From the Department of Zoology, College of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan, Republic of China
Received for publication, November 27, 2000, and in revised form, March 22, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously shown that hepatocyte
growth factor (HGF) selectively increases the expression of integrin
Hepatocyte growth factor
(HGF),1 also known as scatter
factor, is a multifunctional growth factor that elicits mitogenic,
motogenic, and morphogenic activities in various cell types (1). The
diverse biological effects of HGF are transmitted through activation of its transmembrane receptor encoded by the c-met
proto-oncogene (2, 3). Although a number of growth factors are known to modulate cell motility, HGF is unique because of the intensity with
which it stimulates motility and induces the epithelial-mesenchymal (E-M) transition. The scatter response of Madin-Darby canine kidney (MDCK) cells to HGF stimulation has been used extensively as a model to
study the E-M transition, characterized by the loss of epithelial
polarity, the disruption of E-cadherine-mediated cell-cell adhesions,
and the acquisition of a migratory mesenchymal cell phenotype (4-6).
Upon HGF stimulation, the scatter of MDCK cells can be visualized first
as centrifugal spreading of cell colonies (after 2-4 h) followed by
cell-cell dissociation (after 4-6 h) and subsequent cell migration
(from 6 h) (4, 7).
Several intracellular signaling pathways have been implicated to act
downstream of the HGF receptor to mediate scatter response. For
example, both phosphatidylinositol 3-kinase (PI3K) and small GTPase Ras
have been shown to be essential for cell dissociation and migration
following stimulation of MDCK cells with HGF (4, 5, 8). Although
GTP-bound Ras interacts with PI3K and may contribute to its activation
(9, 10), recent studies suggested that Ras and PI3K might act on
different signal transduction cascades to facilitate HGF-induced cell
scattering (6, 11). It has been shown that PI3K acts upstream of Tiam1,
an activator of the small GTPase Rac, to modulate both
E-cadherine-mediated cell adhesion and cell migration (6). More
recently, Zondag et al. (12) showed that the expression of
oncogenic Ras permanently suppresses Rac activity through
down-regulation of Tiam1 expression, which leads to up-regulation of
the Rho activity and the E-M transition of MDCK cells. In addition,
inhibition of HGF-induced cell scattering by dominant negative Ras or
the specific inhibitor for extracellular signal-regulated kinase (ERK)
kinase (MEK) has implicated an essential role for the Ras/ERK cascade
in cell scattering (13-15).
Integrin Materials--
Recombinant human HGF and epidermal growth factor
(EGF) were purchased from R&D Systems, Inc. Fetal bovine serum and
LipofectAMINE Plus were purchased from Life Technologies, Inc. G418,
Bisindolylmaleimide, genistein, PD98059, LY294002, cycloheximide, and
phorbol-12-myristate-13-acetate (PMA) were purchased from Calbiochem
(San Diego, CA). PP1 was purchased from BIOMOL Research Laboratories,
Inc. (Plymouth Meeting, PA). EZ-Link sulfo-NHS-biotin and
avidin-immobilized agarose beads were purchased from Pierce. The
polyclonal (AB1944) and monoclonal (clone P1E6) anti-integrin
Cell Culture and Transfections--
MDCK II 3B5 cells were
maintained in Dulbecco's Modified Eagle Medium (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum and cultured at
37 °C in a humidified atmosphere of 5% CO2 and 95% air
atmosphere. To induce cell scattering and analyze the expression of
integrin
To express constitutively active MEK1, dominant negative MEK1, or HVH2,
MDCK cells were grown on 60-mm dishes and co-transfected with pSV2neo
and a plasmid encoding constitutively active MEK1, dominant negative
MEK1, or HVH2 in 1:20 ratio using LipofectAMINE Plus according to the
manufacturer's instructions. 24 h later, the medium was replaced
with the fresh medium with 10% serum supplemented with 0.5 mg/ml
neomycin G418. 7 days later, the neomycin-resistant cells were
serum-staved and treated with or without 25 ng/ml HGF. 12 h later,
the cells were lysed and subjected to immunoblotting analysis for MEK
expression, ERK phosphorylation, and integrin Biotinylation and Immunoblotting--
For cell surface
biotinylation, MDCK cells were grown for 2 days to allow cell colony
formation. After washed in phosphate-buffered saline (PBS), cells were
incubated in PBS containing 500 µg/ml sulfo-NHS-biotin at room
temperature for 30 min, washed once with cold PBS, and incubated in PBS
containing 0.1 M glycine (pH 7.4) on ice for 15 min. After
several washes in PBS, the cells were lysed in Nonidet P-40 lysis
buffer containing protease inhibitors. Aliquots (500 µg) of lysates
were incubated with avidin-immobilized agarose beads for 1 h at
4 °C. After being washed three times in Nonidet P-40 lysis buffer,
