 |
INTRODUCTION |
The apical junctional complex of epithelial cells is a specialized
set of cell-cell contacts consisting of the tight junction and the
adherens junction (1). The more apically located tight junction serves
as a selective barrier to the paracellular diffusion of solutes and
thereby separates the apical and basolateral fluid compartments of the
epithelial cell monolayer (2-4). By limiting the lateral movement of
plasma membrane lipids and proteins, the tight junction also
contributes to the maintenance of cellular polarity, physically defines
the border between the apical and basolateral plasma membrane surfaces
(2-4), and is essential for the polarized functions of differentiated
epithelial cells such as secretion, absorption, and responsiveness to
extracellular signals. At the molecular level, the tight junction is
formed by the interaction between extracellular domains of the
transmembrane protein occludin from neighboring cells (5-8) and an
intracellular protein complex that includes occludin and the
cytoplasmic proteins ZO-1 (9), ZO-2 (10, 11), cingulin (12, 13), 7H6
antigen (12-15), symplekin (16), and rab13 (17). The adherens
junction, which lies immediately below the tight junction toward the
basal side of the cell (1), is formed by the
calcium-dependent, homophilic binding of E-cadherin
extracellular domains from adjacent cells (18, 19). The adherens
junction is responsible for intercellular adhesive function (20), is a
requirement for the generation of epithelial cell polarity by tight
junctions (21-24), and plays a key role in regulating cell-cell
interactions involved in tissue morphogenesis and differentiation (25).
The E-cadherin-mediated adherens junction is formed by the specific
association of a number of cytoplasmic proteins with the cytoplasmic
tail of E-cadherin (26, 27): these include
-catenin (28, 29),
-catenin (30), plakoglobin (31-33), and p120CTN
(34-36). Other cytoplasmic molecular components of the adherens junction include actin,
-actinin (37), vinculin (38), radixin (39),
tenuin (40), Src (41), and fascin (42).
The tight junction and the adherens junction structures are highly
dynamic in that their function, assembly, and/or disassembly can be
controlled by a variety of intracellular signals that ultimately influence cell-cell interactions in a physiologically appropriate manner (43-48). For example, the permeability properties of the tight
junction and/or adhesive properties of the adherens junction can be
altered by intracellular calcium, protein kinase C activity, and
certain lipid-mediated cell signaling cascades (43, 49). The Rac and
Rho members of the Ras superfamily of small GTPases appear to provide
another common regulatory connection between tight junctions and
adherens junctions because these signaling molecules can control
intercellular adhesion, permeability, and apical junction assembly
(50-53). The actin cytoskeleton, which forms the characteristic
perijunctional ring underlying the adherens junction (54) and has been
shown to associate with the tight junction (55), represents a
structural link between the tight junction and the adherens junction.
One potential molecular connection between the actin cytoskeleton and
the apical junction may be through the fascin actin-bundling protein
(56, 57) which also binds
-catenin to form a biochemically
distinct non-cadherin complex (42), although a functional role for
fascin in the hormonal control of apical junction dynamics has not been characterized.
Mammary-derived cells represent a biologically useful system to explore
hormone-activated signaling pathways that potentially influence and
mediate cell-cell interactions. We have established that
glucocorticoids can induce tight junction formation in a receptor-dependent manner in both nontumorigenic and
tumorigenic rodent mammary epithelial cells (58-62), which implicates
this lactogenic steroid (63, 64) as a key physiological signal for the
increased junctional complex formation and mammary intercellular contacts that occur during the onset of lactation (65). In the rat Con8
mammary tumor epithelial cell line, which was derived from a
7,12-dimethylbenz(a)anthracene-induced rat mammary
adenocarcinoma (66, 67), glucocorticoids induce the remodeling of the
apical junction and a polarized phenotype that results in the
localization of the ZO-1 tight junction protein from the cytoplasm to
the cell periphery at the site of cell-cell contacts. This regulated
response induces the barrier function of the tight junction (60, 61), which decreases paracellular permeability and stimulates the
transepithelial electrical resistance
(TER).1 Given the known
transcriptional mechanism of action of glucocorticoid receptors
(68-73), we propose that the regulated expression of a specific set of
glucocorticoid responsive gene products act as molecular switches
and/or structural components that functionally control the formation of
intercellular junctions to induce a epithelial-like phenotype.
Although, in general, relatively little is known about the regulated
expression and/or activity of apical junction-associated structural or
regulatory proteins by extracellular stimuli. In this study, we
demonstrate that the glucocorticoid down-regulation of fascin protein
expression is a critical event for this steroid to induce the
organization of the apical junctional complex which provides, for the
first time, a direct functional link between a steroid regulated gene
and the control of cell-cell interactions.
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EXPERIMENTAL PROCEDURES |
Cell Culture, Glucocorticoid Treatment, and Measurement of
Transepithelial Electrical Resistance--
Con8 rat mammary
epithelial cells (61) were grown on standard tissue culture plates or
Anopore membrane filters (0.2 µm, Nalgene Nunc International,
Naperville, IL) in Dulbecco's modified Eagle's medium/F-12
supplemented with 10% calf serum and penicillin/streptomycin (BioWhittaker, Walkersville, MD), and maintained at 37 °C and 5%
CO2. Treatment of Con8 cells with glucocorticoids was done by the addition of the glucocorticoid agonist, dexamethasone (Sigma), at a final concentration of 10
6 M (prepared
as a 10
3 M stock in ethanol) to normal growth
media unless otherwise indicated. The formation of tight junctions was
monitored by measuring TER on filter grown cells using an EVOM
Epithelial Voltohmmeter (World Precision Instruments, Sarasota, FL) as
described previously (60, 61). The EVOM provides an alternating square
wave current of ±20 µA through the monolayer. The TER was calculated
from the measured resistance and normalized by the area of the
monolayer. The background TER of blank Transwell filters was subtracted
from the TER of cell monolayers.
Immunofluorescence Staining and Western Blotting--
For
immunofluorescence, Con8 cells were grown on Anopore membranes and the
TER was measured before and after dexamethasone treatment. To prepare
cells for immunofluorescence, cells were fixed with 100% methanol at
20 °C for 30 min, and subsequently dried with 100% acetone at
20 °C for 5 min. Filters were incubated with a blocking buffer
(1% non-fat dry milk in 0.5% Triton X-100, 5 mM EDTA,
0.15 M NaCl, and 20 mM HEPES, pH 7) before
incubation with the primary antibodies. Rabbit anti-occludin antibodies
(8) and anti-
-catenin (gifts of Barry M. Gumbiner, Sloan Kettering), rabbit anti-fascin antibodies (42), rabbit anti-actin antibodies (Sigma), and mouse anti-c-Myc epitope antibodies (Zymed
Laboratories Inc., San Francisco, CA) were all used at a 1:1000
dilution. Secondary fluorescein isothiocyanate-conjugated anti-rabbit
and Texas Red-conjugated anti-mouse antibodies were purchased from
Molecular Probes, Inc. (Eugene, OR) and used at a 1:100 dilution.
