From the School of Medicine, Yale University, New
Haven, Connecticut 06520,
ACLARA Biosciences, Mountain View,
California 94043, and ** Beth Israel Deaconess Medical Center, Boston,
Massachusetts 02215
Received for publication, November 1, 2000
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ABSTRACT |
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Cells derived from the inner medullary
collecting duct undergo in vitro branching tubulogenesis to
both the c-met receptor ligand hepatocyte growth factor (HGF) as well
as epidermal growth factor (EGF) receptor ligands. In contrast, many
other cultured renal epithelial cells respond in this manner only to
HGF, suggesting that these two receptors may use independent signaling
pathways during morphogenesis. We have therefore compared the signaling pathways for mIMCD-3 cell morphogenesis in response to EGF and HGF.
Inhibition of the p42/44 mitogen-activated protein kinase (MAPK)
pathway with the mitogen-activated protein kinase kinase (MKK1)
inhibitor PD98059 (50 µM) markedly inhibits HGF-induced cell migration with only partial inhibition of EGF-induced cell motility. Similarly, HGF-dependent, but not
EGF-dependent, branching morphogenesis was more greatly
inhibited by the MKK1 inhibitor. Examination of EGF-stimulated cells
demonstrated that extracellular-regulated kinase 5 (ERK5) was activated
in response to EGF but not HGF, and that activation of ERK5 was only
60% inhibited by 50 µM PD98059. In contrast, the MKK
inhibitor U0126 markedly inhibited both ERK1/2 and ERK5 activation and
completely prevented HGF- and EGF-dependent migration and
branching process formation. Expression of dominant negative ERK5
(dnBMK1) likewise inhibited EGF-dependent branching process
formation, but did not affect HGF-dependent branching process formation. Our results indicate that activation of the ERK1/ERK2 signaling pathway is critical for HGF-induced cell
motility/morphogenesis in mIMCD-3 cells, whereas ERK5 appears to be
required for EGF-dependent morphogenesis.
In renal epithelial cells, cell morphogenesis is an important
process in embryonic development and repair of injured tubules. Two
cell types commonly utilized to examine renal epithelial morphogenesis are murine mIMCD-3 collecting duct cells and canine MDCK tubular cells.
In mIMCD-3 cells, both the c-met receptor ligand hepatocyte growth
factor (HGF)1 and the
epidermal growth factor receptor (EGFR) ligands, transforming growth
factor- Studies examining the importance of MAPK activation in cell motility in
mammalian cells have had conflicting results. Some researchers conclude
that cell motility involves only pathways independent of the MAPK
cascade (13-16), whereas other studies indicate that MAPK activity is
required for cell motility (17-20). This discrepancy may be due to
differences in the cell types studied and/or growth factors utilized.
For example, Anand-Apte et al. (21) examined PDGF- and
fibronectin-stimulated migration in fibroblasts and determined that
they were differentially regulated by Rac and MAPK. Their results
indicated that the fibronectin pathway requires ERK1/2 activity for
cell motility but the PDGF pathway does not. They concluded that
different receptor classes in response to different ligands
differentially utilize Rac and ERK pathways in cell migration.
There have been few studies to date examining the role of MAPK
activation specifically in HGF- and/or EGF-dependent cell
migration and tubulogenesis. One study of MAPK signaling in EGF-induced cell motility argues that MAPK activation may not be critical for this
response in fibroblasts (22). These investigators found that two
truncation mutants of the EGFR (lacking the tyrosine residues
C-terminal to the kinase domain), which activate ERK1/2 do not support
normal EGF-dependent cell migration, suggesting that
activation of the MAPK pathway is not sufficient for cell migration. In
a three-dimensional assay, Wang et al. (23) found that T4-2
breast carcinoma cells expressing abnormally high levels of the EGFR
had an exaggerated MAPK response to EGF associated with an overly
proliferative, invasive morphology (23). This abnormal morphogenesis
was completely suppressed by the MEK inhibitor PD98059. In contrast,
Polk et al. (24) found no effect of MAPK inhibition on
EGF-induced epithelial cell motility. An investigation of the role of
MAPK signaling in HGF-stimulated MDCK cells recently demonstrated that
the scattering response (a phenotype unique to HGF/c-met, which
requires dissolution of existing cell-cell interactions followed by
random cell migration) and tubulogenesis (a prolonged assay dependent
on both cell morphogenesis and division) was prevented by the MEK
inhibitor PD98059, suggesting that MAPK activation plays an important
role in morphogenesis in these cells (25).
