1 Laboratoire de Régulations Cellulaires et Oncogénése
UMR146, Institut Curie Section de Recherche, Centre Universitaire Paris-Sud,
91405 Orsay, France
2 Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX,
UK
3 Laboratoire de Compartimentation et Dynamique Cellulaires UMR144, Institut
Curie Section de Recherche, 26 rue d'Ulm, 75248 Paris, France
* Author for correspondence (e-mail: Brigitte.Boyer{at}curie.u-psud.fr )
Accepted 12 April 2002
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Summary |
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Key words: EMT, Signal transduction, Cell dissociation, Cell motility
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Introduction |
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The rat bladder carcinoma line NBT-II undergoes EMT in response to growth
factor stimulation (EGF, FGF-1 and TGF)
(Vallés et al., 1990
;
Gavrilovic et al., 1990
).
Cell-cell dissociation is preceded by the internalization of desmosomal
components and their progressive loss from cell-cell contact areas. It has
been previously demonstrated that the Ras proto-oncogene is required during
the EGF-induced NBT-II desmosome breakdown
(Boyer et al., 1997
). Ras is
known to participate in several signalling pathways leading to activation of
the phosphoinositide-3-kinase (PI3K)
(Rodriguez-Viciana et al.,
1994
; Rodriguez-Viciana et
al., 1997
; Kotani et al.,
1994
), RalGDS and Raf/MAPK phosphorylation cascades
(Treisman, 1996
). In addition,
Ras has been shown to activate the small GTP-binding protein Rac
(Van Aelst and D'Souza-Schorey,
1997
).
PI3K is required during the hepatocyte growth factor/scatter factor
(HGF/SF)-induced scattering of epithelial MDCK cells
(Royal and Park, 1995). More
recent studies have shown the involvement of the MAPK pathway during the
HGF/SF- and PI3K-induced scattering of MDCK cells
(Potempa and Ridley, 1998
;
Khwaja et al., 1998
). For
example, keratinocyte cell dispersion depends upon, at least, the sustained
activation of MAPK (McCawley et al.,
1999
). Members of the Rho family of small GTPases also exhibit
specific roles during epithelial cell scattering. The small GTPase Rac is
required downstream of HGF/SF and Ras, as a dominant-negative Rac1 inhibits
SF- or Ras-induced MDCK cell scattering
(Potempa and Ridley, 1998
;
Ridley et al., 1995
), although
activated Rac1 alone does not induce this response
(Ridley et al., 1995
;
Hordijk et al., 1997
). By
contrast, activation of RhoA inhibits MDCK cell spreading and scattering
(Ridley et al., 1995
). Ras and
Rac seem to be involved in cell dissociation by reducing the stability of
adherens junctions in primary BRK cells
(Quinlan, 1999
). In apparent
contradiction, other studies have demonstrated a role for Rac in enhancing the
formation of adherens junctions in MDCK cells
(Ridley et al., 1995
;
Takaishi et al., 1997
) and in
keratinocytes (Braga et al.,
1997
). Although the role of signalling molecules downstream of Ras
in destabilizing adherens junctions has been extensively investigated, the Ras
effectors responsible for desmosome breakdown have still to be
characterized.
The Ras pathway has also been implicated in the cytoskeletal rearrangements
and cellular motility that accompany cell dispersion. Among the Ras effectors,
PI3K mediates the Ras control of the cytoskeleton
(Rodriguez-Viciana et al.,
1997) and is responsible for actin rearrangement induced by
activated FAK and by Rac1 and Cdc42 (Keely
et al., 1997
). Furthermore, the increased motility observed in
response to HGF treatment of the human breast epithelial cell line 184BJ is
blocked by the PI3K inhibitor wortmannin
(Day et al., 1999
), and
carcinoma invasion promoted by
6ß4 integrin involves PI3K activity
(Shaw et al., 1997
). The
Raf/MAPK pathway promotes the migration of fibroblastic cells and is necessary
for Ras- and TC21-induced transformation and metastasis of NIH3T3 cells
(Webb et al., 1998
;
Rosario et al., 1999
).