Stained cells were mounted with SlowFadeTM Light Antifade
reagent (Molecular Probes, Inc.) and stored at 4 °C before visualization.
For Western blot analyses, cells were rinsed twice in
phosphate-buffered saline (BioWhittaker), and extracted directly in SDS-PAGE (polyacrylamide gel electrophoresis) sample buffer (50 mM Tris-HPO4, pH 6.8, 2.5 mM EDTA,
15% sucrose, 2% SDS, and 50 mM dithiothreitol) containing
protease inhibitors (5 mM phenylmethylsulfonyl fluoride, 5 µg/ml pepstatin A, 1 µg/ml TLCK, 10 µg/ml leupeptin, 20 µg/ml
aprotinin, 50 µg/ml antipain, 2 mM benzamidine, 50 µg/ml soybean trypsin inhibitor, and 2.5 mM
iodoactamide). Samples were boiled for 15 min and cooled to room
temperature before the addition of 1 M iodoacetic acid to a
final concentration of 125 mM. SDS-PAGE was performed using
the Mini-protein II cell electrophoresis unit (Bio-Rad) according to
the manufacturer's guidelines. After electrophoresis, proteins were
electrophoretically transferred to nitrocellulose membranes (Micron
Separations Inc., Westboro, MA) using the Mini Trans-Blot
electrophoretic transfer cell (Bio-Rad) according to manufacturer's
guidelines. The blots were incubated with blotting buffer (5% non-fat
dry milk, 0.15 M NaCl, 0.2% Triton X-100) before probing
with a 1:1000 dilution of primary antibodies (anti-occludin, anti-
-catenin, anti-E-cadherin, anti-
-catenin, anti-fascin, or
anti-c-Myc epitope). The mouse monoclonal anti-fascin antibody (gift of
DAKO Corp., Carpinteria, CA) was obtained as a crude supernatant of
hybridoma media and was not diluted prior to use. Horseradish
peroxidase-conjugated anti-rabbit and anti-mouse antibodies (Bio-Rad)
were used as secondary probes, and the blots were developed by an ECL
kit (Amersham Life Sciences, Inc.).
Immunoprecipitation of Junctional Complex Proteins--
For the
examination of co-immunoprecipitated proteins, each 100-mm2
plate of confluent Con8 cells was extracted in 5 ml of 1% Tween 20 buffer (1% Tween 20, 0.15 M NaCl, 5 mM EDTA,
20 mM HEPES, pH 7.4) in the presence of protease inhibitors
(5 mM phenylmethylsulfonyl fluoride, 5 µg/ml pepstatin A,
1 µg/ml TLCK, 10 µg/ml leupeptin, 20 µg/ml aprotinin, 50 µg/ml
antipain, 2 mM benzamidine, 50 µg/ml soybean trypsin
inhibitor, and 2.5 mM iodoactamide). Extraction was carried
out on ice for 30 min and all samples were precleared by centrifugation
at 12,000 × g for 15 min before immunoprecipitation. Co-immunoprecipitation was performed with Protein A-Sepharose CL-4B
(Pharmacia Biotech Inc.) in the presence of rabbit anti-
-catenin or
preimmune serum at 4 °C for 4 h. Immunoprecipitates were washed five times in the same immunoprecipitation buffer, followed by a final
wash with phosphate-buffered saline. Co-immunoprecipitated proteins
were solubilized immediately in SDS-PAGE sample buffer and prepared for
SDS-PAGE as described above for Western blotting.
Assay for Cytoskeletal-associated Actin Filaments--
The
Triton X-100 extraction method (74) was employed to determine level of
F-actin and the amount of cytoskeleton-associated fascin and
-catenin. The mammary tumor cells were grown to confluency and
treated with or without 1 µM dexamethasone for 96 h.
The cells were then trypsinized into a single-cell suspension, counted
using a hemacytometer, and then 2 million cells collected into a pellet by centrifugation. After aspirating the media, each cell pellet was
detergent extracted by suspension in 1 ml of lysis buffer (0.5% Triton
X-100, 20 mM PIPES, pH 6.8, 2 mM
MgCl2, 50 mM KCl, 5 mM EGTA, 5 mM dithiothreitol, 1 mM ATP, and 1 µg/ml each
of leupeptin, pepstatin, and aprotinin) and then immediately
centrifuged at 8,700 × g in a microcentrifuge for 3 min. The resulting pellets (containing the Triton X-100-resistant
proteins) were suspended in 100 µl of 20 mM phosphate
buffer (14.8 mM NaH2PO4 and 5.2 mM K2HPO4, pH 7.0, and 1 µg/ml
each of leupeptin, pepstatin, and aprotinin). A parallel set of cells
was centrifuged and then directly suspended in 100 µl of 20 mM phosphate buffer and represents the total cell extracts.
For the Western blots, the Triton X-100-resistant proteins and total
cell extracts were dissolved in an equal volume of 2 × SDS-PAGE
sample buffer (described above) and the proteins electrophoretically
fractionated by SDS-PAGE.
Stable Con8 Cell Lines Expressing Full-length,
NH2-terminal Portion and COOH-terminal Portion
Myc-Fascin--
The full-length mouse fascin tagged with a 6X c-Myc
epitope at its NH2 terminus was cloned into the mammalian
expression vector pcDNA3 (Invitrogen), which drives the inserted
cDNA by the CMV promoter and contains the neomycin resistance gene.
Mammalian expression vectors for the NH2-terminal fragment
of mouse fascin containing amino acids 1-313 and the COOH-terminal
fragment of mouse fascin containing amino acids 281 to 493 each tagged
with a 6X c-Myc epitope at the NH2 terminus were cloned
into the pCS2+MT mammalian expression vector. The fascin
sequences in this expression vector are driven by the simian CMV IE94
promoter and the vector backbone of pCS2 is pBluescript KS+ (75,
76). Plasmid DNA was expanded in XL-1 Escherichia coli
cells and purified with a plasmid purification kit (Qiagen, Inc.,
Chatsworth, CA). The pcDNA3-fascin, pCS2+MT
NH2-terminal fascin, and the pCS2+MT COOH-terminal fascin DNA were introduced into Con8 cells at 24 h after plating of cells at 30% confluency. One set of cells was also transfected with
the pCS2+MT vector alone. In transfections with the pCS2+MT based
vectors, the cells were co-transfected with the neomycin resistance
gene driven by the CMV promoter. Transfection of cells was performed
using LipofectAMINE reagent according to the manufacturer's protocol
(Life Technologies, Inc., Gaithersburg, MD). Potential expressors were
selected at 600 µg/ml G418 (Life Technologies, Inc.), which was
cytotoxic to >99% of parental Con8 cells. Single clones were
isolated, expanded, and Myc-fascin expression was assessed by antigen
blotting with either the anti-fascin or anti-Myc epitope antibodies.
