(Received for publication, June 15, 1995; and in revised form, September 8, 1995)
From the
Hepatocyte growth factor/scatter factor (HGF/SF) is a
multifunctional cytokine that induces mitogenesis, motility, invasion,
and morphogenesis of several epithelial and endothelial cell lines in
culture. The receptor for HGF/SF has been identified as the Met
tyrosine kinase. To investigate the signaling pathways that are
involved in these events, we have generated chimeric receptors
containing the extracellular domain of the colony-stimulating factor-1
(CSF-1) receptor fused to the transmembrane and intracellular domains
of the Met receptor (MET). Madin-Darby canine kidney (MDCK) epithelial
cells expressing the CSF-MET chimera dissociate and scatter in response
to CSF-1. However, cells expressing a mutant CSF-MET receptor
containing a phenylalanine substitution for tyrosine 1356 were unable
to scatter or form branching tubules following stimulation with CSF-1.
Tyrosine 1356 is essential for the recruitment of multiple substrates
including the p85 subunit of PI3-kinase, phospholipase C, and
Grb2. In this study, we have investigated the role of PI3-kinase and a
downstream target of PI3-kinase, pp70
, in the induction
of MDCK cell scatter in response to HGF/SF. Our results demonstrate
that following stimulation with HGF/SF, activation of PI3-kinase but
not pp70
is essential for MDCK cell scatter.
Cell motility is a fundamental process required during normal
embryonic development, wound healing, inflammatory responses, and tumor
progression toward metastasis(1) . Hepatocyte growth
factor/scatter factor (HGF/SF) ()is a multifunctional
cytokine with activities on a wide variety of normal and neoplastic
cells. HGF/SF is a mitogen, dissociation, and motility factor for many
epithelial cells (2, 3, 4) and stimulates
tubulogenesis of tubular epithelial cells (5) as well as the
invasion of carcinoma cells(6) . In vivo, HGF/SF is a
potent angiogenic factor (7, 8) and is involved in
organ regeneration (9) and
tumorigenesis(10, 11) . A high affinity receptor for
HGF/SF has been identified as the product of the met proto-oncogene(12, 13) , which encodes a receptor
tyrosine kinase originally isolated as the tpr-met oncogene(14, 15) . The Met receptor is
predominantly expressed in epithelial cells in culture (15, 16, 17) and in epithelium in vivo (18, 19). (
)The mature form of the Met receptor is a
heterodimeric protein of 190 kDa, which consists of a 45-kDa
extracellular
-subunit linked by disulfide bonds to a 145-kDa
-subunit that spans the membrane and contains the catalytic kinase
domain(16, 20, 21, 22) . Binding of
HGF/SF induces activation of the kinase and auto/transphosphorylation
of the receptor (13) on specific tyrosine residues in the
chain(12, 23) .
Phosphorylated tyrosine residues
within receptor tyrosine kinases provide binding sites for molecules
containing SH2 and PTB domains that act to transduce extracellular
signals to the interior of the cell (24) . Following
stimulation of cells with HGF/SF, several proteins are phosphorylated,
activated, and/or associated with the Met receptor. These include p120
GTPase-activating protein, mitogen activated protein kinase, Src,
phospholipase C, phosphatidylinositol 3-kinase (PI3-kinase),
Grb2(25, 26, 27, 28, 29, 30) ,
Ras(31) , focal adhesion kinase(32) ,
-catenin,
plakoglobin(33) , and the Shc adaptor protein(34) . The
Met receptor tyrosine kinase is highly phosphorylated on two tyrosine
residues (1234 and 1235) within the kinase domain (35, 36) that are essential for the catalytic activity
of the receptor(29, 35) . In addition, tyrosine 1356
within the carboxyl terminus of the
-subunit of the Met receptor
is phosphorylated (29) and is essential for the recruitment of
multiple substrates including the p85 subunit of PI3-kinase,
phospholipase C
, and
Grb2(29, 30, 37, 38) . Although a
role for many of these signal transduction pathways has been
established in cell mitogenesis, their role in dissociation and scatter
of epithelial cells is unknown.