Moreover, the MAPK pathway is known to affect the actin-myosin system and
leads to an increased motility of COS-7 cells
(Klemke et al., 1997
).
Finally, the Rho family members of small GTPases are implicated in the
cytoskeletal reorganization that occurs during cell migration
(Ridley et al., 1995
;
Ridley et al., 1992
;
Ridley and Hall, 1992
;
Nobes and Hall, 1995
;
Nobes and Hall, 1999
). Rac1
activation induces the formation of lamellipodia, and active Cdc42 promotes
the protrusion of filopodia, leading to increased motility of Swiss3T3 cells
(Nobes and Hall, 1995
).
Accordingly, activation of Cdc42 and Rac1 promotes motility and invasion of
mammary epithelial cells on collagen matrices
(Keely et al., 1997
). Rac1 may
regulate cell migration not only by influencing actin organization but also by
potentiating MAPK activation (Leng et al.,
1999
).
In this report, we show that the Raf/MAPK pathway and Rac play crucial roles during EGF- and Ras-induced NBT-II cell dispersion, whereas PI3K and RalGDS activations are dispensable for cell scattering. Activation of MAPK is sufficient to promote cell dissociation by destabilising desmosomal junctions but is unable to induce cell motility. On the other hand, Rac is necessary for EGF- and Ras-induced NBT-II cell scattering but alone cannot promote cell dissociation. The full programme of EMT (i.e., cell dissociation and motility) is achieved by the simultaneous activation of MAPK and Rac, suggesting that the two pathways downstream of Ras are coordinately regulated during EMT events in NBT-II cells.
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Materials and Methods |
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Cell culture, microinjection and immunofluorescence
NBT-II cells were maintained in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal calf serum (FCS). A stable RasV12-expressing clone was
obtained by transfection of the NBT-II cell line by the calcium phosphate
precipitation method. This clone, designated Ras32, was routinely cultured in
DMEM containing 10% FCS and geneticin at 400 µg/ml (G418 sulfate; Gibco
BRL, Gaithersburg, MD). Microinjection experiments were performed as described
previously (Boyer et al.,
1997). Briefly, cells were microinjected into the nucleus with 100
ng/µl of plasmids 2 days after seeding. Cells were incubated at 37°C
for 4 hours prior to stimulation with EGF at 30 ng/ml. At least 200 cells were
injected on each coverslip. After overnight incubation, cells on coverslips
were rinsed in phosphate-buffer saline (PBS) before a 5 minute fixation at
-20°C in methanol/acetone. Coverslips were dried and processed for
immunofluorescence studies as described previously
(Boyer et al., 1997
). Digitized
pictures were recorded and processed by Adobe Photoshop software. The
percentage of cells having lost their desmosomes from the cell periphery was
estimated by counting the number of cells with desmoplakin (DP)
immunoreactivity within the cytoplasm as compared to cells expressing DP in a
punctate pattern along the cell periphery
(Boyer et al., 1989
).
When needed, cells were treated with FTase2 at 25 µM, UO126 and PD98059 at 100 µM and LY80052 at 20 µM for 48 or 24 hours before fixation in methanol/acetone or addition of EGF for an additional 15 hours before fixation. Cells were processed for immunofluorescence studies as described above. Cell viability in the presence of each agent was assessed by cell morphology under an inverted microscope. At the concentrations used for each drug, no toxic effect was observed on cells.
Ras-injected cells were immunostained with monoclonal rat anti-Ras antibody
followed by FITC-conjugated anti-rat antibody. Wild-type MEK1 and
MEK1SSDD-injected cells were visualized with rabbit anti-MEK1 antibody and
FITC-conjugated anti-rabbit antibody. Myc-tagged CD2-p110-, Rac1V12-,
Rac1N17-, Cdc42V12- and Cdc42N17-injected cells were immunolabelled with the
9E10 mouse monoclonal anti-myc antibodies and FITC-conjugated anti-mouse
antibodies. The RalAV23- and RalBV23-injected cells were visualized with mouse
anti-RalA and rabbit anti-RalB antibodies respectively, followed by
FITC-conjugated anti-mouse or anti-rabbit antibodies. RasV12-, RasV12A38-,
RasV12C40-, RasV12G37-, RasV12S35- and RasV12E38-injected cells were
immunostained with rat monoclonal anti-Ras antibodies followed by
FITC-conjugated anti-rat antibodies. The presence of desmosomes at the cell
periphery was monitored by immunofluorescent staining with anti-desmoplakin
antibodies and Texas-Red-coupled anti-mouse antibodies, as described
previously (Boyer et al.,
1989).