Multiple fascin expressing clonal cell lines derived from the parental
Con8 cells were analyzed in parallel to control for clonal variation.
The mammary tumor cells expressing high levels of full-length
Myc-fascin are f/3.1, f/3.8, and f/3.11, of the
NH2-terminal portion of Myc-fascin are FN12 and FN36, and of the COOH-terminal portion of Myc-fascin are FC10 and FC12. The
f/3.4, f/3.3, and f/3.2 cells do not express Myc-fascin although the
original transfected contained the full-length Myc-fascin gene. The C1
cells were transfected with the pCS2+MT expression vector without any
fascin sequences.
Cell Adhesion Assay--
Parental Con8 and mouse
fascin-expressing and control transfected Con8 cell lines that were
treated with or without dexamethasone (10
6 M
for 4 days) were evaluated using an aggregation assay. To obtain cells
for aggregation assays, each 100-mm2 plate of confluent
cells was rinsed twice in phosphate-buffered saline, and subsequently
incubated with 0.25% trypsin (Sigma) in Dulbecco's modified Eagle's
medium/F-12 for 1 h at 37 °C in a 5% CO2
atmosphere. After an hour of trypsinization, all cells were detached
from the culture plate and subsequently collected by centrifugation at
10 × g for 15 min in 45 ml of fresh Dulbecco's modified Eagle's medium/F-12. Cells were resuspended gently by pipetting in 5 ml of fresh growth media (Dulbecco's modified Eagle's medium/F-12 containing 10% calf serum). The presence of 10% calf serum in the cell suspension inhibits the activity of residual trypsin.
Cells were either examined immediately after resuspension or allowed to
rest on the bottom of a 50-ml conical tube for 30 min with 5-min
intervals of gentle swirling. Cell aggregates were examined by light
microscopy using a × 10 objective lens and a dark field condenser setting.
 |
RESULTS |
Time Course and Dose Response of the Glucocorticoid Down-regulation
of Fascin Protein in Rat Mammary Epithelial Tumor Cells--
We have
previously established that glucocorticoids stimulate tight junction
formation in Con8 mammary epithelial tumor cells (61). Given the well
characterized effects of glucocorticoids on gene expression (68-73),
it seemed likely that this steroid induces the formation of tight
junctions by altering the expression of a select subset of proteins
that are associated with and that may regulate the overall junctional
complex. Furthermore, the formation of intercellular junctions is
intimately linked to the cytoskeleton, and we have observed that
dexamethasone causes a rearrangement of Con8 mammary tumor cells from
round-shaped cells that grow on top of each other to cuboidal shapes
characteristic of an epithelial monolayer (60, 61). Thus, potential
targets of glucocorticoid signaling may include proteins that interact with both the tight junction and the adherens junction as well as the
cytoskeleton. Recently, fascin, an actin-bundling protein (56, 57), was
found to interact with the adherens junction protein
-catenin (42),
and therefore provided a candidate regulatory protein involved in the
glucocorticoid control of junctional complex formation. To explore this
possibility, the level of fascin protein produced was compared with the
kinetics of tight junction formation in Con8 mammary tumor cells
treated with the synthetic glucocorticoid dexamethasone over a 4-day
time course. At the indicated times in dexamethasone, the generation of
TER in cell monolayers grown on Anopore membrane filters was used to
monitor tight junction formation, and a parallel set of Western blots
was probed for fascin protein or for the tight junction protein
occludin. As shown in Fig. 1a,
dexamethasone induced a significant increase in the TER that continued
to rise throughout the time course. In contrast, the TER of cells not
treated with glucocorticoids remained essentially unchanged. Western
blots showed that total cellular fascin protein began to decrease at
approximately 48 h and was markedly reduced (>80%) by 72 h
of dexamethasone treatment (Fig. 1a). During this time
period in which fascin levels are reduced, occludin protein levels
remained unchanged. The overall time frame of induced TER and fascin
protein down-regulation is consistent with a functional relationship
between these processes.

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Fig. 1.
Time course and dose response of the
glucocorticoid down-regulation of fascin protein. a,
confluent monolayers of Con8 cells grown on Anopore membrane filter
supports were treated with 1 µM dexamethasone
(Dex), and at the indicated days in culture, the formation
of tight junctions was monitored in triplicate sets of cell cultures by
measurements of the TER. Western blots of a parallel set of cell
cultures were probed for occludin and fascin protein expression.
b, Con8 cells grown on Anopore membrane filter supports were
treated with the indicated concentration of dexamethasone for 4 days
and the TER was determined in triplicate sets of cell cultures. Western
blots of electrophoretically fractionated total cell extracts from a
parallel set of cell cultures treated 4 days with 10 9
M, 10 8 M, or 10 7
M dexamethasone were probed for fascin protein
expression.
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As a complementary approach to confirm the correlation between the
glucocorticoid down-regulation of fascin with the formation of tight
junctions, the production of fascin and the induction of TER was
analyzed in mammary tumor cells treated with various doses of steroid
for 4 days. As shown in Fig. 1b, half-maximal down-regulation of fascin protein and the half-maximal stimulation of
TER was observed at approximately 10
8 M
dexamethasone, which correlates with the Kd of
glucocorticoid-receptor occupancy for these cells (67). The maximal
effect of dexamethasone on both fascin down-regulation and the
stimulation of TER was observed at 10
7 M
(Fig. 1b), with higher concentrations of dexamethasone
having no additional effects (data not shown). A similar
dose-dependent response on fascin down-regulation and
stimulation of TER was observed in mammary tumor cells treated for
shorter durations with dexamethasone. Thus, the down-regulation of the
45-kDa fascin expressed in mammary tumor cells correlated with the
formation of tight junctions in a dose- and time-dependent
manner that is consistent with a glucocorticoid
receptor-dependent process.
Glucocorticoid Withdrawal Reverses the Rearrangement of the Apical
Junction and the Down-regulation of Fascin Protein--
To test
whether glucocorticoids are required for the maintenance of the
organized apical junction and for the continued down-regulation of
fascin, we examined the effects of dexamethasone withdrawal on the
overall rearrangement of junctional proteins and on fascin protein
production. Con8 mammary tumor cells were treated with dexamethasone
for 6 days, at which time the TER is strongly induced. Subsequently,
the culture medium was replaced with fresh medium without added
glucocorticoids for 4 days and the TER returned to basal levels. The
subcellular localization of the tight junction protein, occludin, and
the adherens junction protein,
-catenin, were examined by indirect
immunofluorescence microscopy. Dexamethasone withdrawal effectively
reversed the glucocorticoid-induced rearrangement of both occludin and
-catenin (Fig. 2a,
panels ± versus +). Both occludin and
-catenin
redistributed from the junctional complex at the cell periphery back to
an unorganized pattern indistinguishable from the untreated cells
upon withdrawal of dexamethasone (Fig. 2a, panels ± versus
).

