From the Department of Biological Sciences and the Walther Cancer Institute, University of Notre Dame, Notre Dame, Indiana 46556-0369
Received for publication, January 29, 2003, and in revised form, February 26, 2003
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ABSTRACT |
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Epithelial cell scattering encompasses the
dissolution of intercellular junctions, cell-cell dissociation, cell
spreading, and motility. The Rac1 and ARF6 GTPases have been
shown to regulate one or more of these aforementioned processes. In
fact, activated Rac1 has been shown to promote cell-cell adhesion as
well as to enhance cell motility, leading to conflicting reports on the
effect of Rac1 activation on epithelial cell motility. In this study, we have examined the activation profiles of endogenous Rac1 and ARF6
during the sequential stages of epithelial cell scattering. Using Madin-Darby canine kidney cells treated with hepatocyte growth
factor/scatter factor or cell lines stably expressing activated v-Src,
we show that Rac1 and ARF6 exhibit distinct activation profiles during
cell scattering. We have found that an initial ARF6-dependent decrease in the levels of Rac1-GTP is
necessary to induce cell-cell dissociation. This is followed by a
steady increase in Rac1 and ARF6 activation and cell migration. In sum, this study documents the progression of ARF6 and Rac1 activities during
epithelial cell scattering.
Epithelial tissues are composed of highly organized sheets of
polarized cells that are poorly motile. Cell-cell adhesion is pivotal
for the maintenance of structural integrity and function of epithelial
tissues. Within the epithelial sheet, polarized cells are held together
by intercellular junctions such as the tight junctions and the
adherens junctions (1, 2). The adherens type junctions are principally
responsible for homotypic cell-cell adhesion and are composed of a
transmembrane protein, E-cadherin, that is linked to the actin
cytoskeleton via a family of cytoplasmic proteins, the catenins. The
disassembly of the adherens junctions has been shown to promote a loss
of cell polarity and the acquisition of a more fibroblast-like or
mesenchymal phenotype (3, 4). Such a transition, referred to as an
epithelial to mesenchymal transition, is a hallmark feature of
processes such as tumor cell invasion and wound healing (4-6). A
similar change in morphology also occurs during some normal
developmental processes, for example, the formation of the neural crest
(7). Epithelial to mesenchymal transitions are characterized by
cell scattering, a process involving the dissolution of cell junctions
followed by cell-cell dissociation and acquisition of a migratory phenotype.
There are several mechanisms for regulation of adherens junction
stability. v-Src-mediated tyrosine phosphorylation of the cadherin-catenin complex correlates with decreased cell-cell adhesion, although it has yet to be proven that phosphorylation is directly responsible for these observed effects (4, 8, 9). In addition to
stabilizing existing adherens junctions, the actin cytoskeleton can
drive the formation of new cadherin-based cell-cell contacts by
promoting the appositioning of adjacent cell membranes (10). Thus,
actin polymerization represents another means by which adherens
junction stability may be regulated. The Rho-family GTPases Rac, Rho,
and Cdc42 have also been implicated in regulation of cadherin-based
cell-cell adhesion (11). Like all GTPases of the Ras superfamily, these
proteins cycle between their active GTP- and inactive GDP-bound
forms. Activated Rac1 promotes actin accumulation and cadherin-based
adhesion at cell contact sites (12-15). Rho and Cdc42 have been shown
to have similar roles as Rac, although their effects have been less
consistent (12, 15).