For biochemical assays and migration experiments, NBT-II cells were
transiently cotransfected with the indicated expression vectors and EGFP
reporter construct by the PEI (polyethylenimine) method
(Boussif et al., 1995).
Briefly, NBT-II cells were plated on a 35 mm dish, transfected with 5 µl of
PEI and 2.5 µg of the desired contructs, along with 0.5 µg of EGFP
reporter construct. Cells were allowed to incorporate the DNAs for 5 hours,
washed and analyzed 24 hours later.
Cell migration assay
To analyse cell migration, two conditions were used: cells in clusters and
isolated cells. Cells in clusters were microinjected with 100 ng/µl of the
constructs of interest along with 50 ng/µl of the construct coding for EGFP
reporter gene. For motility of isolated cells, the constructs of interest and
the EGFP construct were transiently overexpressed by the PEI method (see
above), and 24 hours later, cells were trypsinized and plated under low
confluence conditions. Four hours after microinjection or 10 hours after
plating, cells were placed on the motorized stage of a Leica inverted
microscope equipped with a chamber providing a controlled temperature and
CO2 concentration and connected to a Princeton MicroMax CCD camera
(Princeton Instruments Inc., Trenton, NJ). Phase contrast and epifluorescence
images were collected and analysed with the Metamorph software (Metamorph
Imaging System, Universal Imaging Corporation, West Chester, PA) running on a
PC workstation. The motility of cells was evaluated by tracking their movement
over 15 hours with images recorded every 4 minutes. For each overexpressed
construct, 40 to 60 cells were analysed in at least three independent
experiments.
MAPK and Rac activity assays
The assay of MAPK activity was performed as described previously
(Boyer et al., 1997), using
myelin basic protein (MBP) as an exogenous substrate for MAPK.
For the Rac activity assay, bacteria transformed with the GST-PAK2 construct were activated with 100 µM IPTG for 3 hours at 37°C. After centrifugation, the bacterial pellets were resuspended in 10 ml prelysis buffer [50 mM Tris, pH 7.5, 1% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 10 µg/ml aprotinin and leupeptin and 0.1 mM phenylmethylsulfonidefluoride (PMSF)] and lysed by adding lysosyme (0.1 volume of a 10 mg/ml solution in 25 mM Tris-HCl, pH 8.0) for 30 minutes at 4°C. DNA was degraded by sonication at 4°C. Lysates were centrifuged at 15,000 g for 15 minutes, and the cleared lysates were frozen at -80°C. Twenty five µg of lysate was incubated with 30 µl of glutathione-sepharose beads (Pharmacia Biotech, Uppsala) at 4°C for 30 minutes before processing for pull-down experiments.
When needed, NBT-II cells were activated with 30 ng/ml of EGF for 90 minutes or transiently transfected with the constructs of interest 24 hours before pull-down experiments. Cells were washed three times with cold TBS and scraped in 100 µl of lysis buffer (50 mM Tris, pH=7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 100 µM PMSF, 100 µM sodium orthovanadate and 500 µM sodium fluoride). Lysates were centrifuged at 15,000 g for 3 minutes at 4°C, and the supernatants were incubated with GST-PAK2 beads for 60 minutes at 4°C. The beads and proteins bound to the fusion protein were washed with a Tris buffer (50 mM pH 7.2) containing 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 0.1 mM PMSF, eluted in Laemmli sample buffer, separated by gel electrophoresis and analysed for Rac1 expression by western blot using Rac1 monoclonal antibodies (Transduction Laboratories).