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Fig. 2.
Reversibility of glucocorticoid-induced
translocation of occludin and -catenin and
fascin down-regulation by dexamethasone withdrawal.
a, Con8 cells were treated without ( ) or with (+) 1 µM dexamethasone or were first treated with dexamethasone
and then the steroid removed by replacing the culture medium (±). The
localization of occludin or -catenin was examined by indirect
immunofluorescence microscopy. The scale bar represents 50 µm. b, Western blots of Con8 cells treated without ( ) or
with (+) 1 µM dexamethasone or of cells first treated
with dexamethasone and then the steroid removed (±) were probed for
expression of -catenin, -catenin, cingulin, or fascin.
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A parallel set of Con8 mammary tumor cells were examined for the
protein expression of fascin,
-catenin,
-catenin, and cingulin. As shown in Fig. 2b, dexamethasone withdrawal (lower
panel, lane ±) reversed the dexamethasone-mediated
down-regulation of fascin protein under conditions in which the
expression of tight junction and adherens junction proteins remained
constant. Fascin expression was almost restored to pretreatment levels
by glucocorticoid withdrawal. It is worth mentioning that the lane
for ± was slightly under loaded which is noticeable on the
-catenin blot. Therefore, it appears that the glucocorticoid
regulation of junctions assembly and disassembly correlated closely
with the expression levels of fascin protein. In addition, the
reversibility of the glucocorticoid-induced junctions formation
implicates this steroid as a maintenance factor rather than a terminal
differentiation signal.
Ectopic Expression of Full-length Fascin Prevents the
Glucocorticoid Induction of Transepithelial Electrical Resistance in
Mammary Tumor Cell Monolayers--
Conceivably, the down-regulation of
fascin is a critical functional event in the glucocorticoid signaling
pathway in that fascin may inhibit key cellular events that lead to the
formation of tight junctions. To directly investigate this possibility, we tested whether the ectopic constitutive expression of fascin would
prevent the dexamethasone induction of tight junction formation. Con8
mammary tumor cells were stably transfected with cDNA of full-length mouse fascin containing a Myc epitope tag at the
NH2 terminus (77). This fascin mammalian expression vector
contains the neomycin resistance gene (Fig.
3a) and stable clonal cell lines that express different levels of Myc-fascin were obtained by
selection using a lethal dosage of G418. Based on the open reading
frame, the Myc-fascin cDNA is expected to encode an approximate 65-kDa protein, taking into account the 10-kDa increase in molecular weight due to the Myc epitope. However, during our initial screening for expressing clones, we consistently observed that the Con8 mammary
tumor cells expressed Myc-fascin as an 80-kDa protein species. Fig.
3a shows a Western blot characterizing the ectopically expressed Myc-fascin from one of the transfected cell lines (f/3.1). Cell extracts from f/3.1 cells were electrophoretically fractionated and Western blots were probed with c-Myc monoclonal antibodies, fascin
polyclonal antibodies, or fascin monoclonal antibodies. As a control, a
Western blot of a cell extract isolated from non-transfected Con8 cells
was also incubated with c-Myc monoclonal antibodies. As shown in Fig.
3a, the c-Myc antibody recognized an 80-kDa protein that is
not present in the non-transfected Con8 cells. Similarly migrating
forms of Myc-fascin were detected in the f/3.1 cells using the
polyclonal or monoclonal fascin antibodies (Fig. 3a). Endogenous fascin is not apparent in the blots because of the short
time of exposure to the chemiluminescence reagents before development
of the x-ray film. Taken together, these results verify that the 80-kDa
protein observed in transfected Con8 cells is the ectopically
expressed, Myc-tagged mouse fascin. In this regard, transfection of
this same Myc-fascin expression vector, or a fascin expression vector
with a COOH-terminal Myc epitope, also produced an 80-kDa protein
species in a human tumor cell
line.2

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Fig. 3.
Ectopic expression of a Myc epitope-tagged
form of full-length mouse fascin in Con8 cells prevents the
glucocorticoid induction of transepithelial electrical resistance.
a, the Myc-fascin expression vector encodes the full-length
mouse fascin with an amino-terminal Myc epitope tag linked to the
pcDNA3.1 expression vector. Western blots of electrophoretically
fractionated cell extracts from nontransfected Con8 cells or a
Myc-fascin transfected Con8 subcloned denoted f/3.1 were probed with
anti-Myc primary antibodies (myc). Parallel Western blots of
the f/3.1 cell extracts were also probed with polyclonal
(poly) or monoclonal (mono) antibodies to mouse
fascin. b, individual subclones of transfected Con8 mammary
tumor cells were grown on Anopore membrane filter supports and treated
with or without 1 µM dexamethasone for 4 days. The f/3.1,
f/3.8, and f/3.11 subclones produce varying levels of constitutively
expressed Myc-fascin, whereas, the f/3.4, f/3.3, and f/3.2 subclones do
not produce Myc-fascin and represent transfection controls. The
monolayer TER was monitored in each cell culture and Western blots of
parallel sets of cells were probed using Myc epitope or SGK-specific
primary antibodies.
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Several individual single cell-derived subclones of transfected Con8
cells were screened for expression of Myc-fascin and for the ability of
glucocorticoids to induce tight junction formation as assessed by the
development of TER. In the three clonal transfected cell lines that
express high levels of Myc-fascin (f/3.1, f/3.8, and f/3.11),
dexamethasone failed to stimulate the monolayer TER under conditions in
which the level of ectopically expressed fascin remained constant (Fig.
3b, upper panel versus middle panel). In contrast, in three
recovered transfected cell lines that express little or no Myc-fascin
(f/3.4, f/3.3, and f/3.2), the TER was glucocorticoid inducible
indicating the proper formation of tight junctions. Overall, the level
of Myc-fascin expression correlated with the degree of inhibition on
glucocorticoid-induced TER. To confirm that the transfected cell clones
remained generally glucocorticoid responsive, the dexamethasone
stimulated expression of the endogenous serum-glucocorticoid inducible
protein kinase (SGK) was examined in the recovered transfected cell
lines. We have previously established that SGK is under direct
glucocorticoid receptor transcriptional control due to a glucocorticoid
response element in its promoter (78, 79). As shown in Fig.