One possible explanation for these conflicting results is that
activation of MAPK may be a ubiquitous requirement for cell morphogenesis, but that different growth factor receptors utilize different MAPK pathways. For example, it has been recently demonstrated that the EGFR utilizes ERK5 (also called Big MAPK (BMK1)) for mitogenic
signaling rather than ERK1/2 (26). In the present study, we utilized
mIMCD-3 cells to compare signaling pathways downstream of the c-met and
EGFR in cell migration and early branching morphogenesis assays. Both
receptors were found to require activation of the PI3K and PLC Cell Culture--
Immortalized mIMCD-3 (27) cells were grown in
DMEM/F12 media supplemented with 10% fetal calf serum. The response of
these cells to HGF and EGF has been extensively studied in both cell migration assays and three-dimensional matrix assays (1, 8, 11, 28).
All chemicals were purchased from Sigma Chemical Co. unless otherwise noted.
Protein Analysis--
Subconfluent mIMCD-3 cells were
serum-starved for 24 h prior to stimulation with either HGF (40 ng/ml, Sigma) or EGF (20 ng/ml, Upstate Biotechnology Inc.) for 10 min
at 37 °C. The cells were then washed twice with ice-cold
phosphate-buffered saline, scraped in ice-cold 0.5% Igepal lysis
buffer (137 mM NaCl, 20 mM Trizma base, 1 mM MgCl, 1 mM CaCl, 1 mM sodium
orthovanadate, 10% glycerol, 0.5% Igepal CA-630, 1 mM
phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.5 µg/ml
pepstatin A, and 25 µg/ml antipain) and vortexed vigorously.
Nonsolubilized debris was removed by microcentrifugation at 13,000 × g for 10 min at 4 °C, and the supernatant was
collected. In the MEK inhibitor experiments, the cells were
preincubated with the appropriate concentration of PD98059 (Calbiochem)
or U0126 (Promega) for 20 min prior to stimulation with growth factor. Protein content was determined using the Bio-Rad assay, and 40 µg of
each supernatant was resolved by SDS-polyacrylamide gel electrophoresis
and transferred to Immobilon (Millipore). Activated ERK1 and ERK2 were
probed using the antibody to phospho-MAPK (New England BioLabs) at a
1:1000 dilution and a chemiluminescence detection system (ECL, Amersham
Pharmacia Biotech). ERK5 was detected in mIMCD-3 cells by cell lysis as
described above followed by immunoprecipitation with anti-ERK5 antibody
(StressGen, Canada). To determine the phosphorylation state of ERK5,
anti-ERK5 immunoprecipitates were resolved on 7.5% SDS-PAGE and
immunoblotted with either the ERK5 antibody (to detect
phosphorylation-dependent gel mobility retardation) or an
antibody specific for MEK5-phosphorylated ERK5 (BIOSOURCE International, Camarillo, CA).
Kinase Assay--
For kinase assays of endogenous ERK5, near
confluent mIMCD-3 cells were serum-starved for 48 h and then
treated with or without stimuli (HGF, 40 ng/ml, or EGF, 20 ng/ml, for
10 min), in the presence or absence of 50 µM PD98059 or
20 µM UO126. Cells were then washed once in ice-cold
phosphate-buffered saline, collected in 500 µl of lysis buffer (20 mM Tris-Cl (pH 7.5), 5 mM EGTA, 25 mM Cell Migration--
Cell migration assays were performed using a
modified Boyden chamber assay as previously described (8). Briefly, a
48-well bottom plate (Neuro Probe, Cabin John, MD) was filled
with DMEM/F12 media containing the appropriate growth factor. The well
was overlaid with a rat tail collagen type I (Collaborative
Biomedical)-coated polycarbonate filter with 8-µm pores (Nucleopore).