To investigate the signaling
pathways that are involved in these events, we have generated chimeric
receptors containing the extracellular domain of the CSF-1 receptor
fused to the transmembrane and intracellular domains of the Met
receptor. Madin-Darby canine kidney (MDCK) epithelial cells dissociate,
scatter, and form branching tubules in response to HGF/SF. MDCK cells
expressing the CSF-MET chimera dissociate and scatter in response to
CSF-1(39) ; however, MDCK cells expressing a mutant CSF-MET
receptor containing a phenylalanine substitution for tyrosine 1356
(Y1356F) are unable to scatter or form branching tubules following
stimulation with CSF-1(29) . This has suggested that one or
more of the substrates that bind to Tyr are required for
the induction of MDCK cell scatter following stimulation with HGF/SF.
Our results demonstrate that following stimulation with HGF/SF,
activation of PI3-kinase but not phospholipase C
or
pp70
, a downstream target of PI3-kinase, is essential for
MDCK cell scatter.
Figure 1: E-cadherin, desmoplakins I/II, and ZO-1 remain insoluble following CSF-1 stimulation of MDCK cells expressing the Y1356F CSF-MET mutant receptor. Colonies of MDCK cells expressing the Y1356F CSF-MET mutant receptor were serum-starved for 24 h and further incubated for 24 h in the same medium (a-d), in medium containing HGF/SF (5 units/ml) (e-h), or in medium containing CSF-1 (50 ng/ml) (i-l). Cells were extracted in situ with CSK buffer and fixed with 1.75% formaldehyde in PBS as described under ``Experimental Procedures.'' Cells were then processed for indirect immunofluorescence with antibodies against E-cadherin (b, f, and j), desmoplakins I/II (c, g, and k), and ZO-1 (d, h, and l) as primary antibodies, followed by rhodamine-coupled anti-mouse antibodies (for E-cadherin and ZO-1) and fluorescein isothiocyanate-coupled anti-rabbit antibodies (for desmoplakins I/II) as secondary antibodies.
Figure 3:
The redistribution of E-cadherin and
desmoplakins I/II, cell spreading, and scatter in HGF/SF-stimulated
MDCK cells are inhibited by wortmannin. A, colonies of
serum-starved MDCK cells were incubated for 3.5 h in medium containing
0.1% MeSO (a-d), preincubated in medium
containing 0.1% Me
SO for 30 min followed by stimulation
with HGF/SF (10 units/ml) for 3 h (e-h), or preincubated
in medium containing 500 nM wortmannin (in 0.1%
Me
SO) for 30 min followed by stimulation with HGF/SF for 3
h (i-l). Cells were extracted in situ with CSK
buffer, fixed with 1.75% formaldehyde in PBS, and processed for
indirect immunofluorescence with antibodies against E-cadherin (b, f, and j), desmoplakins I/II (c, g, and k), and ZO-1 (d, h, and l). B, colonies of serum-starved MDCK
cells were incubated for 7.5 h in medium containing 0.1%
Me
SO (a), preincubated in medium containing 0.1%
Me
SO for 30 min followed by stimulation with HGF/SF (10
units/ml) for 7 h (b), preincubated in medium containing 1
µM wortmannin (in 0.1% Me
SO) for 30 min
followed by stimulation with HGF/SF for 7 h (c), preincubated
in medium containing 5 nM staurosporine (in 0.1%
Me
SO) for 30 min followed by stimulation with HGF/SF for 7
h (d), preincubated in medium containing 2 µM U-73122 (e), or 2 µM U-73343 (f)
(in 0.1% Me
SO) for 30 min followed by stimulation with
HGF/SF for 7 h. Cells were fixed in 0.2% glutaraldehyde in
PBS.
MDCK cells expressing a CSF-MET chimera dissociate and scatter in a similar manner in response to CSF-1 or HGF/SF(39) . However, MDCK cells expressing a mutant CSF-MET receptor containing a phenylalanine substitution for tyrosine 1356 (Y1356F) fail to scatter following stimulation with CSF-1(29) . We show here that junctional complexes remained intact and in an insoluble form at cell-cell interfaces in CSF-1-stimulated MDCK cells expressing the Y1356F CSF-MET mutant (Fig. 1, i-l).