3b (lower panel), in each of the recovered clonal
cell lines, SGK expression was induced by glucocorticoids. Therefore,
the inability of glucocorticoids to induce tight junction formation in
the high Myc-fascin expressors was not due to a general defect in
glucocorticoid receptor signaling.
For the remainder of the experiments, the f/3.1 clone was utilized
because this cell line produced the highest constitutive level of
Myc-fascin. The f/3.2 clone expressed an insignificant level of
Myc-fascin and therefore was employed as one of the transfection control cells for comparison to the f/3.1 clone. As discussed below,
the C1 cell clone represents the other transfection control cell line
used in certain experiments. As shown in Fig.
4, ectopic expression of Myc-fascin in
the f/3.1 clone inhibited the dexamethasone-stimulated TER over a 4-day
time course, whereas the TER was rapidly stimulated in f/3.2 cells in a
manner similar to that of parental Con8 mammary tumor cells (see Fig.
1a). Indirect immunofluorescence microscopy using Myc
antibodies showed the localization of Myc-fascin throughout the cells
in both 4-day dexamethasone-treated and untreated f/3.1 cells (Fig. 4).
In contrast, Myc-fascin was not detected by immunofluorescence staining
in either untreated (Fig. 4) or dexamethasone-treated (data not shown)
f/3.2 cells. Taken together, these results demonstrated that ectopic
expression of the full-length fascin gene inhibited the
glucocorticoid-stimulated formation of tight junctions and further
implicates the down-regulation of fascin as an essential intermediate
step in the glucocorticoid signaling pathway that controls the
junctional complex in mammary tumor cells.

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Fig. 4.
Ectopic expression of Myc-fascin inhibits the
dexamethasone induction of tight junction formation.
Top panel, the f/3.1 and f/3.2 subclones of
transfected Con8 mammary tumor cells were grown on Anopore membrane
filter supports, treated with or without 1 µM
dexamethasone (Dex), and the monolayer TER was measured at
the indicated days in culture. Lower panel, the localization
of Myc epitope-fascin protein was examined in f/3.1 and f/3.2 cells
treated with or without 1 µM dexamethasone for 4 days by
indirect immunofluorescence microscopy using Myc epitope antibodies.
The scale bar represents 25 µm.
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Ectopic Expression of Amino-terminal or Carboxyl-terminal Fragments
of Fascin Fail to Prevent the Glucocorticoid Induction of
Transepithelial Electrical Resistance--
Previous studies have shown
that the COOH-terminal 216 amino acids of fascin (residues 277 to 493)
can bind in vitro to actin (56, 57, 77), whereas, analysis
by the yeast two-hybrid assay revealed that the fascin coding region
corresponding to amino acids 324-453 functionally interact with
-catenin (42). Although the precise structural boundaries involved
in each interaction have not been defined, the fascin molecule can be
approximately dissected into an NH2-terminal fragment that
does not bind either actin or
-catenin and a COOH-terminal fragment
that binds to both actin and
-catenin. Therefore, Con8 mammary tumor
cells were transfected with expression vectors that encode either the amino-terminal 313 amino acids (FN construct) or the carboxyl-terminal 213 amino acids (FC construct) of the fascin coding region that contains both the actin and the
-catenin-binding sites. These constructs overlap by 32 amino acids between residues 281 and 313 and
each contain six copies of the Myc epitope tag at the amino terminus
(Fig. 5a). The Con8 cells were
stably transfected with each construct as well as with an empty pSC2+MT
expression vector and individual cell colonies isolated from each
population of G418-resistant cells. Two single cell-derived subclones
of cells transfected with either the NH2-terminal fascin
fragment (FN12 and FN36 cells) or the COOH-terminal fascin fragment
(FC10 and FC12 cells) as well as one of the vector-transfected cell lines (C1 cells) were examined for expression of the Myc-tagged fascin
domains by Western blots using the Myc antibodies and for the
glucocorticoid stimulation of TER. As shown in Fig. 5a, the transfected FC10 and FC12 cell lines produced a 34-kDa Myc-tagged COOH-terminal fascin protein and the transfected FN12 and FN36 cell
lines produced a 45-kDa Myc-tagged NH2-terminal fascin
protein, compared with the 80-kDa full-length Myc-fascin expressed in
the f/3.1 cells. The ectopically expressed COOH-terminal and
NH2-terminal fascin proteins correspond to the predicted
sizes based on the fascin amino acid sequence in each construct and the
11-kDa size of the Myc epitope tag. As expected, the vector-transfected
control cells, C1, do not express any Myc epitope-tagged fascin
proteins.

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Fig. 5.
Stable ectopic expression of amino-terminal
and carboxyl-terminal Myc-fascin fragments and effects on the
transepithelial electrical resistance. a, the FN
expression construct encodes the NH2-terminal 313 amino
acids of fascin with a 6X Myc-epitote tag on the amino terminus and
driven by the CMV promoter. The FC expression construct encodes the
COOH-terminal 213 amino acids of fascin with a 6X Myc epitope tag on
the amino terminus and driven by the CMV promoter. Con8 mammary tumor
cells were co-transfected with a CMV driven neomycin resistance gene
together with the FN construct, FC construct, or a pCS2+MT empty vector
and individual single cell-derived colonies were collected. FC10 and
FC12 cells express COOH-terminal Myc-fascin, FN12, and FN36 express
NH2-terminal Myc-fascin, f/3.1 cells produce full-length
Myc-fascin (see Fig. 3), and the C1 cells are a vector-transfected
control cell line. Western blots of electrophoretically fractionated
cell extracts from each of these transfected cells were probed with
anti-Myc primary antibodies. b, individual subclones of
transfected Con8 cells were grown on Anopore membrane filter supports
and treated with or without 1 µM dexamethasone
(DEX) for 4 days and the monolayer TER was monitored in each
cell culture as described under "Experimental Procedures."
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The ability of glucocorticoids to stimulate the TER was monitored in
each of these transfected mammary tumor cells after 96 h treatment
with or without 1 µM dexamethasone. As shown in Fig. 5b, in contrast to inhibitory effects of full-length
Myc-fascin (Fig. 3), the ectopic expression of either the COOH-terminal
or NH2-terminal fragments of fascin, in two different
transfected cell lines, failed to disrupt the dexamethasone stimulation
of TER (Fig. 5b). The overall range of the
glucocorticoid-induced TERs in the transfected cells approximated that
observed in the vector control C1 cells (Fig. 5b) and other
stable transfected control cell lines (Fig. 3). Thus, the ability of
constitutively expressed fascin to inhibit the glucocorticoid
regulation of tight junction function does not simply require the
portion of the protein containing both the actin and
-catenin-binding sites.