the beads were subjected to immunoblotting with polyclonal
anti-integrin
For immunoblotting, whole cell lysates or avidin-precipitated complexes
were boiled for 3 min in SDS sample buffer, subjected to
SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose (Schleicher & Schuell). Immunoblotting was performed with anti-integrin Antibody Inhibition of HGF-induced Cell Scatter and Cell
Migration--
MDCK cells were allowed to grow as discrete colonies on
60-mm dishes and then treated with 25 ng/ml HGF in the presence or the
absence of 25 µg/ml monoclonal anti-integrin
For the cell migration assay, the MDCK cells were treated with 25 ng/ml
HGF for 12 h, collected by trypsinization, and suspended in
serum-free medium at 5 × 105 cells/ml with or without
25 µg/ml monoclonal anti-integrin Northern Hybridization--
The total cellular RNA from MDCK
cells that had been treated with or without 25 ng/ml HGF for 12 h
was extracted by TRIzol reagent (Life Technologies, Inc.) following the
manufacturer's instructions. To analyze the mRNA level of integrin
Immunofluorescence and Nuclear Staining--
MDCK cells
(104) were grown on glass coverslips in 24-well plates for
2 days to allow cell colony formation. After an 18-h serum starvation,
the cells were treated with HGF or EGF in serum-free medium for 15 min
or 6 h, fixed with 4% paraformaldehyde at room temperature for 20 min, and permeabilized for 1 h by several changes of PBS
containing 0.5% Triton X-100. For ERK staining, the cells were
incubated with polyclonal anti-ERK (1:200 dilution in PBS) for 1 h
at room temperature. After washed in PBS, the cells were incubated with
fluorescein isothiocyanate-conjugated secondary antibody for
1 h. For nuclear staining, the cells were incubated with 0.5 µg/ml Hoechst 33258 (Sigma) for 10 min at room temperature. Preparations were then washed in PBS, mounted in anti-fading solution (20 mM n-propyl gallate in 80% glycerol), and
analyzed by an immunofluorescent microscopy (Leica).
To characterize the induction of integrin 2 in Madin-Darby canine kidney (MDCK) cells. In
this study, we have further investigated the signal transduction
pathways responsible for the event and its role in HGF-induced cell
scattering. We found that the level of integrin
2
1 expression induced by HGF correlated
with the extent of cell scattering and that a functional blocking
antibody against integrin
2 at the concentration of 25 µg/ml partially (40%) inhibited the HGF-induced cell scattering.
However, in the presence of the specific phosphatidylinositol 3-kinase
inhibitor LY294002 or the selective Src family kinase inhibitor PP1,
although cells retained their response to HGF for increasing integrin
2 expression, they failed to scatter, indicating that
increased expression of integrin
2 alone is not
sufficient for cell scattering. Moreover, epidermal growth factor,
which induced a transient (1 h) activation of extracellular
signal-regulated kinase (ERK) in MDCK cells, only slightly increased
integrin
2 expression and failed to trigger cell
scattering. Conversely, HGF induced a sustained (at least 12 h)
activation of ERK in the cells. Expression of constitutively active ERK
kinase (MEK) in MDCK cells led to increased expression of integrin
2 even in the absence of HGF stimulation. In contrast,
expression of ERK phosphatase or dominant negative MEK inhibited
HGF-induced integrin
2 expression. Taken together, our
results suggest that the increased expression of integrin
2
1 by HGF is at least partially required
for cell scattering and that the duration of MEK/ERK activation is
likely to be a crucial determinant for cells to activate integrin
2 expression and cell scattering.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 is known to serve as the
major receptor for collagen on MDCK cells (16) and play a crucial role
in HGF-elicited cell motility and tubulogenesis (7, 17). We have
previously demonstrated that HGF selectively enhances the expression of
integrin
2 and to a lesser extent
3 in
MDCK cells (7). In this study, we have further investigated the signal
transduction pathways responsible for increased integrin
2 expression and its role in HGF-induced cell scattering.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 and the polyclonal anti-integrin
1
(AB1952) were purchased from Chemicon (Temecula, CA). The polyclonal anti-ERK (sc-94) was purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). The polyclonal anti-phosphoERK was purchased from New
England Biolabs, Inc. (Beverly, MA). The cDNA of human integrin
2 was purchased from the American Type Culture
Collection. The plasmids encoding constitutively active MEK1
(S218E/S222E), dominant negative MEK1 (S218A/S222A), and ERK
phosphatase HVH2 were kindly provided by Dr. Kun-Liang Guan (University
of Michigan, Ann Arbor, MI) and described previously (18, 19).