Figure 2:
PI3-kinase is activated following HGF/SF
stimulation of MDCK cells and is inhibited by wortmannin in vitro and in vivo. A, MDCK cells starved for 48 h were
stimulated with HGF/SF (10 units/ml) for different time intervals, and
cell lysates were then immunoprecipitated with the anti-phosphotyrosine
antibody PY20. Immune complexes were adsorbed using protein A-Sepharose
and subjected to a PI3-kinase assay. The products of the reaction were
analyzed by thin layer chromatography, visualized by autoradiography,
and quantified by a PhosphorImager. B,
phosphotyrosine-containing proteins from lysates of MDCK cells were
immunoprecipitated with the PY20 antibody and assayed for PI3-kinase
activity in vitro in the presence of either 0.1%
MeSO or various concentrations of wortmannin for 10 min
prior to the kinase reaction. C, serum-starved MDCK cells were
preincubated with either 0.1% Me
SO or wortmannin (10 nM to 10 µM) for 30 min at 37 °C, followed by
stimulation with HGF/SF (10 units/ml) for 1 min at 37 °C. Cells
were lysed, and phosphotyrosine-containing proteins were
immunoprecipitated with the PY20 antibody. Immune complexes were then
processed as described above. The position of phosphatidylinositol
3-phosphate (PI(3)P) is indicated.
To examine the role of PI3-kinase in vivo, serum-starved MDCK cells were pretreated for 30 min
at 37 °C with either wortmannin or its solvent (MeSO
0.1%) and stimulated with HGF/SF, and PI3-kinase was immunoprecipitated
with anti-phosphotyrosine antibodies and assayed in vitro.
Wortmannin, at concentrations of 100 nM and 1 µM,
reduced the level of PI3-kinase activity in HGF/SF-stimulated MDCK
cells by 35 and 65% respectively (Fig. 2C). To evaluate
the effect of wortmannin on HGF/SF-induced cell scatter, colonies of
serum-starved MDCK cells were pretreated for 30 min at 37 °C with
wortmannin (10 nM-1 µM) and then stimulated with
HGF/SF. MDCK cells, following stimulation with HGF/SF, changed shape
and flattened by 3 h (Fig. 3A, e), whereas
MDCK cells in the presence of 500 nM wortmannin remained as
tight colonies (Fig. 3A, i). Moreover, in the
presence of 1 µM wortmannin, MDCK cells stimulated with
HGF/SF for 7 h had flattened (Fig. 3B, c) but
were inhibited in their ability to scatter when compared with control
HGF/SF-stimulated MDCK cells (Fig. 3B, b). The
ability of wortmannin to inhibit both cell spreading and scatter was
concentration-dependent and correlated with its ability to inhibit
PI3-kinase activity in vivo (data not shown).
To establish which processes were inhibited by wortmannin, we examined the effect of wortmannin on the redistribution of junctional complex proteins. The spreading and changes in morphology observed in MDCK cells, following HGF/SF stimulation for 3 h, were accompanied by a reduction in the amount of insoluble E-cadherin and desmoplakins I/II at cell-cell interfaces (Fig. 3A, f and g). At this time however, the concentration of insoluble ZO-1 at cell-cell interfaces (Fig. 3A, h) was comparable to that observed in control unstimulated MDCK cells (Fig. 3A, d). A redistribution of ZO-1 was only apparent when cells began to scatter (data not shown). However, in the presence of 500 nM wortmannin, all three proteins from junctional complexes were retained in an insoluble compartment at the plasma membrane (Fig. 3A, j-l). Thus, the ability of wortmannin to inhibit MDCK cell spreading and scatter correlated with the retention of insoluble junctional complexes and tight cell-cell interactions, suggesting that PI3-kinase activity is required for the redistribution of junctional complex proteins and cell dissociation induced by HGF/SF in MDCK cells.