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Results |
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To confirm that the Raf/MAPK pathway, but not the PI3K pathway, is required
during NBT-II cell dissociation, we next tested the effect of their
pharmacological inhibition. When cells were pretreated for 24 hours with the
MEK inhibitors PD089059 or UO126, which specifically prevent MEK1 from
promoting both threonine and tyrosine phosphorylation of MAPK
(Pang et al., 1995;
Dudley et al., 1995
), EGF
stimulation of NBT-II cells was no longer able to promote desmosome disruption
(Fig. 2Ae,f). By contrast,
incubation of cells with LY294002, a specific PI3K inhibitor, had no effect on
EGF-induced desmosome breakdown (Fig.
2Ac). However, when cells were incubated with 3 µM UO126, a
concentration that prevented EGF-induced cell dissociation in only 10% of
cells, addition of 20 µM LY294002 protected more than 90% of cells from
EGF-induced desmosome breakdown (data not shown). Thus, when MEK activity was
reduced below the level required for NBT-II cell dissociation, PI3K was able
to play a role in desmosome internalization. As a control, a farnesyl
transferase inhibitor that prevents the activation of Ras by inhibiting its
membrane localisation was tested under the same experimental conditions
(Fig. 2Ad). The effects of
PD089059 and LY294002 were also examined in Ras32, a stable
RasV12-overexpressing clone of NBT-II cells. Ras32 cells exhibit a
fibroblast-like phenotype that is clearly different from the epithelial
morphology of the parental NBT-II cells
(Fig. 2B). Immunofluorescence
analysis of Ras32 cells revealed a cytoplasmic punctate pattern of DP
immunostaining, with few immunofluorescent dots at the cell surface, which is
indicative of desmosome disruption (Fig.
2Ag). Twenty-four hours after addition of PD089059 or UO126 to the
culture medium, desmosomes were reformed at intercellular junctions
(Fig. 2Aj,k) whereas, under the
same conditions, LY025052 had no effect on DP localization
(Fig. 2Ah). However, the
intensity of intracellular DP immunostaining was markedly higher in
LY025052-treated Ras 32 cells than in their untreated counterparts, suggesting
that PI3K could be involved in preventing desmosomal proteins from
degradation. The same effect was observed in parental NBT-II cells
(Fig. 2, compare b with c). As
a control, addition of FTase2 relocalized DP to cell-cell junctions
(Fig. 2Ai).
|
Another way to confirm that Ras induces NBT-II cell dissociation through
the Raf/MAPK pathway was to test the effects of partial loss-of-function Ras
mutants that allow characterization of the various effector pathways
downstream of Ras (White et al.,
1995). RasV12C40 selectively activates the PI3K pathway; RasV12G37
is specific for RalGDS effector, whereas RasV12S35 and RasV12E38 selectively
activate the Raf/MAPK pathway
(Rodriguez-Viciana et al.,
1997
; White et al.,
1995
). Transient overexpression of RasV12 led to DP
internalization in 100% of cells. A similar result was obtained after
microinjection of RasV12S35 and RasV12E38, whereas transient expression of
RasV12C40 and RasV12G37 did not induce DP internalization
(Fig. 3). These data confirmed
the specific contribution of the Raf pathway to cell dissociation. As a
control, microinjection of RasV12A38, an inactive Ras mutant that does not
interact with an effector
(Rodriguez-Viciana et al.,
1997
), had no effect on DP internalization
(Fig. 3).
|
Altogether, these results demonstrate that the Ras-Raf-MAPK pathway is necessary and sufficient to promote desmosome breakdown in NBT-II cells.
MEK1-induced cell dissociation is a prerequisite for Rac1-mediated
cell motility
To determine whether active MAPK also participates in the migration
response of cells to EGF, we used two assays. In the first one, we
microinjected the desired constructs into clustered cells along with a EGFP
plasmid, and four hours later, their motility was recorded on a motorized
Leica microscope. In the second one, cells transfected with the different
constructs were trypsinized and plated at low density to obtain individual
cells, thus bypassing the cell dissociation step. Cell motility was then
measured for the isolated cells. In both assays, overexpression of RasV12
alone was sufficient to induce cell motility, suggesting that Ras was endowed
with both cell dissociation- and motility-promoting activities. Whereas
MEK1SSDD induced desmosome breakdown in NBT-II cells, its overexpression did
not promote cell migration (Fig.