Earlier work from our laboratory has shown that the
ARF61 GTPase, via its effect
on membrane traffic and the actin cytoskeleton, promotes the
disassembly of adherens junctions and membrane ruffling, respectively,
and thereby facilitates the acquisition of a motile phenotype (16). We
have shown that ARF6 is activated in response to treatment of cells
with HGF/scatter factor, whereas dominant-negative ARF6 expression
abolishes Src-induced cell scattering. These findings implicate an
important role for ARF6 during cell scattering. More recently, we have
also shown that activation of ARF6 is coupled to the down-regulation of
Rac1 activity during adherens junction disassembly (17). Thus, ARF6-GTP
serves to facilitate adherens junction disassembly and epithelial
scattering in part by decreasing the cellular pool of Rac1-GTP. On the
other hand, the activation of Rac1 has also been linked to promoting
cell migration. For example, gain of a motile phenotype in response to
treatment of cells with hepatocyte growth factor or by wounding results
in the activation of Rac1 (6, 18, 19). In addition, the expression of
ARNO, an ARF6-GEF, has been shown to facilitate cell motility in
part by activating Rac1 (20). Taken together, activation of Rac1 in
epithelia is associated with opposite outcomes: one, the promotion of
cell-cell junctions and enhancement of the epithelial phenotype, and
two, the formation of lamellipodia during epithelial cell migration. It
is apparent, therefore, that an intricate and spatial coordination of
the activity levels of the Rac1 and ARF6 GTPases must occur during
epithelial cell scattering.
In this study, we have examined the activation profiles of endogenous
Rac1 and ARF6 during the sequential stages of HGF- and Src-induced
epithelial cell scattering. We have found that during cell scattering,
an initial decrease in the levels of active Rac1 is necessary to induce
cell-cell dissociation. This transient down-regulation of Rac1 is
ARF6-dependent. Subsequently, a steady increase in Rac1 and
ARF6 activation and cell migration is observed. We present a model that
brings together disparate observations and delineates the progression
of ARF6 and Rac1 activities during epithelial cell scattering.
Cell Culture and Transfection--
The MDCKpp60v-Src
cell line was kindly provided by W. Birchmeier. Cells were grown at
41 °C on glass coverslips or tissue culture plastic as described
previously (21) and allowed to form small colonies of ~40-60
cells/colony. To induce cell scattering, MDCKpp60v-Src cells
were incubated at the permissive temperature of 35 °C for time
periods as indicated. MDCK type I cells (Clontech,
a gift of A. Zahraoui) were grown as described previously (16).
Transfection of cells with cDNA plasmids were carried out using the
LipofectAMINE transfection reagent according to the manufacturer's
instructions. HA (hemagglutinin)-tagged Rac1 and FLAG-tagged
nm23-H1( Microscopy--
MDCKpp60v-Src cells were allowed to
scatter, and stages of cell scattering were monitored by time-lapse
microscopy. All phase images were obtained using an inverted Zeiss
Axiovert TV-135 microscope equipped with a cool CCD camera (Princeton
Instruments, Trenton, NJ). Time-lapse imaging was a carried out for a
maximum of 4 h in a temperature-controlled environment
(~35 °C) with 5% CO2 maintained in an insulated
plexiglass chamber encasing the microscope. Cells were returned to the
cell culture incubator, and after an additional 4 and 12 h, cells
were visualized by phase-contrast microscopy. Image acquisition and
processing were carried out with MetaMorph imaging software
(Universal Imaging Corp.). For immunofluorescence microscopy, MDCK
cells were fixed, permeabilized, and processed for immunofluorescence
as described (16). Anti-HA rabbit polyclonal antibodies were from
Covance, anti-FLAG mouse monoclonal was from Stratagene, and rhodamine
phalloidin was from Molecular Probes. Anti-E-cadherin mouse monoclonal
antibody was a generous gift from William Gallin. Stained cells were
analyzed using a Bio-Rad confocal scanning laser system.