The top compartment was connected, and 1.5 × 104
cells suspended in DMEM/F12 were added to the top of each well. After
4 h of incubation at 37 °C, the filters were removed and stained with Diff-Quik (Baxter Healthcare), and the cells remaining on
the top of the membrane mechanically were scraped off. Cells that had
passed through the pores were counted to determine the number of
cells/mm2 of membrane. Each well represents an n
value of 1, and each experiment was repeated at least three separate
times. p values were determined using an unpaired
Student's t test.
For experiments using signaling inhibitors, cells were trypsinized and
counted as before, then separated into vehicle control and inhibitor
aliquots. Cells were then rocked in suspension in the presence of the
appropriate inhibitor for 10 min (50 µM PD98059, 20 µM U0126, 100 nM wortmannin (Sigma), or 1 µM U73122 (Calbiochem)) prior to loading on the
collagen-coated membrane. Inhibitor at the appropriate concentration
was also included in the bottom well of the migration assay. The
inhibitor doses for wortmannin and U73122 were chosen based on our
previous experience with these inhibitors in mIMCD-3 cells (9).
Branching Morphogenesis--
mIMCD-3 cells were trypsinized, and
isolated cells were resuspended in type I collagen and cultured in the
presence or absence of the desired growth factor as previously
described (29). After a 24-h period of incubation at 37 °C 30-40
single cells were scored for the number of branching tubular processes.
Quantitation of branching process formation was performed by counting
40 random individual cells and dividing the number of branches by the
total number of cells counted. Each well represents an n
value of 1, and each experiment was repeated at least three separate times.
Expression of ERK5 in mIMCD-3 Cells--
pcDNA3 expression
vectors (Invitrogen, San Diego, CA) encoding for wild-type or dominant
negative BMK1 (ERK5) were kindly provided by Dr. J. D. Lee.
Dominant negative BMK1 contains a threonine to alanine substitution at
position 218 and a tyrosine to phenylalanine mutation at position 220 in the MEK5 dual phosphorylation site (30). mIMCD-3 cells were
transfected with 4 µg of the appropriate ERK5-expressing plasmid and
1 µg of pEGFP (CLONTECH) in 90-mm plastic dishes
using LipofectAMINE Plus as previously described (29). 24 h
following transfection, cells were trypsinized and branching
morphogenesis assays performed as described above. Cells transiently
expressing the appropriate ERK5 construct or the empty pCDNA3
vector were detected and counted as described above using inverted
fluorescence microscopy to identify only GFP-positive cells. The 60×
confocal images shown in Fig. 5A were acquired with a
Bio-Rad MRC1024ES attached to a immunofluorescence microscope
Inhibition of ERK1/2 Activation Differentially Inhibits HGF- and
EGF-dependent Cell Migration--
To determine whether
HGF- and EGF-dependent morphogenesis required activation of
the extracellular signal-regulated kinase (ERK) pathway, we utilized
the MEK1 inhibitor PD98059 in both cell migration and branching
morphogenesis assays. A dose-response curve for inhibition of ERK1/2
phosphorylation was performed in the presence of either HGF or EGF
stimulation (Fig. 1A). At 50 µM, PD98059 inhibited HGF-induced phosphorylation of ERK2
by 100% (the small degree of basal ERK2 phosphorylation seen in these cells was inhibited as well) and EGF-induced ERK2 phosphorylation by
92%. This dose was then chosen for further experiments. In later
experiments, the more potent MEK1/2 inhibitor U0126 was utilized (31).