Ectopic Fascin Expression Prevents the Glucocorticoid-induced
Rearrangement of
-Catenin and Occludin to the Cell Periphery and
Disrupts the Regulated Reorganization of the Actin
Cytoskeleton--
To determine whether the ectopic expression of
fascin disrupts the formation of both tight junctions and adherens
junctions, the glucocorticoid-regulated rearrangement of the adherens
junction protein
-catenin and the tight junction protein occludin
was examined in the parental and transfected mammary tumor cell clones. Myc-fascin expressing f/3.1 cells, control f/3.2 cells, and
untransfected Con8 cells were treated with or without dexamethasone for
a 72-h time course and the localization of the apical junction proteins was analyzed by indirect immunofluorescence microscopy. Glucocorticoids induce
-catenin to redistribute from a disorganized staining pattern
mostly away from the cell periphery to a distinct "cobblestone" junctional-like pattern in parental mammary tumor cells (Fig. 6, left column) and in the
transfection control f/3.2 cell clone (Fig. 6, right
column). In contrast, constitutive expression of Myc-fascin in the
f/3.1 cell clone prevented this reorganization of
-catenin to the
cell periphery (Fig. 6, middle column). Similar to the
disruptive effects on adherens junction proteins, ectopic expression of
Myc-fascin also prevented the localization of tight junction proteins.
As shown in Fig. 7 (panels a
and a'), the dexamethasone stimulated rearrangement of
occludin to the cell periphery observed in f/3.2 cells was inhibited in
f/3.1 cells which produce high levels of Myc-fascin (Fig. 7,
panels b and b'). Taken together, these results
demonstrated that ectopic expression of fascin interfered with the
ability of glucocorticoids to induce a global rearrangement of the
tight and adherens junction proteins, and hence the formation of the
junctional complex.

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Fig. 6.
Ectopic expression of fascin prevents the
glucocorticoid-induced rearrangement of
-catenin to the junctional complex.
Transfected f/3.1 high Myc-fascin expressing cells (b-b''')
or f/3.2 transfection control cells (c-c''') as well as the
parental Con8 mammary tumor cells (a-a''') were treated with
1 µM dexamethasone for the indicated times and the
localization of -catenin was examined by indirect immunofluorescence
microscopy. The scale bar represents 50 µm.
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Fig. 7.
Ectopic expression of fascin prevents the
glucocorticoid-induced rearrangement of occludin and actin to the
junctional complex. Upper panels, transfected f/3.1
high Myc-fascin expressing (b, b') and f/3.2 transfection
control (a, a') mammary tumor cells were treated with or
without 1 µM dexamethasone for 3 days. The localization
of occludin was examined by indirect immunofluorescence microscopy.
Lower panels, transfected f/3.1 high Myc-fascin expressing
(d, d') or f/3.2 transfection control (e, e')
mammary tumor cells as well as the parental Con8 cells (c,
c') were treated with or without 1 µM dexamethasone
for 3 days and actin localization examined by indirect
immunofluorescence microscopy. The scale bar represents 25 µm.
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The effect of ectopic fascin expression on the organization of the
actin cytoskeleton was examined by indirect immunofluorescence staining
for actin. As shown in Fig. 7 (panels c versus d),
constitutive expression of Myc-fascin did not alter the actin
cytoskeleton staining pattern in cells that were not treated with
glucocorticoids. However, in f/3.1 cells (Fig. 7, panel d'),
the expression of Myc-fascin prevented the dexamethasone-induced
rearrangement of the actin cytoskeleton that was observed in both
parental and f/3.2 cells (Fig. 7, panels c' and
e'). A similar result was obtained with phalloidin staining
which confirms that the observed differences are due to polymerized
actin (data not shown). Thus, ectopic expression of fascin did not
change the basal actin cytoskeleton organization, but rather it
disrupted the glucocorticoid-induced signal that controls the membrane
rearrangement that leads to junctional complex formation.
Because fascin binds and bundles actin filaments as well as binds to
-catenin in a non-E-cadherin complex (56, 57, 77), the level of
cytoskeletal-associated F-actin, fascin, and
-catenin were
biochemically examined in the Myc-fascin transfected f/3.1 cells
compared with the C1 vector-transfected control cells. Cells were
treated with or without dexamethasone for 4 days and level of total
Myc-fascin, actin, or
-catenin in the whole cell extracts were
compared with the level of each protein detected in a Triton X-100-resistant cell pellet, which represents the cytoskeletal associated actin filaments (74). As shown in Fig.
8, most of the actin expressed in the
presence or absence of glucocorticoids can be detected in the Triton
X-100-resistant cell pellet and the ectopic expression of Myc-fascin
did not alter the actin content of this fraction. Also, the same amount
of
-catenin was detected in the Triton X-100-resistant fraction
regardless of the level of Myc-fascin or the steroid treatment (Fig.
8). Approximately 50% of the expressed Myc-fascin fractionated with
the detergent-resistant cell pellets which likely results from its
binding to actin. These results suggest that the constitutive
expression of fascin did not disrupt the apical junctions or the
glucocorticoids regulated organization of the actin cytoskeleton by an
alteration in the cytoskeletal associated actin filaments or the
interaction of
-catenin with the cytoskeleton.

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Fig. 8.
Characterization of the
cytoskeletal-associated actin filaments and association of
-catenin with the cytoskeleton in Myc-fascin
expressing and control transfected cells. The f/3.1 cells
expressing Myc-fascin and the vector control C1 mammary tumor cells
were grown to confluency and treated with or without 1 µM
dexamethasone (DEX) for 96 h. After trypsinization into
single-cell suspensions, 2 million cells per condition were collected
by centrifugation, extracted with a Triton X-100 containing buffer, and
the detergent-resistant cell pellet (Triton X-100 resistant)
fractionated by SDS-polyacrylamide gel electrophoresis. A second 2 million cell pellet collected from each condition was
electrophoretically fractionated without detergent extraction (total
cell extracts). The Western blots were probed for Myc-fascin (using Myc
antibodies), actin, and -catenin using the appropriate primary
antibodies.
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The Levels and Complex Formation of Junctional Proteins Are Not
Altered by the Ectopic Expression of Fascin--
The expression levels
and complex formation of junctional proteins were examined in the
transfected mammary tumor cells. Cells that express high (f/3.1) or
insignificant (f/3.2) levels of ectopic Myc-fascin were treated with
dexamethasone over a 7-day time course and the total cellular proteins
were analyzed by Western blotting. As shown in Fig.