2, MDCK cells were seeded at
105/60-mm dish and allowed to grow as discrete colonies for
2 days. For HGF stimulation, the culture medium was replaced by fresh medium containing 5% serum and 25 ng/ml HGF. In some experiments, HGF
was substituted by EGF (100 ng/ml) or PMA (100 nM). After various times, the morphology of cell colonies were photographed under
a phase contrast microscope before cell lysis in 1% Nonidet P-40 lysis
buffer containing protease inhibitors as described previously (20). To
measure the phosphorylation of ERK, 105 MDCK cells were
plated on 60-mm dish in medium with 10% serum for 24 h and
followed by a 24-h serum starvation. These cells were treated with 25 ng/ml HGF or 100 ng/ml EGF in serum-free medium for various times
before cell lysis in 1% Nonidet P-40 lysis buffer containing protease inhibitors.
2 expression.
2.
2 (1:1500), anti-integrin
1 (1:1500), anti-ERK (1:1500), or anti-phosphoERK
(1:1500) using the PerkinElmer Life Sciences chemiluminescence system
for detection.
2 (clone
P1E6). 12 h later, cells were washed in PBS, fixed in methanol for
10 min, and stained by modified Giemsa stain (Sigma) for 1 h. The percentage of scattered cells was measured in the total number of
counted (~1000) cells from 50 colonies under a light microscope. A
cell was judged as a scattered cell when it has lost contact with its
neighbors and exhibited a fibroblast-like phenotype.
2 (P1E6) antibody.
The cells were incubated with the antibody at 37 °C for 30 min
before loading them to a Neuro Probe 48-well chemotaxis chamber (Cabin
John, MD). Cells in the upper chamber were allowed to migrate through a
porous (8-µm pore size) membrane toward the lower chamber containing
10 µg/ml collagen as an attractant for 5 h. The migrated cells
were fixed, stained, and enumerated as described previously
(21).
2 in MDCK cells, an equal amount (25 µg) of total
cellular RNA was fractionated through 1% agarose gels containing 2.2 M formaldehyde and transferred to nitrocellulose membrane
(Schleicher & Schuell). A fluorescein-labeled DNA probe from human
integrin
2 was prepared using Renaissance random primer
fluorescein labeling kit (PerkinElmer Life Sciences). The hybridization
was carried out at 55 °C. After several washes, the membrane was
incubated with peroxidase-conjugated anti-fluorescein (1:1000) at room
temperature for 1 h, and the PerkinElmer Life Sciences
chemiluminescence system was used for detection.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
expression by HGF, MDCK cells were allowed to grow as discrete colonies
and then exposed to 25 ng/ml HGF for various times before harvest.
Coincident with the scatter of cell colonies (Fig.
1B), the levels of integrins
2 and
1 were gradually increased and
reached 3.9- and 3.3-fold, respectively, 24 h after HGF
stimulation (Fig. 1A). In contrast, the level of focal
adhesion kinase, a key intracellular mediator in integrin signaling,
was not affected by HGF stimulation. To measure the level of integrin
2 on cell surface, cell surface proteins were
biotinylated, precipitated by avidin-immobilized agarose beads, and
immunoblotted with anti-integrin
2 (Fig. 1C). Consistent with its increase in whole cell lysates, integrin
2 on cell surface was also increased by HGF stimulation
(25 ng/ml) and remained constant for 48 h. However, HGF at a low
concentration (5 ng/ml) appeared to fail to promote the expression of
integrin
2 on the cell surface. To examine whether the
HGF induction of integrin
2 expression is regulated at
transcriptional level, total RNA extracted from MDCK cells was analyzed
by Northern hybridization using human integrin
2
cDNA as a probe (Fig. 1D). The result showed that a 12-h
treatment of HGF apparently (~3-fold) increased the level of integrin
2 transcripts, indicating that the promoter activation
of the integrin
2 gene is involved in its increased expression induced by HGF.