Chemotaxis of some cell types
transduced by the PDGF- and epidermal growth factor receptors
requires phospholipase C
(51, 56) ; however, the
inhibition of protein kinase C, a downstream target of phospholipase
C
, did not inhibit MDCK cell scatter in response to
HGF/SF(57) . To investigate the involvement of phospholipase
C
in HGF/SF-induced MDCK cell scatter, we used an inhibitor of
phospholipase C (U-73122; IC
of 1-2 µM) (56, 58, 59) and the protein kinase C
inhibitor staurosporine (IC
of 0.7
nM)(60) . Colonies of serum-starved MDCK cells were
pretreated with Me
SO (0.1%), staurosporine (1
nM-20 nM), U-73122 (0.25-2 µM), or
an inactive analogue of U-73122 (U-73343; 0.25-2 µM)
for 30 min at 37 °C. MDCK cells in the presence of staurosporine (Fig. 3B, d, and data not shown), the
phospholipase C inhibitor U-73122 (Fig. 3B, e,
and data not shown), the inactive analogue U-73343 (Fig. 3B, f, and data not shown), or
Me
SO (0.1%) (Fig. 3B, b) scattered
following stimulation with HGF/SF for 7 h. Therefore, in contrast to
PI3-kinase, phospholipase C
and protein kinase C are not essential
for MDCK cell spreading and scatter.
Figure 4:
pp70 is activated following
HGF/SF stimulation of MDCK cells and is inhibited by wortmannin or
rapamycin. A, serum-starved MDCK cells were stimulated with
HGF/SF (10 units/ml) for different time intervals, and proteins from
cell lysates were immunoprecipitated with antibodies against
p70
. Immune complexes were adsorbed using protein
A-Sepharose and subjected to SDS-polyacrylamide (8%) gel
electrophoresis, transferred onto nitrocellulose and immunoblotted with
an anti-p70
antibody (top), or subjected to an in vitro kinase assay using S6 peptide as a substrate as
described under ``Experimental Procedures'' (bottom). The results are reported as counts incorporated into
S6 peptide. B, serum-starved MDCK cells were preincubated with
either 0.1% Me
SO or various concentrations of wortmannin
for 30 min at 37 °C, followed by further incubation in
Me
SO or by stimulation with HGF/SF (10 units/ml) for 20 min
at 37 °C. Cells were lysed, pp70
was
immunoprecipitated, and immune complexes were then processed as
described above. C, serum-starved MDCK cells were preincubated
with either 0.1% ETOH or various concentrations of rapamycin for 30 min
at 37 °C, followed by further incubation in ETOH or by stimulation
with HGF/SF (10 units/ml) for 20 min at 37 °C. Cells were lysed and
pp70
-immune complexes were processed as described
above.
To establish whether pp70 is essential for MDCK cell
dissociation and scatter induced by HGF/SF, serum-starved MDCK cells
were pretreated with the macrolide antibiotic rapamycin, which inhibits
the PI3-kinase-related protein RAFT1 (65) and as a consequence
its downstream target pp70
(66, 67, 68) . HGF/SF-induced pp70
phosphorylation (Fig. 4C, top) and in vitro kinase activity (Fig. 4C, bottom) were inhibited equally by rapamycin (1-50 ng/ml)
or wortmannin (1-10 µM) (Fig. 4B).
However, preincubation of MDCK cells with 20 ng/ml of rapamycin for 30
min followed by stimulation with HGF/SF did not inhibit scattering of
MDCK cells (Fig. 5i). Moreover, rapamycin pretreatment
did not inhibit the redistribution or solubilization of E-cadherin,
desmoplakins I/II, and ZO-1 following stimulation of MDCK cells with
HGF/SF (Fig. 5, j-l). Therefore, although
pp70
is activated following stimulation with HGF/SF and
is a possible downstream target of PI3-kinase in MDCK cells, it is not
required for scatter of MDCK cells in response to HGF/SF.
Figure 5:
Inhibition of pp70
activation in HGF/SF-stimulated MDCK cells by rapamycin does not block
cell spreading and dissociation. Colonies of serum-starved MDCK cells
were incubated for 24 h in medium containing 0.1% ETOH (a-d), preincubated in medium containing 0.1% ETOH for
30 min and then stimulated by HGF/SF (10 units/ml) for 24 h (e-h), or preincubated in medium containing 20 ng/ml of
rapamycin for 30 min and then stimulated with HGF/SF for 24 h (i-l). Cells were extracted in situ with CSK
buffer and fixed with 1.75% formaldehyde in PBS and then processed for
indirect immunofluorescence with antibodies against E-cadherin (b, f, and j), desmoplakins I/II (c, g, and k), and ZO-1 (d, h, and l).