9a (top panel,
anti-Myc blot), Myc-fascin levels in f/3.1 cells remained constant at a
high level throughout the 7-day dexamethasone treatment, while f/3.2
cells expressed virtually no Myc-fascin. During the same 7-day time
course, the expression levels of occludin, E-cadherin,
-catenin, and
actin remained unchanged in both f/3.1 and f/3.2 cell clones. Under
these conditions, the cells maintained their glucocorticoid
responsiveness as indicated by the strong induction of SGK protein
expression (Fig. 9a, bottom panel). The formation of
-catenin protein complexes with E-cadherin,
-catenin, and fascin
protein was examined in the transfected cell clones by Western blot
analysis of coimmunoprecipitated proteins. Cell extracts of
dexamethasone-treated and untreated cells were first immunoprecipitated
with
-catenin antibodies in 1% Tween 20 to preserve the
protein-protein interactions of
-catenin. Western blot analysis of
the electrophoretically fractionated immunoprecipitates revealed that
ectopic expression of Myc-fascin did not alter the ability of
-catenin to complex with E-cadherin or
-catenin (Fig. 9b,
upper three panels). Similar amounts of E-cadherin and
-catenin
were observed to co-immunoprecipitate with
-catenin in both
dexamethasone-treated and untreated f/3.1 and f/3.2 cells. Furthermore,
anti-Myc blots showed that the Myc-fascin protein co-immunoprecipitated
with
-catenin from f/3.1 cells, but not from f/3.2 cells (Fig.
9b, bottom panel). These results demonstrate that the
constitutive expression of Myc-fascin does not alter the expression
levels or complex formation of the adherens junction proteins.

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Fig. 9.
Ectopic expression of fascin does not alter
the expression levels and complex formation of junctional
proteins. a, transfected f/3.1 Myc-fascin expressing
and f/3.2 transfection control mammary tumor cells were treated with 1 µM dexamethasone for 0, 3, 5, and 7 days. Parallel sets
of Western blots of total cell extracts were probed for the production
of the Myc epitope, occludin, E-cadherin, -catenin, actin, and SGK.
b, the transfected f/3.1 and f/3.2 mammary tumor cells were
treated with or without 1 µM dexamethasone for 4 days and
-catenin immunoprecipitates isolated from the corresponding cell
extracts. The Western blots of electrophoretically fractionated
immunoprecipitated protein were probed for E-cadherin, -catenin,
-catenin, and the Myc epitope.
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The Glucocorticoid Induction of Intercellular Adhesion Is Inhibited
by the Ectopic Expression of Fascin--
To test whether the ectopic
expression of Myc-fascin alters intercellular adhesion through the
adherens junction, a cell aggregation assay was performed on parental
and transfected mammary tumor cells. Dexamethasone-treated (1 µM for 4 days) or untreated cells were collected by
trypsinization in the presence of calcium, which preserves E-cadherin
function, and subjected to sheer force by gently pipetting through a
round-tip pipette until only small cell aggregates remained. As shown
in Fig. 10, in the absence of dexamethasone, each of the three tested cell lines formed small aggregates of 50 cells or less. The cell aggregates were loosely attached to each other, the cell borders were rounded and intercellular space was prominently seen as bright lines under the dark field microscope setting. In dexamethasone-treated cultures, parental cells
and the f/3.2 transfection control cells formed large compact aggregates (200-1000 cells) in which the cell borders were not readily
observed even at the periphery of the cell mass. There were no
observable differences in cell aggregate size and characteristics between the parental and f/3.2 cells (Fig. 10, panels a' and
c'). In addition, when the parental and f/3.2 cells were
allowed to further aggregate for 15 min after the initial cell adhesion
assay, cell aggregates consisting of thousands of cells were formed
(data not shown). In contrast, glucocorticoids failed to stimulate
intercellular adhesion in f/3.1 cells, which constitutively produce
Myc-fascin (Fig. 10, panel b'). Dexamethasone-treated f/3.1
cells remained as small, loosely attached aggregates that were
indistinguishable from cell aggregrates of f/3.1 cells not treated with
steroid and thereby demonstrates that the ectopic expression of
Myc-fascin disrupted the glucocorticoid activation of cell-cell
adhesion.

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Fig. 10.
Ectopic expression of fascin prevents the
glucocorticoid activation of cell-cell adhesion. Transfected f/3.1
high Myc-fascin expressing (b, b') or f/3.2 transfection
control (c, c') mammary tumor cells as well as the parental
Con8 cells (a, a') were treated with or without 1 µM dexamethasone for 4 days and intercellular adhesion
assessed as described in the text. Representative views of the cell
adhesion assay are shown and the scale bar represents 100 µm.
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DISCUSSION |
The regulation of cell-cell interactions and junctional complex
dynamics is essential for epithelia to reversibly adapt to proliferative signals and to respond to physiological controls during
tissue growth and differentiation. In this study, we have discovered
that glucocorticoids, a key hormonal regulator of mammary gland
differentiation, down-regulated the production of fascin protein, an
actin-bundling protein that interacts with
-catenin, concomitant
with the induction of tight junctions formation and cell-cell adhesion
in Con8 rat mammary epithelial tumor cells. The constitutive expression
of fascin disrupted the ability of glucocorticoids to trigger the
formation of the apical junctional complex and induce intercellular
adhesion. These results implicate the down-regulation of fascin as a
key intermediate step in the glucocorticoid signaling pathway that
controls mammary cell-cell interactions and provides, for the first
time, a direct functional link between a steroid-regulated gene and the
control of the apical junctions.
We propose that the selective regulation of fascin production in
mammary tumor cells is necessary for the coordinate regulation of tight
and adherens junctions formation by glucocorticoids. It is tempting to
speculate that fascin acts as an inhibitor of junctional complex
formation, and thus the glucocorticoid down-regulation of fascin
eliminates an inhibitory signal. The glucocorticoid-mediated decrease
in total cellular fascin protein levels temporally and dose-dependently correlated with the generation of
functional tight junctions as indicated by the development of
transepithelial electrical resistance, as well as with the localization
of tight junction protein occludin and the adherens junction protein
-catenin to the cell periphery. Under these conditions,
glucocorticoids did not significantly alter the total cellular protein
levels of tight junction and adherens junction proteins such as
occludin, cingulin, ZO-1 (61),
-catenin, and E-cadherin. Thus,
glucocorticoids appear to induce the assembly of both the tight and
adherens junctions from pre-existing junctional components. The role of
fascin down-regulation in the glucocorticoid-regulated formation of
epithelial junctions was directly tested by the ectopic expression of
full-length mouse fascin in Con8 rat mammary tumor cells.
Characterization of stable clonal cell lines that produce various
levels of ectopically expressed full-length Myc-fascin showed a direct
correlation between the expression level and the degree of inhibition
of glucocorticoid-induced transepithelial electrical resistance. These
results show that the inhibition of junctional complex formation is
specific to fascin expression and is not due to random clonal
variation. The constitutive production of exogenous fascin was
sufficient to prevent the glucocorticoid-induced development of
transepithelial electrical resistance and the reorganization of
occludin and
-catenin to the tight and adherens junctions,
respectively. In transfected cells that produce a high level of ectopic
fascin, expression of junctional proteins and formation of the
E-cadherin·
-catenin·
-catenin complex was unaltered.