View larger version (57K):
[in a new window]
Fig. 1.
Induction of integrin
2
1
expression and cell scattering by HGF. A, MDCK cells
were allowed to grow as colonies and incubated with 25 ng/ml HGF for
various times as indicated. Whole cell lysates were prepared and
analyzed by immunoblotting with anti-integrin
2,
anti-integrin
1, or anti-focal adhesion kinase
(FAK). Quantification of protein expression was carried out
by a densitometer, which is expressed as -fold increase relative to the
level of cells without HGF treatment. B, the scatter of MDCK
cell colonies upon 25 ng/ml HGF stimulation was chased under the same
microscopic field and photographed at the indicated times.
C, MDCK cells were incubated with 25 ng/ml HGF for various
times or incubated with HGF at various concentrations for 24 h and
then subjected to cell surface biotinylation, as described under
"Experimental Procedures." Cell lysates were prepared and incubated
with avidin-immobilized agarose beads. Integrin
2 in
avidin-precipitated complexes was detected by immunoblotting with
anti-integrin
2. Values are expressed as -fold increase
relative to the level of cells without HGF stimulation. D,
MDCK cell colonies were incubated with (+) or without (
) 25 ng/ml HGF
for 12 h. Total cellular RNA was extracted and analyzed by
Northern hybridization using human integrin
2 cDNA
as a probe. Ethidium bromide-stained agarose gel was shown to indicate
an equal loading of total RNA. The positions of the 28 and 18 S
ribosomal RNAs are indicated. The values are expressed as -fold
increase relative to the level of cells without HGF stimulation.
To examine the role of de novo synthesized integrin
2
1 in HGF-induced cell scattering, a
functional blocking antibody against integrin
2 was
applied in cell scatter assays (Fig.
2A). We quantified the extent
of cell scattering by counting the number of cells that have lost
contact with their neighbors in a total of 50 colonies containing
~1000 cells. Our results indicated that the HGF-induced cell
scattering was partially (~40%) inhibited by the integrin
2 blocking antibody at the concentration of 25 µg/ml.
However, at the same concentration, this antibody was able to
efficiently inhibit HGF-induced cell migration toward collagen in a
chemotaxis chamber (Fig. 2B), indicating a successful
inhibition of integrin
2 function by this antibody.
Thus, these results indicated that increased expression of integrin
2 induced by HGF contributes only partially to cell
scattering.
|
PMA, a strong activator for conventional and novel subgroups of protein
kinase C (PKC), has previously been shown to induce integrin
2 expression in osteosarcoma cells (22) and breast cancer cells (23). To examine whether PKC mediates the effect of HGF on
integrin
2 induction and cell scattering, MDCK cells were treated with HGF or PMA in the presence of a broad range PKC
inhibitor, bisindolylmaleimide. As shown in Fig.
3A, the effect of PMA on
promoting both integrin
2 expression and cell scattering was significantly inhibited by bisindolylmaleimide, supporting an
essential role for PKC in PMA-induced cellular effects. In contrast,
bisindolylmaleimide did not appear to inhibit HGF-induced cell
scattering and only modestly (~25%) decreased the level of integrin
2 induced by HGF, indicating that the effect of HGF on
promoting integrin
2 expression and cell scattering is
mainly PKC-independent.
|
To identify signal transduction pathways required for HGF to induce
integrin 2 expression, inhibitors including genistein (a
general tyrosine kinase inhibitor), PP1 (a selective Src family kinase
inhibitor), PD98059 (a specific MEK inhibitor), LY294002 (a specific
PI3K inhibitor), and cycloheximide (a translation inhibitor) were
applied to the experiments (Fig. 3B). Of these inhibitors,
genistein, PD98059, and cycloheximide were found to efficiently inhibit
the effect of HGF on integrin
2 induction. In contrast,
PP1 and LY294002 had no such effect. These results indicate that MEK,
but not Src or PI3K, is required for HGF to induce integrin
2 expression. It should be noted that in the presence of
PP1 or LY294002, although cells retained their response to HGF for
increasing integrin
2 expression, they failed to
scatter, indicating that the elevated expression of integrin
2 alone is not sufficient for cell scattering.