GTPase Activation Assays--
Cells were grown in 6-well dishes
in islands (~40% confluency). After inducing cell scattering, cells
were quickly rinsed in ice-cold phosphate-buffered saline and incubated
with 0.5 ml of lysis buffer (25 mM Tris-HCl, pH 7.4, 300 mM sucrose, 25 mM NaF, 10 mM
Na4P2O7, 2 mM
Na3VO4, 0.5% Triton X-100) plus protease inhibitors for 30 min on ice with gentle rocking. Cells were then scrapped and centrifuged for 5 min at 14,000 rpm at 4 °C. To
determine the levels of Rac1 GTP, 400 µl of the cell lysates were
incubated with PAK(CRIB)-GST beads as described (23), and the levels of active Rac1, Rac1-GTP, were detected by Western blotting using specific
Rac1 monoclonal antibodies (Transduction Laboratories). In parallel,
the level of ARF6-GTP in 400 µl of cell lysates was measured using
the ARF6-GTP pull-down assay recently described (24). Additionally, 20 µl of cell lysates were examined for the distribution of total Rac1
and ARF6 by Western blot analysis.
v-Src activation is a potent inducer of epithelial cell
scattering. Thus, to monitor the different phases of
epithelial cell scattering, we made use of an MDCK cell line stably
transfected with a temperature-sensitive v-Src mutant
(MDCKpp60v-Src) (21). At the non-permissive temperature of
41 °C, the cells assemble into compact and polarized colonies, as
shown in Fig. 1. When switched to the
permissive temperature of 35 °C, the cell colonies undergo
scattering within hours. For our investigations, cells were
seeded at low confluency and allowed to develop into small colonies
(~40-60 cells/colony) prior to incubation at 35 °C for increasing
time periods. Cell scattering was monitored by phase-contrast
microscopy. After 30 min of Src activation, colony compaction was
reduced, and cells within the colony were observed to "loosen"
cell-cell contacts. By 1 h, the cells were clearly less adherent,
and the colonies exhibited intercellular spaces. Such changes have been
reported in past studies and are thought to be caused by an initial
collapse in cell-cell adhesion (26). After 4 h of Src activation,
cells appeared more flat and spread, and a centrifugal detachment of
cells was observed. Most of the cells at the edges of the disrupted
colony formed extensive lamellipodia. After 8 h of Src activation,
cells within a colony were completely dispersed, and finally, at
16 h of Src activation, cells were migratory and showed a more
fibroblast-like phenotype.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Kpn) expression plasmids have been described previously (17,
22).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Sequential stages of scattering of
MDCKpp60v-Src cells. Cells were grown to form small
compact colonies at 41 °C. Cells were then switched to 35 °C for
varying time periods as indicated and visualized by time-lapse
phase-contrast microscopy for a maximum of 4 h. Cells were
returned to the cell culture incubator, and phase images were taken 4 or 12 h later. The image at the 8-h time point is shown at a lower
magnification to portray a larger field of a dispersed colony of cells.
Arrows indicate intercellular spaces, and
arrowheads point to lamellipodia.
We proceeded to monitor the activation profiles of ARF6 and Rac1 during cell scattering. The levels of active, GTP-bound Rac1 were measured using the PAK-GST pull-down assay. We observed that in polarized cell colonies, the levels of Rac1-GTP were high. These results are in agreement with previous studies showing that confluent monolayers of epithelial cells have high levels of active Rac1 (13, 14). Upon initial activation of Src, the levels of active Rac1 decreased as the cells started to scatter. After 1 h of Src activation, the levels of Rac1-GTP were decreased by ~50%. However, during subsequent stages of cell scattering, Rac1-GTP levels steadily increased. The total levels of Rac1 were not significantly altered during cell scattering. From the above observations, we conclude that there is a transient decrease followed by a subsequent rise in Rac1 activation during Src-induced epithelial cell scattering.