A similar dose-response curve demonstrated 100% inhibition of ERK1/2
phosphorylation at a dose between 10 and 50 µM U0126
(Fig. 1B). Based on this result, 20 µM U0126
was used in subsequent experiments.
To examine the importance of ERK1/2 activation in the cell migratory
response to HGF and EGF, a modified Boyden chamber assay was performed.
Inhibition of the p42/44 ERK pathway with 50 µM PD98059
blocked HGF-induced cell migration by 75% (Fig.
2A) while causing only 37%
inhibition of EGF-induced cell migration (control: 10.2 ± 1.3 cells/mm2; 50 µM PD98059: 20.2 ± 2.4;
HGF: 114.9 ± 8.5; HGF + 50 µM PD98059: 36.0 ± 4.2; EGF: 228 ± 12.5; EGF + 50 µM PD98059:
145.7 ± 5). Of note, in different experiments, the inhibitory
effect of PD98059 on EGF-dependent cell migration varied
between 0 and 50% depending on the supplier.
In prior studies, we and others have demonstrated that activation of
the phosphoinositide 3-kinase (PI3K) and phospholipase C EGF Activates ERK5 in mIMCD-3 Cells--
The recent observation
that activation of the EGFR results in activation of ERK5 and that ERK5
plays a critical role in EGF-dependent cell proliferation
(26) led us to test whether EGF activates ERK5 in mIMCD-3 cells and
whether this alternate MAPK member might be important in
EGF-dependent cell migration. The activation of ERK5 occurs
following MEK5 phosphorylation of ERK5 on both threonine and tyrosine,
similar to that seen for MEK1 phosphorylation of ERK1/2 (30, 32, 33).
To investigate the ability of either HGF and/or EGF to activate ERK5,
we initially utilized a gel shift assay. We detected a marked gel shift
of a fraction of the immunoprecipitated ERK5 following EGF but not HGF
stimulation (Fig. 3A). This
reduced gel mobility is consistent with the classically described
decrease in gel mobility of ERK1/2 following its threonine/tyrosine
dual site phosphorylation by MEK1 and with the results of Kato
et al. (26) examining ERK5 activation by EGF. With prolonged
exposure of the immunoblot, we could observe a modest level of
phosphorylated ERK5 detected in the HGF-stimulated cells (data not
shown). The EGF-induced gel shift was not prevented by pretreatment
with 50 µM PD98059 but was completely inhibited by the
more potent MEK inhibitor U0126. We further investigated this
differential activation of ERK5 by HGF and EGF using an antibody
specific for the threonine-tyrosine dual phosphorylation site on ERK5
and detected phospho-ERK5 in mIMCD-3 lysates following EGF stimulation
that was completely inhibited by U0126 and partially inhibited by
PD98059 (Fig. 3B). In contrast, HGF failed to stimulate ERK5
phosphorylation (Fig. 3B).
Examination of ERK5 kinase activity revealed a significant activation
of ERK5 by EGF and not by HGF (Fig. 3C). When the data from
four kinase assays was quantified, we observed a 3-fold increase in
ERK5 activation by EGF and no significant activation by HGF (Fig.
3D). EGF-induced activation was 62% inhibited by PD98059, whereas UO126 blocked EGF-dependent activation of ERK5 by
97%. As noted for the effects on cell migration, inhibition of ERK5 activation by PD98059 varied greatly in these assays. Thus, ERK1 and -2 were activated by both HGF and EGF, whereas only EGF significantly activates ERK5. Furthermore, U0126 markedly inhibits the activation of
both MAPK signaling pathways, whereas PD98059, used at doses that
completely inhibit ERK1/2 activation, is a less potent inhibitor of
ERK5 activation in mIMCD-3 cells.