As suggested in Fig. 3, the HGF induction of integrin 2
expression is mainly dependent on the MEK pathway. However, in addition to HGF, many other growth factors such as EGF are known to potently activate MEK and its downstream targets, ERK 1 and 2. To examine whether EGF induces integrin
2 expression and/or cell
scattering, MDCK cells were incubated with EGF or HGF for 24 h. As
shown in Fig. 4A, although EGF
induced a slight increase in integrin
2 expression and
lamellipodium formation around cell colony, it failed to trigger cell
scattering. Next, we measured the phosphorylation of ERK upon HGF or
EGF stimulation, which serves as an indicator for ERK activation (Fig.
4B). The phosphorylation of ERK induced by EGF was
transient; it declined by 30 min and returned to the basal level
by 1 h post-stimulation. In contrast, HGF induced a sustained
phosphorylation of ERK, which lasted for at least 12 h
post-stimulation. Furthermore, the sustained phosphorylation of ERK
induced by HGF coincided with its persistent nuclear accumulation (Fig.
4C).
|
To further examine whether sustained activation of ERK leads to
integrin 2 expression, constitutively active MEK was
expressed in MDCK cells (Fig.
5A). As expected, the
expression of constitutively active MEK resulted in ERK activation, and
importantly, even in the absence of HGF stimulation, it enhanced the
expression of integrin
2. Conversely, the expression of
an ERK phosphatase HVH2 or a dominant negative MEK suppressed both
HGF-induced ERK activation and integrin
2 expression
(Fig. 5B). These results together suggest that sustained
activation of the MEK/ERK signal cascade may be required and sufficient
for induction of integrin
2 expression.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have used MDCK cells as a model to investigate
the role of up-regulation of integrin 2
1
in HGF-induced cell scattering and the signal transduction pathways
responsible for this up-regulation. We showed that the level of
integrin
2
1 expression increased by HGF
stimulation correlated with the extent of cell scattering (Fig. 1) and
that block of HGF-induced integrin
2 expression by
inhibitors including genistein, PD98059, and cycloheximide all
accompanied an inhibition in cell scattering (Fig. 3B).
However, only 40% of HGF-induced cell scattering could be inhibited by
the functional blocking antibody against integrin
2
(Fig. 2A), suggesting that increased integrin
2
1 may contribute only partially to cell
scattering likely through promoting the third phase of cell scattering
(i.e. cell migration). In addition, our results presented
here also suggest that increased expression of integrin
2 alone may not be sufficient for MDCK cells to scatter. We showed in Fig. 3B that in the presence of PP1 or
LY294002, although cells retained their response to HGF for increasing
integrin
2 expression, they failed to scatter.
Although the ERK signaling pathway has been implicated to be essential
for HGF-induced cell scattering (13-15), here we propose for the first
time that the duration of MEK/ERK activation is likely to be the major
determinant for cells to activate integrin 2 expression
and cell scattering upon HGF stimulation. Treatment of MDCK cells with
HGF led to prolonged activation of ERK as judged by the phosphorylation
of ERK and its accumulation in the nucleus (Fig. 4). In contrast, EGF,
which does not cause MDCK cells to scatter, led to transient activation
of ERK. Moreover, the expression of constitutively active MEK, which
led to sustained activation of ERK, increased the expression of
integrin
2 even in the absence of HGF stimulation (Fig.
5A). In contrast, the expression of ERK phosphatase or
dominant negative MEK blocked HGF-induced integrin
2
expression (Fig. 5B). Together, these data support a model in which the different consequences of transient versus
sustained activation of ERK are because sustained activation leads to
persistent nuclear accumulation of ERK, resulting in phosphorylation of
transcription factors and changes in gene expression.
We showed in Fig. 1 that the increased expression of integrin
2 by HGF in MDCK cells was a consequence of increased
2 mRNA likely because of transcriptional activation
of the integrin
2 gene. The mechanism of transcriptional
regulation of the canine integrin
2 gene by HGF is
currently unknown. However, characterization of the 5'-flanking region
of the human integrin
2 gene revealed that the promoter
region contains consensus binding sites for several transcription
factors, including Sp1, AP1, and AP2 (24). The binding of
phosphorylated Sp1 proteins to two Sp1-binding sites in the core
promoter region of the human integrin
2 gene has been
shown to be required for full promoter activity (25). It is known that
ERK activation increases AP1 activity via c-Jun activation and
increased c-Fos synthesis, leading to an increase in c-Jun/c-Fos
heterodimerization and DNA binding (26, 27). In addition, a recent
study showed that ERK was able to phosphorylate Sp1 and induced its DNA
binding activity (28). Therefore, it is possible that persistent
nuclear accumulation of ERK induced by HGF stimulation may target Sp1
and AP1, leading to activation of the integrin
2 gene.