4B,C). This result demonstrates that cell dissociation alone is
not sufficient to promote cell motility and suggests that a component of the
EGF-induced Ras-dependent transduction machinery distinct from the MEK/MAPK
pathway is responsible for inducing cell motility.
|
In a search for signalling molecules that cooperate with the active
MEK/MAPK signal to induce EMT, we examined Rac involvement, as small GTPase
proteins of the Rho family have been shown to induce actin cytoskeleton
remodeling (Nobes and Hall,
1995; Nobes and Hall,
1999
) and have been implicated in processes involving cell
dissociation and motility (Potempa and
Ridley, 1998
). When microinjected into clustered cells, Rac1V12
did not induce cell dissociation in more than 15% of the cells (data not
shown), unlike the high rate of cell dissociation induced by MEK1SSDD (60%).
Accordingly, coexpression of MEK1SSDD and Rac1V12 induced 60% of cells to
dissociate, a figure similar to that obtained with MEK1SSDD alone. This result
confirmed that Rac1 does not contribute to cell dissociation. Cdc42 was also
tested and did not promote DP internalization in more than 13% of cells (data
not shown). However, cells overexpressing Rac1V12 together with MEK1SSDD
exhibited an elongated morphology that was clearly different from that of
cells injected with MEK1SSDD alone (Fig.
4A). This result suggested that expression of Rac1 resulted in
dramatic changes towards a fibroblast-like phenotype often associated with
cell motility. Therefore, we asked whether Rac1 activity could be sufficient
to promote cell migration. Coexpression of Rac1V12 with MEK1SSDD in clustered
cells or overexpression of Rac1V12 in isolated cells was sufficient to induce
cells to migrate at a speed similar to that of RasV12-expressing cells,
suggesting that the Ras-mediated scattering activity was fully reproduced by
coexpression of active MEK and Rac. Cdc42V12 expression did not promote cell
motility of MEK1SSDD-expressing cells or of isolated cells
(Fig. 4B,C).
We also tested whether activated p110 with or without active RalA could substitute for Rac1V12 in the induction of motility. Coexpression of active p110 with MEK1SSDD did not elicit cell motility (speed of transfected cells: 8.4±5.2 µm/h; speed of control cells: 7.2±3.4 µm/h). Moreover, coexpression of active p110 with MEKSSDD and active RalA had no effect on the speed of locomotion of clustered cells (speed of transfected cells: 12.4±3.6 µm/h; speed of control cells: 9.6±3.2 µm/h). Consistently, coexpression of active p110 and active RalA in isolated cells did not promote cell movement (speed of transfected cells: 12.2±5.6 µm/h; speed of control cells: 7.9±4.4 µm/h). Conversely, inhibition of P13K activity by LY80052 did not block EGF- or Ras-induced migration (data not shown). Altogether, these results suggested that the motility-activating role of Rac was not dependent upon P13K activity. They also demonstrated that P13K, alone or in association with RalA, was not endowed with a migration-promoting activity.
Rac is activated under conditions leading to EMT
In order to investigate whether EGF and Ras were able to activate Rac in
NBT-II cells, we used a Rac pull-down assay that relies on the capacity of
activated Rac to bind to its cellular target PAK2 (p21 activated protein
kinase). EGF promoted a robust and sustained activation of Rac, starting 15
minutes after addition of EGF and progressively increasing thereafter
(Fig. 5A). For comparison, MAPK
activity was induced earlier, starting 5 minutes after addition of EGF, and
persisted over time. Moreover, transient overexpression of RasV12 induced Rac
activation (Fig. 5B). The
potential activation of Rac by the Ras-P13K pathway was investigated by
pull-down assay. Whereas overexpression of Rac1V12 and RasV12 induced a strong
Rac activation, the amount of active Rac bound to PAK-GST beads was almost
undetectable in cells expressing active p110, suggesting that P13K is not a
good inducer of Rac activation in NBT-II cells
(Fig. 5B). This result
correlated with the absence of migration of NBT-II cells overexpressing active
p110 (Fig. 4B) and suggested
furthermore that EGF- or Ras-dependent activation of Rac was not mediated by
P13K activity. Furthermore, the Ras-Raf-MAPK signalling pathway was not likely
to be responsible for Rac activation, as active c-Raf and active MEK1 had no
effect on Rac binding to PAK-GST (Fig.