HGF/SF is a multifunctional cytokine that stimulates
dissociation, scatter, and morphogenesis of epithelial
cells(2, 4, 5) . The response of MDCK cells
to HGF/SF can be visualized first as cell spreading (after 2-3
h), when cells are still associated and present within the colony (Fig. 3A), followed by cell dissociation (after
4-6 h) and cell scatter (from 6 h) (Fig. 1). Breakdown of
cell contacts is a prerequisite for cell dissociation, and we have
shown that dissociation of MDCK cells in response to HGF/SF is
concomitant with the loss of stable insoluble junctional complexes at
sites of cell contact. Interestingly, following HGF/SF stimulation of
MDCK cells, a redistribution of E-cadherin and desmoplakins I/II, which
are components of adherens junctions and desmosomes, is observed prior
to the redistribution of ZO-1, a component of tight junctions ( Fig. 1and 3A). This may reflect the ability of HGF/SF
to stimulate the phosphorylation of -catenin and
plakoglobin(33) , which could contribute to the destabilization
of adherens junctions and desmosomes but not tight junctions, in which
these proteins are absent.
MDCK cells expressing a CSF-MET mutant
receptor (Y1356F) fail to stimulate scatter (29) or
redistribute junctional complexes (Fig. 1). Tyrosine 1356 in the
carboxyl terminus of the Met receptor is essential for association with
PI3-kinase, phospholipase C, and
Grb2(29, 30, 37, 38) , suggesting
that at least one of these signaling pathways is required for cell
dissociation and scatter. However, a mutant CSF-MET receptor containing
a substitution of a histidine residue for the asparagine downstream
from tyrosine 1356 (N1358H), which failed to bind only the Grb2 adaptor
protein, stimulated MDCK cell scatter in response to ligand, (
)demonstrating that association of Grb2 with the Met
receptor is not essential. Because binding sites for PI3-kinase (51, 52) or phospholipase C
(51) in the
PDGF receptor-
are important for PDGF-BB-induced chemotaxis and
because phospholipase C
is involved in epidermal growth
factor-mediated chemotaxis(56) , we have investigated the role
of these signaling pathways in HGF/SF-induced cell dissociation and
scatter of MDCK epithelial cells. We have shown that PI3-kinase plays
an essential role in HGF/SF-mediated scatter of MDCK cells. In the
presence of wortmannin, a potent inhibitor for PI3-kinase, MDCK cells
stimulated with HGF/SF for 3 h remained as tight colonies and retained
insoluble E-cadherin, desmoplakins I/II, and ZO-1 at cell-cell
interfaces. Conversely, control MDCK cell colonies contained cells with
a flattened appearance, which showed a redistribution of junctional
complex proteins involving E-cadherin and desmoplakins I/II, whereas,
as discussed above, ZO-1 from tight junctions was maintained while
cells were in contact (Fig. 3A)(69) . Moreover,
concurrent with the ability of wortmannin to inhibit the spreading of
MDCK cells 3 h after HGF/SF stimulation, wortmannin also inhibited the
scatter of MDCK cells at 7 h (Fig. 3B). In contrast, an
inhibitor of phospholipase C (U-73122) or protein kinase C
(staurosporine) at concentrations equal or higher than their respective
IC
did not inhibit scatter of MDCK cells in response to
HGF/SF (Fig. 3B).