Furthermore, the MEK inhibitor PD98059 inhibits the up-regulation of
integrin
2 expression induced by PMA in MDCK cells (data
not shown) or by phorbol dibutyrate in human leukemia K562 cells (29),
suggesting that the MEK/ERK cascade may be generally required for
extracellular stimuli to activate the integrin
2 gene.
Although the role of the ERK singling pathway in cell proliferation has
been well established (30, 31), increasing evidence has suggested that
the duration of ERK activation may be a crucial determinant for cells
to proceed certain cellular functions other than proliferation. For
example, sustained ERK activity has been shown to be required for nerve
growth factor-induced neuronal differentiation of the phaeochromocytoma
PC12 cells (32), fibroblast growth factor-induced angiogenesis of the
endothelial cells on chick chorioallantoic membrane (33), and
PMA-induced macrophage-like differentiation of the myeloid leukemia
TF-1a cells (34). In this study, we demonstrate that HGF-mediated
sustained activation of ERK is required for MDCK cell scattering,
associated with morphological transformation to a mesenchymal cell
phenotype. The mechanism by which MDCK cells maintain ERK in an active
status for a long period upon HGF stimulation is currently unclear.
York et al. (32) showed that in PC12 cells, the early phase
of nerve growth factor-stimulated ERK activation is mediated by the
small GTPase Ras, but the sustained phase of the ERK activation is due
to the activation of another small GTPase, Rap1. Thus, it will be of interest to examine whether Rap1 is involved in HGF-induced sustained activation of ERK. Alternatively, the de novo expression of
integrin 2
1 stimulated by HGF may
contribute to the sustained phase of ERK activation. It is known that
integrin-mediated cell adhesion to extracellular matrix proteins
activates the ERK cascade and often potentiates the effect of growth
factors on activating the cascade (35). Eliceiri et al. (33)
showed that the sustained phase but not the initial phase of ERK
activation by the fibroblast growth factor depends on integrin
v
3 in endothelial cells. The potential
role of de novo synthesized integrin
2
1 in HGF-induced sustained activation of
ERK cascade is currently under investigation.
It is known that in cells transformed by oncogenic Ras or Raf, the
relative ability of external stimuli to activate ERK is usually
diminished. However, we showed in this study that the expression of
constitutively active MEK in MDCK cells weakly activated ERK compared
with HGF and did not negatively impact on subsequent activation of ERK
by HGF (Fig. 5A). The explanation for this discrepancy could
be simply the low expression of constitutively active MEK in our
selected MDCK cells, in which a fraction of ERK remains inactive and
susceptible to HGF stimulation. This assumption was based on our
observation that consecutive culture of active MEK-expressed MDCK
cells, especially those with higher expression level of it, resulted in
apoptosis.2 These results
suggest that severely aberrant activation of ERK by constitutively
active MEK may be cytotoxic to MDCK cells and that only those with a
lower expression level of active MEK can survive. Alternatively, it is
possible that in addition to the Ras/Raf/MEK pathway, other signal
transduction pathways may collaborate to activate ERK upon HGF
stimulation. For example, ERK has been shown to be activated by protein
kinase C and
via Ras-independent pathways (36, 37) and
inactivated by protein phosphatases such as PP2A (38) and ERK
phosphatases (39) in other systems. Therefore, it is plausible that
activation of protein kinase C and/or inactivation of ERK/MEK
phosphatases by HGF may further activate ERK in MDCK cells expressing
constitutively active MEK. Recently, the protein-tyrosine phosphatase
SHP-2 was shown to be required for HGF-induced activation of ERK in
MDCK cells (40), although the detailed mechanism is currently unclear.
