mammary
epithelial cells prevented the complete steroid-induced reorganization
of apical F-actin structures and
-catenin localization under
conditions that ablate tight junction formation. Moreover, expression
of a dominant negative RhoA protein precociously organized F-actin
structures and
-catenin localization at the lateral apical surface
in the absence of steroid, with almost no cytoplasmic staining of actin
or
-catenin. We propose that the steroid-induced down-regulation of
RhoA or expression of dominant negative RhoA allows an apical F-actin
organization that permits
-catenin accumulation at the lateral
plasma membranes, for example by generating a cytoskeletal scaffold to
which
-catenin, and possibly ZO-1, can bind at points of cell-cell
contacts. Alternatively, the dominant negative RhoA may cause the
adherens and tight junction proteins to organize at the lateral plasma
membrane and thus provide anchorage for actin filaments.
In the mammary epithelial tumor cells used in our study, ectopic expression of the constitutively active RhoA had no effect on stress fiber formation already produced by Con8 cells. This result suggests that the hormonal manipulation of tight junction dynamics in mammary epithelial tumor cells does not alter the general cellular adhesion properties. In MDCK cells, overexpression of either the dominant negative or the constitutively active RhoA increased stress fibers (42). In another study, ectopically expressed RhoA had a biphasic effect in that low levels stimulated actin stress fibers, whereas high levels of RhoA reduced stress fiber formation and caused further disruption of the overall actin cytoskeleton (73). These observations suggest that differences in the role of RhoA in stress fiber formation may be the result of either cell type-specific differences between MDCK cells and the mammary tumor cells used in our study and/or the nonhormonal approaches used to manipulate tight junction formation in MDCK cells.
Increased RhoA signaling has been reported in several malignant
carcinomas such as breast cancer (74), testicular germ cell tumors (75,
76), and in human pancreatic cancer cells (77). Conversely,
monoterpenes, which are being tested in clinical trials as breast
cancer therapy, have been shown to inhibit the isoprenylation of RhoA
and likely RhoA functionality (78). The Con8 cells used in our study
are a tumorigenic mammary epithelial cell line (79), suggesting that
utilization of RhoA is distinct from nontumorigenic cells such as the
MDCK cell lines. We have documented that glucocorticoids induce an
overall "differentiated-like" phenotype in the Con8 mammary tumor
cells, which includes the remodeling of the apical junctional complex
and formation of tight junctions, a G1 cell cycle arrest,
the down-regulation of transforming growth factor- production, and
the stimulated formation of a polarized cell monolayer (34-36, 79,
80). Our study demonstrates that the glucocorticoid down-regulation of
RhoA is an integral event in the emergence of this cellular phenotype.
Furthermore, our results imply that small GTPases, such as RhoA and
Ras, are involved in the in vivo regulation of the tight
junctions in the mammary gland, for example during late pregnancy and
lactation when glucocorticoid levels are high (81, 82). We are
currently examining the mechanism by which glucocorticoids
down-regulate RhoA protein and are determining the potential in
vivo significance of the down-regulation of RhoA and effects on
its signaling targets.
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ACKNOWLEDGEMENTS |
---|
We thank Bridget O'Keeffe for helpful suggestions during the course of this work. We also thank Rebecca Berdeaux, James Chan, Joseph Kim, Minnie Wu, and Cindy Huynh for technical assistance.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK-42799 (G. L. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
and Cell Biology, 591 LSA, University of California at Berkeley, Berkeley, CA 94720-3200. Tel.: 510-642-8319; Fax:
510-643-6791; E-mail: glfire@uclink4.berkeley.edu.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M213121200
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ABBREVIATIONS |
---|
The abbreviations used are: ZO, zonula occludens; MDCK cells, Madin-Darby canine kidney cells; Sgk, serum and glucocorticoid-inducible protein kinase; TER, transepithelial electrical resistance.
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