5C). Accordingly, MEK and P13K inhibitors did not inhibit Rac
activation induced by EGF or RasV12 (Fig.
5D), thus confirming that Rac activation by EGF or Ras is
independent of the MAPK and P13K pathways in NBT-II cells.
|
Partial loss-of-function mutants of Ras activate Rac
We also examined whether the partial loss-of-function mutants of Ras
trigger cell motility. When overexpressed in NBT-II cell clusters, RasV12S35
and RasV12E38 promoted cell movement, with a speed of locomotion that was not
significantly different from that of RasV12-expressing cells. In contrast,
neither RasV12C40 nor RasV12G37 mutants were able to elicit cell migration
(Fig. 6A). As a control, the
RasV12A38 mutant had no effect on cell motility. On the other hand, when
coexpressed with MEK1SSDD, which promotes cell dissociation, all Ras mutants
were able to induce cell motility (Fig.
6B). Altogether, these data suggested that the partial
loss-of-function mutants of Ras were endowed with a motility-promoting
function. To test whether this function could be caused by Rac activation, a
Rac pull-down assay was done on extracts of NBT-II cells transfected with the
various partial loss-of-function mutants of Ras. All of them elicited robust
Rac activity, similar to that produced by RasV12 expression
(Fig. 6C). This result
demonstrated that all partial loss-of-function mutants of Ras were able to
activate Rac.
|
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Discussion |
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The precise mechanisms whereby the MAPK pathway controls the integrity of
intercellular junctions remain elusive. However, the Ras signal has been
demonstrated to be upstream of the transcription factor Slug
(Boyer et al., 1997), the
expression of which has been shown to induce desmosome breakdown in NBT-II
cells (Savagner et al., 1997
).
Nevertheless, a direct link between MAPK activity and induction of Slug
expression remains to be investigated. Disruption of the other types of
epithelial junctions is also expected to depend on the ability of the Ras/MAPK
pathway to activate the transcription of target genes.
In the NBT-II system of epithelial cell scattering, there is no evidence
that other known effectors of Ras contribute to desmosome breakdown and cell
dissociation. Several lines of evidence ruled out the involvement of PI3K in
the dissolution of cell-cell junctions: microinjection of RasV12C40 and p110
contructs did not lead to desmosome breakdown and, conversely, addition of the
PI3K inhibitor LY80052 to EGF-stimulated NBT-II cells or to RasV12-expressing
transfectants did not restore desmosomes at the cell surface. Our results are
in agreement with recent data showing that MAPK but not PI3K activity is
responsible for the disruption of desmosomes and adherens junctions in
EGF-treated ovarian cancer cells
(Ellerbroek et al., 2001).
Apart from PI3K, neither Rac nor RalGDS displayed a desmosome-dissociating
activity, as inferred from the fact that microinjection of Rac1V12 or
RasV12G37, RalAV23 or RalBV23 did not lead to DP internalization. Therefore,
the Raf/MAPK signal appeared as the only downstream effector of Ras capable of
dissociating NBT-II cells. However, RasV12 expression induced 100% of cells to
dissociate, whereas MEK1SSDD expression led to DP internalization in only 60%
of cells. This difference may reveal the hypothetical existence of a
cell-dissociating Ras effector distinct from Raf. Alternatively, it may simply
result from the greater efficiency of the RasV12 construct, compared with
MEK1SSDD.