In this study, we have found that PMA was able to induce the E-M
transition similar to that induced by HGF (Fig. 3). However, unlike
HGF, the effect of PMA on induction of the E-M transition is mainly
PKC-dependent (Fig. 3). In addition, stable expression of
constitutively active MEK in MDCK cells has been shown to lead to the
E-M transition (41), suggesting that sustained activation of ERK
signaling cascade may be sufficient for triggering this process. Zondag
et al. (12) reported recently that expression of oncogenic
Ras leads to the E-M transition of MDCK cells through regulating the
balance between the activation state of two GTPase Rho family proteins,
Rac and Rho. They showed that oncogenic Ras-mediated sustained ERK
signaling decreases Rac activity through transcriptional down-regulation of the Rac-specific exchange factor Tiam1 and, by
contrast, increases Rho activity through an unknown mechanism. It
remains to be tested whether HGF, PMA, and constitutive active MEK act
in the same fashion as oncogenic Ras to induce the E-M transition.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. K.-L. Guan for the plasmid encoding constitutively active MEK, dominant negative MEK, and phosphatase HVH2.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Council, Taiwan Grant NSC90-2311-B-005-063 (to H.-C. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
886-4-22854922; Fax: 886-4-22851797; E-mail: hcchen@nchu.edu.tw.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M010669200
2 C.-C. Liang and H.-C. Chen, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HGF, hepatocyte growth factor; E-M, epithelial-mesenchymal; MDCK, Madin-Darby canine kidney; PI3K, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; MEK, ERK kinase; EGF, epidermal growth factor; PMA, phorbol-12-myristate-13-acetate; PBS, phosphate-buffered saline; PKC, protein kinase C.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Zarnegar, R., and Michalopoulos, G. K. (1995) J. Cell Biol. 129, 1177-1180[Medline] [Order article via Infotrieve] |
2. | Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A. M.-L., Kmiecik, T. E., Vande Woulde, G. F., and Aaronson, S. A. (1991) Science 251, 802-804[Medline] [Order article via Infotrieve] |
3. | Naldini, L., Vigna, E., Ferracini, R., Longati, P., Gandino, L., Prat, M., and Comoglio, P. M. (1991) Mol. Cell. Biol. 11, 1793-1803[Medline] [Order article via Infotrieve] |
4. | Ridley, A. J., Comoglio, P. M., and Hall, A. (1995) Mol. Cell. Biol. 15, 1110-1122[Abstract] |
5. |
Royal, I.,
and Park, M.
(1995)
J. Biol. Chem.
270,
27780-27787 |
6. |
Sander, E. E.,
van Delft, S.,
ten Klooster, J. P.,
Reid, T.,
van der Kammen, R. A.,
Michiels, F.,
and Collard, J. G.
(1998)
J. Cell Biol.
143,
1385-1398 |
7. |
Lai, J.-F.,
Kao, S.-C.,
Jiang, S.-T.,
Tang, M.-J.,
Chan, P.-C.,
and Chen, H.-C.
(2000)
J. Biol. Chem.
275,
7474-7480 |
8. |
Hartmann, G.,
Weidner, K. M.,
Schwarz, H.,
and Birchmeier, W.
(1994)
J. Biol. Chem.
269,
21936-21939 |
9. | Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J., Waterfield, M. D., and Downward, J. (1994) Nature 370, 527-532[CrossRef][Medline] [Order article via Infotrieve] |
10. | Hu, Q., Klippel, A., Muslin, A. J., Fantl, W. J., and Williams, L. T. (1995) Science 268, 100-102[Medline] [Order article via Infotrieve] |
11. |
Potempa, S.,
and Ridley, A. J.
(1998)
Mol. Biol. Cell
9,
2185-2200 |
12. |
Zondag, G. C. M.,
Evers, E. E.,
ten Klooster, J. P.,
Janssen, L.,
van der Kammen, R. A.,
and Collard, J. G.
(2000)
J. Cell Biol.
149,
775-781 |
13. | Terauchi, R., and Kitamura, N. (2000) Exp. Cell Res. 256, 411-422[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Khwaja, A.,
Lehmann, K.,
Marte, B. M.,
and Downward, J.
(1998)
J. Biol. Chem.
273,
18793-18801 |
15. |
Herrera, R.
(1998)
J. Cell Sci.
111,
1039-1049 |
16. |
Schoenenberger, C.-A.,
Zuk, A.,
Zinkl, G. M.,
Kendall, D.,
and Matlin, K. S.
(1994)
J. Cell Sci.
107,
527-541 |
17. |
Saelman, E. U.,
Keely, P. J.,
and Santoro, S. A.
(1995)
J. Cell Sci.
108,
3531-3540 |
18. |
Sugimoto, T.,
Stewart, S.,
Han, M.,
and Guan, K.-L.
(1998)
EMBO J.