We next examined the levels of GTP-bound ARF6 during Src-induced cell
scattering. To measure the levels of ARF6-GTP, we utilized an ARF6-GTP
pull-down assay recently developed in our laboratory (24). As shown in
Fig. 2B, in confluent
epithelia, ARF6-GTP levels were minimal. However, upon Src activation,
there was an exponential increase in ARF6-GTP. In studies describing a
role for the ARF6-GEF, ARNO, in MDCK cells, it was shown that
overproduction of ARNO did not perturb cell-cell adhesion within the
colony but rather promoted lamellipodia extensions in migrating
epithelial cells via the activation of Rac1 (19). Thus, it is likely
that ARNO functions to activate ARF6 during the latter stages of cell scattering. In fact, the apical distribution of ARNO in polarized epithelia may serve to sequester the protein, which later becomes accessible for ARF6 activation as the cells become non-polarized. Activation of ARF6 in non-polarized cells could serve to translocate at
least a subpopulation of cytoplasmic Rac1 to the cell surface as has
been described previously (22, 27).
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To complement the above investigations, we used a second experimental
system in which the parental clone of MDCK cells described earlier was
treated with HGF. Observation of cells under a phase-contrast microscope revealed that cells exhibited similar morphological alterations during cell scattering except that the kinetics appeared to
be delayed. For instance, the initial stages of cell-cell dissociation and formation of intercellular spaces were more evident at 4 h after HGF treatment (data not shown). The GTPase activation profiles during HGF-induced cell scattering were examined. As shown in Fig. 2,
C and D, the unique profiles for ARF6 and Rac1
activation detected earlier for Src-induced scattering were also
observed upon treatment of cells with HGF. Moreover, the transient
decrease in Rac1 activation coincided with an increase in cellular
levels of ARF6-GTP and dissolution of cell-cell contacts. The decrease in Rac1-GTP levels was significantly inhibited when
MDCKpp60v-Src cells were transfected with the dominant-negative
ARF6(T27N) mutant (Fig. 3A),
suggesting that the decrease in Rac1-GTP levels is dependent on
ARF6.
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Our previous work has shown that expression of a constitutively
activated ARF6 mutant in epithelial cells induces a breakdown of
cell-cell contacts. These effects of ARF6 are mediated by recruitment of nm23-H1, a nucleoside diphosphate kinase, that facilitates junction
disassembly in part by sequestering the Rac1 nucleotide exchange
factor, Tiam 1, leading to decreased levels of Rac1-GTP (17).
Disassembly of cell-cell contacts and down-regulation of Rac1-GTP is
blocked by expression of an nm23-H1 mutant, nm23-H1(Kpn), which
prevents oligomerization and hence the function of wild type nm23-H1
(28). To determine whether the decrease in Rac1-GTP might be due to the
ARF6-regulated recruitment of nm23-H1, we examined the effect of
expressing nm23-H1(
Kpn) on cell scattering and Rac1 activation.
Transfection of cells with plasmid encoding nm23-H1(
Kpn) inhibited
scattering of MDCKpp60v-Src cells at permissive temperatures
(Fig. 3). As observed in Fig. 3B, the expression of
nm23-H1(
Kpn) elicited only a partial "rescue" of Rac1-GTP levels
during the scattering response. This could be because not all the cells
express nm23-H1. Alternatively, it is possible that there are
additional mechanisms that lead to a decrease in activated Rac1 levels
during the onset of cell scattering.
Taken together, the above studies demonstrate that Rac1 activity is
modulated during cell scattering and in part is regulated by ARF6
activation. The unique activation profile of Rac1 appears to be
reflected in the morphological changes and polymerized actin redistribution that occurs during cell scattering. At sites of cell-cell contacts, increased accumulation of actin and Rac1-GTP facilitates cell-cell junction formation (29). Rac1-GTP also promotes
the formation of actin-rich lamellipodia at the leading edge of
migrating cells (6, 18, 19). Indeed, staining of MDCKpp60v-Src
cells with rhodamine-phalloidin during scattering shows increased labeling for polymerized actin at the cell junctions at very early time
points (Fig. 4). However, actin staining
between cells decreases by 1 h. Other studies have also shown that
during the initial stages of the breakdown of cell-cell contacts, the
actin cytoskeleton is disrupted at the adherens junctions (10). Within
4 h after Src activation, actin filaments have been markedly
remodeled, and cells exhibit actin-rich lamellipodia at leading edge
(Fig. 4). This is likely mediated by the increase in Rac1-GTP and
ARF6-GTP levels during the later stages of cell scattering.