ERK5 Inhibition Prevents EGF-dependent Cell Migration
and Branching Process Formation--
We next examined HGF- and
EGF-dependent cell migration using the U0126 compound to
inhibit both ERK1/2 and ERK5 activation. As compared with the
previously demonstrated 37% inhibition by PD98059, preincubation with
U0126 inhibited EGF-dependent cell migration by 82% and
HGF mediated cell migration by 97% (control, 2.82 ± 0.35; U0126,
2.80 ± 0.57; HGF, 30.65 ± 2.46; HGF + U0126, 3.78 ± 0.46; EGF, 54.06 ± 3.52; EGF + U0126, 11.94 ± 0.42) (Fig. 4A). These results suggest
that EGF-dependent cell migration may be largely dependent
on ERK5 activation.
mIMCD3 cells grown in a three-dimensional collagen matrix in the
presence of HGF or EGF/transforming growth factor- Expression of Dominant Negative ERK5 Prevents EGF-induced, but Not
HGF-induced, Morphogenesis--
To more carefully examine the role of
ERK5 in EGF-dependent morphogenesis, we transiently
expressed dominant negative ERK5 (dnBMK1) in mIMCD-3 cells. Using
cotransfection with enhanced GFP to specifically identify cells
expressing dnBMK1, we compared the effects of expression of wild-type
ERK5 and dominant negative ERK5 on HGF- and EGF-dependent
branching process formation. Cotransfection with pCDNA3 + pEGFP was
found to have no effect on either HGF- or EGF-induced branching process
formation (Fig. 5A, left
panels). In contrast, expression of dominant negative ERK5
resulted in a marked decrease in EGF-induced process formation with no
detectable change in HGF-dependent branching (Fig.
5A, right panels). Quantitation of cell processes
using fluorescence microscopy was performed (Fig. 5B) and
confirmed that expression of dnBMK1 caused complete inhibition of
EGF-induced process formation (control (pCDNA3 + pEGFP), 0.54 ± 0.05 processes/cell; wild-type ERK5 (pcDNA3-BMK1 + pEGFP),
0.66 ± 0.12; dominant negative ERK5 (pcDNA3-dnBMK1 + pEGFP),
0.48 ± 0.06; HGF, 1.80 ± 0.11; HGF + wild-type ERK5,
1.53 ± 0.04; HGF + dominant negative ERK5, 1.76 ± 0.12;
EGF, 1.64 ± 0.13; EGF + wild-type ERK5, 1.56 ± 0.12; EGF + dominant negative ERK5, 0.42 ± 0.07). Thus, ERK5 appears to be
critical for the EGF-dependent morphogenesis but plays
little or no role in this response to HGF.
There is a rapidly enlarging literature in regards to a role for
MAPK activation in cell morphogenesis. In yeast, the MAPK pathway has
been found to be critical for the morphogenic events necessary for
hyphae formation and budding, specifically mediated by the
G-protein-coupled Ste20/MEKK upstream activators of MEK and MAPK (34).
In mammalian endothelial cells, proliferin can induce cell motility and
angiogenesis (an event similar to epithelial tubulogenesis) via the
G-protein-linked insulin-like growth factor-II receptor, and Groskopf
et al. (18) have found that proliferin-mediated chemotaxis
is blocked by the MEK inhibitor PD98059. Similarly, Graf et
al. (17) found that either inhibition of MEK with PD98059 or
down-regulation of MAPK expression with antisense oligodeoxynucleotides resulted in a 70-80% inhibition of PDGF-mediated cell migration in
vascular smooth muscle cells.
It has been recently demonstrated that HGF-dependent
tubulogenesis in MDCK cells (a 7- to 10-day assay of multicellular
tubule formation requiring cell division) was inhibited by the MEK1
inhibitor PD98059 (25). Because cell proliferation requires MAPK
activation, we chose to utilize a 4-h cell migration assay and a 24-h
single cell branching morphogenesis assay to eliminate the role of cell division in this process and thereby specifically examine regulation of
cell morphology. In the present study, we find that hepatocyte growth
factor-dependent cell migration and branching process
formation are blocked by PD98059, demonstrating that these morphogenic
responses are dependent on activation of the MEK1 substrates ERK1
and/or ERK2.