17,
1717-1727 |
19. | Guan, K.-L., and Butch, E. (1995) J. Biol. Chem. 270, 7191-7203 |
20. |
Chen, H.-C.,
Chan, P.-C.,
Tang, M.-J.,
Cheng, C.-H.,
and Chang, T.-J.
(1998)
J. Biol. Chem.
273,
25777-25782 |
21. |
Reiske, H. R.,
Kao, S.-C.,
Cary, L. A.,
Guan, J.-L.,
Lai, J.-F.,
and Chen, H.-C.
(1999)
J. Biol. Chem.
274,
12361-12366 |
22. | Nissinen, L., Westermarck, J., Koivisto, L., Kahari, V.-M., and Heino, J. (1998) Exp. Cell Res. 243, 1-10[CrossRef][Medline] [Order article via Infotrieve] |
23. | Rosfjord, E. C., Maemura, M., Johnson, M. D., Torri, J. A., Akiyama, S. K., Woods, V. L., and Dickson, R. B. (1999) Exp. Cell Res. 248, 260-271[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Zutter, M. M.,
Santoro, S. A.,
Painter, A. S.,
Tsung, Y. L.,
and Gafford, A.
(1994)
J. Biol. Chem.
269,
463-469 |
25. |
Zutter, M. M.,
Ryan, E. E.,
and Painter, A. D.
(1997)
Blood
90,
678-689 |
26. | Angel, P., Hattori, K., Smeal, T., and Karin, M. (1988) Cell 55, 875-885[Medline] [Order article via Infotrieve] |
27. | Chiu, R., Boyle, W. J., Meek, J., Smeal, T., Hunter, T., and Karin, M. (1988) Cell 54, 541-552[Medline] [Order article via Infotrieve] |
28. | Merchant, J. L., Du, M., and Todisco, A. (1999) Biochem. Biophys. Res. Commun. 254, 454-461[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Zutter, M. M.,
Painter, A. D.,
and Yang, X.
(1999)
Blood
93,
1600-1611 |
30. |
Pages, G.,
Lenormand, P.,
Allemain, G. L.,
Chambard, J. C.,
Meloche, S.,
and Pouyssegur, J.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8319-8323 |
31. |
Seger, R.,
and Krebs, E. G.
(1995)
FASEB J.
9,
726-735 |
32. | York, R. D., Yao, H., Dillon, T., Ellig, C. L., Eckert, S. P., McCleskey, E. W., and Stork, P. J. S. (1998) Nature 392, 622-626[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Eliceiri, B.,
Klemke, R.,
Stromblad, S.,
and Cheresh, D. A.
(1998)
J. Cell Biol.
140,
1255-1263 |
34. |
Hu, X.,
Moscinski, L. C.,
Valkov, N. I.,
Fisher, A. B.,
Hill, B. J.,
and Zuckerman, K. S.
(2000)
Cell Growth Differ.
11,
191-200 |
35. |
Howe, A. K.,
and Juliano, R. L.
(1998)
J. Biol. Chem.
273,
27268-27274 |
36. |
Ueda, Y.,
Hirai, S.,
Osada, S.,
Suzuki, A.,
Mizuno, K.,
and Ohno, S.
(1996)
J. Biol. Chem.
271,
23512-23519 |
37. |
Takeda, H.,
Matozaki, T.,
Takada, T.,
Noguchi, T.,
Yamao, T.,
Tsuda, M.,
Ochi, F.,
Fukunaga, K.,
Inagaki, K.,
and Kasuga, M.
(1999)
EMBO J.
18,
386-395 |
38. | Alessi, D. R., Gomez, N., Moorhead, G., Lewis, T., Keyse, S. M., and Cohen, P. (1995) Curr. Biol. 5, 283-295[Medline] [Order article via Infotrieve] |
39. |
Muda, M.,
Boschert, U.,
Dickinson, R.,
Martinou, J.-C.,
Martinou, I.,
Camps, M.,
Schlegel, W.,
and Arkinstall, S.
(1996)
J. Biol. Chem.
271,
4319-4326 |
40. |
Maroun, C. R.,
Naujokas, M. A.,
Holgado-Madruga, M.,
Wong, A. J.,
and Park, M.
(2000)
Mol. Cell. Biol.
20,
8513-8525 |
41. |
Schramek, H.,
Feifel, E.,
Healy, E.,
and Pollack, V.
(1997)
J. Biol. Chem.
272,
11426-11433 |