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To compliment our observations of the biphasic activation profile of
Rac1, we have examined the distribution of wild type Rac1 in
MDCKpp60v-Src cells during cell scattering. We find that a
significant pool of Rac1-GTP shifts from cell junctions to the
perinuclear cytoplasm in MDCKpp60v-Src cells at ~1 h after
Src induction at permissive temperatures. At later time points, when
cell-cell contacts are practically non-existent, the majority of the
Rac1 is found at lamellipodia of migrating cells (Fig.
5). Consistent with these observations, studies by Nakagawa et al. (30) showed that upon
Ca+2 chelation, the dissolution of cell junctions is
accompanied by the redistribution of Rac1 to the cytoplasm. Thus, the
spatial distribution of Rac1 in the cell may be important for temporal control of Rac1 activation during epithelial to mesenchymal
transitions.
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What is the significance of the changes in the activities of ARF6
and Rac1 during cell scattering? Significant progress has been made
toward elucidating the role of the actin cytoskeleton either to
facilitate cell-cell adhesion or to promote surface protrusions and
ruffles required for cell migration. The dynamic process of cell
scattering necessitates dynamic remodeling, from intercellular adhesion
to the migratory phenotype. The activation of the ARF6 and Rac1 GTPases
are likely critical determinants that facilitate this transition. In
organized polarized epithelial sheets, ARF6-GTP levels are minimal,
whereas Rac-GTP is high and E-cadherin cycling and turnover is minimal.
This is consistent with our earlier observations that a
dominant-negative ARF6-GDP mutant is localized almost exclusively to
cell-cell junctions in polarized epithelia, and its expression enhances
the epithelial phenotype (16). Addition of migratory stimulus induces
the activation of ARF6. Further acquisition of the migratory phenotype
is facilitated by subsequent increases in ARF6 and Rac1 activities
(Fig. 5).
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From the above investigations, we also infer that the cellular
phenotype can markedly affect the activation profile of individual GTPases. In individual or small clusters of loosely adherent epithelial cells that do not exhibit colony compaction, basal levels of Rac-GTP are lower than those observed in larger and polarized epithelial colonies that exhibit significantly higher cell-cell
contacts.2 Moreover, loosely
adherent cells will likely exhibit a steady increase in Rac1 activation
as opposed to a transient down-regulation of Rac1 that is observed in
larger colonies. These findings also support previous reports that
cadherin engagement or cell-cell adhesion can modulate the
activation profiles of the Rho family GTPases (14).
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ACKNOWLEDGEMENTS |
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We thank Dr. Jill Schweitzer for help with these studies and critical reading of the manuscript, Dr. Philippe Chavrier for helpful discussions, and Kandus Kruger-Passig for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants from the American Cancer Society (RSG number 03-023-01-CSM) and The U. S. Department of Defense (to C. D.-S.).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.
A predoctoral fellow of the Walther Cancer Institute.
§ To whom correspondence should be addressed: Dept. of Biological Sciences, University of Notre Dame, Box 369, Galvin Life Sciences Bldg., Notre Dame, IN 46556-0369. Tel.: 574-631-3735; Fax: 574-631-7413; E-mail: D'Souza-Schorey.1@nd.edu.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M300998200
2 F. Palacios and C. D'Souza-Schorey, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ARF, ADP-ribosylation factor; ARNO, ARF nucleotide-binding site opener; MDCK cells, Madin-Darby canine kidney cells; GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; PAK, p21-activated protein kinase; CRIB, Cdc42/Rac-interactive binding; HGF, hepatocyte growth factor; HA, hemagglutinin.
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