Therefore, the ectopic expression of fascin appears to specifically
disrupt the overall assembly process of the junctional complex that is
usually triggered by glucocorticoids and suggests that fascin acts as a
negative regulator of the glucocorticoid-mediated formation of
epithelial junctions.
The glucocorticoid induction of junctions assembly is accompanied by an
activation of cell-cell adhesion that resulted in the formation of
compact cell aggregates. In contrast, the Myc-fascin expressing cells
remained loosely packed in the presence of dexamethasone. Thus, the
constitutive expression of fascin protein not only blocks the
glucocorticoid-induced formation of junctional complexes, but also
interferes with the glucocorticoid activation of cell-cell adhesion.
Fascin might regulate intercellular adhesion directly by modulating the
function of E-cadherin or indirectly via its effect on actin which has
been implicated to contribute to the adhesive strength of E-cadherin
(80, 81). Fascin, which has been shown to bundle actin filaments (56,
82, 83), could potentially influence cell-cell adhesion and the
assembly of intercellular junctions by modulating the actin
cytoskeleton, a key player in the generation of epithelial cellular
architecture and polarity (24). However, this possibility seems
unlikely because the ectopic expression of fascin did not disrupt the
cytoskeletal-associated actin filaments or the interaction of
-catenin with the cytoskeleton. Although the immediate mechanism of
action of fascin is not clear, the discovery of a role for fascin in
the regulation of cell-cell adhesion and intercellular junctions
formation provides both structural and functional links between the
actin cytoskeleton and the control of intercellular junctions.
Different molecular weight forms of fascin have been reported in a
variety of rodent and human cells, although to date, most of the
characterized cell types express fascin with molecular masses in the
range of 54-58 kDa (56, 82, 84). We have observed that the rat Con8
mammary tumor epithelial cell line expressed a fascin-like protein at
an apparent molecular mass of 45 kDa, which we have shown to be
antigenically related to mouse fascin by competition binding to
anti-mouse fascin antibodies. The polyclonal anti-fascin antibody used
in our experiments was raised against a thioredoxin fusion protein that
contains full-length mouse fascin (42, 77) and the primary antibody
binding to the 45-kDa fascin protein produced in the mammary tumor
cells was completely competed off by the addition of 100 µg of the
fusion protein (data not shown). Conceivably, the processing of fascin
protein into a smaller form may be important in the regulation of
cell-cell adhesion and junctional complex formation. In this regard, it
has been shown that fascin can be proteolytically cleaved to a 30-kDa
form that binds actin filaments in vitro (56). We have also
observed that ectopic expression of the full-length mouse fascin
(tagged with the Myc epitope) yielded a form of mouse fascin that
electrophoretically migrated as a 80-kDa protein. Although an unusually
high molecular weight, this 80-kDa Myc-fascin species is recognized by
both polyclonal and monoclonal anti-fascin antibodies. Moreover, our
preliminary evidence suggests that expression of this full-length
Myc-fascin cDNA, as well as a fascin cDNA with the Myc epitope
tag on the COOH terminus, in human epithelial tumor cells also
generates a similar 80-kDa species.2 The mechanism by which
this high molecular weight form of Myc-fascin is produced is unknown,
although stable protein-protein interactions may potentially account
for the unexpected size. A dimeric form of the tryptic fragments of
purified recombinant fascin has been shown to migrate as a stable
complex in SDS-PAGE, suggesting that fascin indeed forms stable dimers
under denaturing conditions (83).
It is tempting to consider that the interaction of fascin with
components of the apical junction, such as
-catenin, represents a
potential regulatory mechanism by which the expression of high levels
of exogenous fascin might inhibit glucocorticoid-induced cell-cell
adhesion and intercellular junctions formation. Several studies have
shown that the regulation of adherens junction assembly correlated with
a reduced binding of
-catenin to E-cadherin (44-46). However, our
results showed that under conditions in which the ectopic expression of
full-length Myc-fascin disrupted the glucocorticoid induction of
cell-cell adhesion and intercellular junctions formation, the same
level of
-catenin co-precipitated with the E-cadherin immune
complex. A small fraction of Myc-fascin coprecipitates with E-cadherin,
likely through its interaction with
-catenin. It is not clear
whether Myc-fascin and E-cadherin bind to
-catenin in the same
complex or in mutually exclusive complexes, although it has been shown
that fascin competes with E-cadherin for
-catenin binding in an
in vitro assay (42). The ectopic expression of the
carboxyl-terminal 213 amino acids of fascin, which includes the actin
and
-catenin-binding sites, failed to disrupt the glucocorticoid induction of tight junction formation, which suggests that the ability
of fascin to disrupt the glucocorticoid-induced cell-cell interactions
involves potentially complicated structure/function relationships
within the full-length fascin protein. Conceivably, the inhibition of
cell-cell adhesion by exogenous fascin could result from an effect of
fascin on the E-cadherin·
-catenin protein complex, such as to
induce conformational changes that attenuate the adhesive function of
E-cadherin. Alternatively, fascin might indirectly regulate the
function of E-cadherin via the modulation of the various functions of
-catenin or of the cortical actin cytoskeleton. Since
-catenin is
known to interact with the LEF-1 transcription factor (85) and the
adenomatous polyposis coli protein (86), the binding of exogenous
fascin to endogenous
-catenin, and perhaps other cellular
components, may potentially affect the normal activities of
-catenin
in the regulation of its interacting proteins, which might in turn
affect intercellular adhesion and junctions formation.
The coordinate induction of tight junction formation and cell-cell
adhesion through fascin down-regulation might reflect an important
biological switch in mammary cell differentiation. Our results, in
which the ectopic expression of fascin disrupted the glucocorticoid
stimulation of tight junctions and adherens junctions, implicate fascin
as a negative regulator of cell-cell interactions. Thus, the
glucocorticoid down-regulation of endogenous fascin expression is
necessary for the induced formation of the apical junctional complex in
rodent mammary tumor cells. Glucocorticoid receptors can stimulate or
inhibit gene transcription by their selective DNA binding to
glucocorticoid response elements (68-70), and by their ability to
directly bind to and attenuate the function of certain transcription
factors (68, 71-73). If the fascin gene is a direct target of
glucocorticoid signaling, it is likely that the glucocorticoid receptor
inhibits fascin gene transcription by interfering with specific
transcription factors that act on the fascin gene promoter or by
inducing the expression of transcriptional inhibitors that target the
fascin gene. Unraveling the signaling pathway by which glucocorticoid
down-regulates fascin protein expression would be an important step
toward the understanding of glucocorticoid regulation of epithelial
junctions formation, and perhaps the role of intercellular junctions in
the regulation of cell growth and differentiation.