The ability of wortmannin to
inhibit HGF/SF-induced spreading and scatter of MDCK cells correlated
directly with the extent of PI3-kinase inhibition in vivo,
thus supporting a crucial role for PI3-kinase activity in the
dissociation and scatter of MDCK cells. The concentrations of
wortmannin required for the inhibition of cell spreading,
redistribution and solubilization of junctional complex proteins, and
cell scatter were higher than that reported for the inhibition of
diverse biological responses involving membrane ruffling (70) ,
histamine secretion(55) , respiratory
burst(53, 71) , or glucose transport (72) (between 50-100 nM). Although high
concentrations of wortmannin (in the µM range in
vitro) have been reported to inhibit myosin light chain
kinase(55, 73) , an enzyme thought to be involved in
cell motility, treatment of MDCK cells with an inhibitor of myosin
light chain kinase (ML-9) had no effect on HGF/SF-induced MDCK cell
scatter (57) . Moreover, our data demonstrated that the canine
PI3-kinase was sensitive to wortmannin in vitro, with an
IC below 10 nM, which is comparable to other
studies(53, 54, 55, 72) . In
addition, wortmannin is a lipophilic compound and is expected to
inactivate PI3-kinase localized at the plasma membrane. However, the
Met receptor is localized to the basolateral surface of MDCK
cells(74) . Thus the application of wortmannin to the apical
surface of a colony of tightly associated MDCK cells may be unable to
efficiently inactivate the Met-stimulated PI3-kinase localized to the
basolateral compartment.
Wortmannin is also an unstable compound when maintained at 37 °C (54) . Thus, the ability of MDCK cells treated with wortmannin to begin to spread at 7 h post HGF/SF stimulation (Fig. 3B) may reflect the instability of wortmannin. Consistent with this possibility, MDCK cells stimulated with HGF/SF for 7 h remained as tight cell colonies when the medium containing wortmannin and HGF/SF was replaced at 2-h intervals (data not shown). Although MDCK cells showed some signs of toxicity under these conditions, this suggests that PI3-kinase is essential for cell spreading. We therefore conclude that PI3-kinase is required for MDCK cell dissociation and thus, scatter, following stimulation with HGF/SF, but due to the instability of wortmannin, we cannot rule out that other factors are involved in these events.
The pp70 has
been described as a downstream target of PI3-kinase in various cell
types(61, 62) . We show that pp70
is
activated following stimulation of MDCK cells with HGF/SF and that this
activity is also inhibited by wortmannin, suggesting that pp70
is a downstream target of PI3-kinase in MDCK cells (Fig. 4). pp70
is required for the progression
through G1 in response to serum and growth factors in a variety of
cells (66, 67, 68, 75) . However,
although pretreatment of MDCK cells with rapamycin inhibited the
activation of HGF/SF-induced pp70
, this had no effect on
MDCK cell spreading, redistribution of junctional complex proteins, or
cell scatter (Fig. 5). These results suggest that although
PI3-kinase is required for cell dissociation and scatter following
stimulation of MDCK cells with HGF/SF, this is independent from the
activation of pp70
.
The requirement for a functional
PI3-kinase has been implicated in actin reorganization at the plasma
membrane (membrane ruffling) stimulated by PDGF (70, 76) and insulin(76) . Moreover,
PI3-kinase is involved in the activation of the small GTP-binding
protein Rac(77) , which is required for membrane ruffling in
response to growth factors (76, 78) . Interestingly,
microinjection of MDCK cells with a dominant negative mutant of Rac
(N17Rac1) inhibits cell spreading and actin reorganization induced by
HGF/SF(79) , thus supporting a crucial role for Rac in these
events. In addition, Ras is also essential for the dissociation and
scatter of MDCK cells(79, 80) . Expression of a
dominant negative mutant Ras protein (N17Ras) (80) or the
injection of a neutralizing antibody for Ras (Y13-259) (79) block HGF/SF-induced cell dissociation and scatter,
whereas microinjection of an activated Ras (V12H-Ras) in MDCK cells
promotes cell spreading in the absence of HGF/SF(79) .
Furthermore, GTP-bound Ras interacts with PI3-kinase and may contribute
to its activation(81, 82) . Thus, we propose that
activation of PI3-kinase in MDCK cells following stimulation of the Met
receptor by HGF/SF promotes cell dissociation, which is independent
from the activation of pp70, but may involve the small
GTP-binding proteins Rac and Ras. The relationship between PI3-kinase
and these proteins in MDCK cells is currently under investigation.