Even though they were dissociated, MEK1SSDD-expressing NBT-II cells were
not induced to migrate. In other models, the role of MEK/MAPK in mediating
cell migration is controversial. It is necessary for EGF-induced chemotaxis
(Slack et al., 1999), for
PDGF-induced migration of RPE epithelial cells
(Hinton et al., 1998
) and for
collagen and fibronectin-induced haptotactic migration of Rat1 fibroblasts
(Klemke et al., 1997
;
Anand-Apte et al., 1997
). By
contrast, PDGF induction of chemotaxis of Rat1 fibroblasts
(Anand-Apte et al., 1997
) and
EGF stimulation of migration of mouse NR6 cells cannot be mimicked by active
MAPK (Xie et al., 1998
). The
apparent discrepancy between these data may be explained by cell migration not
occurring below a certain threshold of MAPK activation
(Krueger et al., 2001
). In
contrast to active MEK1, RasV12 promoted vigorous cell movements. These
results suggested that cell dissociation and motility are distinct events that
require specific signalling pathways to occur and that RasV12 is likely to
activate an effector molecule specifically involved in cell motility.
In a search for a Ras effector that triggers cell motility, we found that
overexpression of Rac1V12 was sufficient to activate cell movement, provided
that cell-cell contacts were not present at the cell surface. The loss of
desmosomes was achieved by either culturing cells under sparse conditions or
transiently expressing MEK1SSDD. The involvement of the small GTPase Rac in
triggering cell motility has already been reported
(Nobes and Hall, 1999;
Anand-Apte et al., 1997
;
Kjoller and Hall, 2001
;
Bourguignon et al., 2000
;
Sander et al., 1998
). However,
the role of Rac in cell motility is still controversial since downregulation
of Rac by active Ras leads to EMT (Zondag
et al., 2000
), and, conversely, Rac activation in NIH3T3
fibroblasts induces an epithelium-like morphology and inhibits cell migration
(Sander et al., 1999
).
In the NBT-II cell system, it is not possible to test whether Rac acts
alone or cooperates with the MEK/MAPK pathway to induce cell motility. In
COS-7 cells, Rac potentiates the C-Raf-induced cell motility
(Leng et al., 1999) by
promoting the formation of membrane ruffles, whereas MEK is known to affect
the actin-myosin system (Klemke et al.,
1997
) and Rho-dependent actin stress fiber formation
(Sahai et al., 2001
).
Coordinated activation of MEK1 and Rac1 has also been reported to reproduce
the proliferative effect of oncogenic Ras
(Cobellis et al., 1998
). In
NBT-II cells, Rac was strongly activated by EGF or Ras signalling.
Interestingly, all Ras partial loss-of-function mutants activated Rac and
reproduced the effect of active Rac on NBT-cell motility. These results
suggested that the effector molecule that triggers Rac activation does not
contact the GTP-bound Ras at conserved residues in the `effector loop'.
Accordingly, a systematic study of contact sites between small GTP-binding
proteins and partner proteins has revealed the presence of binding
interactions outside the effector loop
(Corbett and Alber, 2001
). The
existence of Ras effectors that use this new type of binding strategy may be
linked to specific functions.
In contrast to what has been described in previous reports, the molecule
mediating Rac activation is not likely to be PI3K
(Keely et al., 1997;
Buhl et al., 1995
;
Nobes et al., 1995
), as active
p110 did not induce Rac activity and did not mimick Rac activation of NBT-II
cell motility. Moreover, LY80052 did not decrease Rac activation elicited by
RasV12 or EGF and did not interfere with RasV12-induced cell motility. Rac may
be activated in a Ras-dependent manner by Rac exchange factors such as Tiam
(Hordijk et al., 1997
;
Bourguignon et al., 2000
) or
Vav2 (Liu and Burridge, 2000
).
Tiam/Rac signalling is dependent on extracellular matrix components
(Sander et al., 1998
), whereas
Vav2-mediated activation of Rac1 is dependent on growth factor stimulation
(Liu and Burridge, 2000
). For
that reason, it is reasonable to speculate that EGF- and Ras-induced
activation of Rac1 in NBT-II cells is mediated by Vav2. Since Tiam1 but not
Vav2 has been demonstrated to be downstream from PI3K
(Sander et al., 1998
), this
could explain why PI3K is not involved in EGF-stimulated cell motility in
NBT-II cells. However, this hypothesis awaits further evidence.
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Acknowledgments |
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References |
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