In contrast to the results observed with HGF, we found that
EGF-dependent morphogenesis in mIMCD-3 cells was only
modestly inhibited by doses of PD98059 that resulted in >90%
inhibition of ERK1/2 activation. Thus it appears that the classical
ERK1/2 MAPK pathway is critical for HGF- but not
EGF-dependent cell migration and branching process
formation. Of note, the dependence of EGF-induced cell migration on
activation of the phosphoinositide 3-kinase and phospholipase C was
nearly indistinguishable from that demonstrated for HGF. To determine a
mechanism for the differential MAPK dependence, we tested the ability
of HGF and EGF to activate the MEK5/ERK5 MAPK pathway that has been
found to be critical for EGF-dependent mitogenesis (26). In
mIMCD-3 cells, EGF was found to activate ERK5 kinase activity 3-fold,
whereas HGF failed to activate ERK5.
In work by Kamakura et al. (33) using COS cells transfected
with tagged ERK5, EGF activation of ERK5 was found to be fully inhibited by both PD98059 and U0126. In the present study examining the
kinase activity of native ERK5 in mIMCD-3 cells, we found that
concentrations of PD98059 that are sufficient to fully inhibit EGF-dependent ERK1/2 activation only inhibited ERK5
activation by 60%. In contrast, the alternate MEK inhibitor U0126
fully inhibited both pathways. The difference in our result as compared
with the work by Kamakura and coworkers may be due to differences in
cell type, differences in the native versus overexpressed
protein, or differences in the PD98059 itself. It has been our
experience that the apparent Ki for MEK varies with
different lots of PD98059. Of note, in one series of experiments, ERK5
activation was more fully inhibited in our cells by Our observations, i.e. that EGF (but not HGF) activates ERK5
and that 50 µM PD98059 (which inhibits ERK1/2 activation
fully but ERK5 activation by only 60%) inhibited
HGF-dependent morphogenesis in a much greater fashion than
EGF-dependent morphogenesis, suggested to us that EGF
utilizes the MEK5/ERK5 MAPK pathway for initiation of
morphogenesis. To test this possibility, we expressed dominant negative ERK5 in mIMCD-3 cells. In our experience, LipofectAMINE transfection of mIMCD-3 or MDCK cells typically results in ~25-35% transfection efficiency. Therefore, to identify those cells that were
expressing the dnBMK1 construct, we performed cotransfection with a
plasmid expressing GFP, allowing us to quickly identify and count
transfected cells based on fluorescence. By utilizing one-fourth of the
amount of cDNA for GFP as compared with dnERK5, cells expressing
GFP are expected to be cotransfected with the dnBMK1 construct. We
found that, although expression of enhanced GFP (EGFP) with the
pcDNA3 control vector or wild-type ERK5 had no effect on HGF- or
EGF-dependent branching process formation, expression of
dominant negative ERK5 in mIMCD-3 cells selectively prevented
EGF-induced branching process formation without inhibiting HGF-mediated
morphogenesis. Unfortunately, background fluorescence of the chemotaxis
membrane prevented utilization of this approach for examination of cell
migration assays. However, based on our results with U0126, we believe
that both EGF-mediated cell migration and branching process formation
in mIMCD-3 cells are ERK5-dependent.
In fibroblasts, Slack et al. (35) have recently described
that EGF-mediated migration was blocked by either PD98059 or U0126, suggesting that inhibition of ERK1/2 alone may be sufficient to prevent
migration in these cells. Interestingly, examination of their results
reveals that, although PD98059 inhibited their
EGF-dependent cell migration index from 1.9 to 1.2, U0126
produced a 2-fold greater inhibition from 1.9 to 0.2, again consistent
with a role for a non-ERK1/2 MAPK family member in
EGF-dependent fibroblast migration. Their detection of
significant inhibition of EGF-mediated fibroblast migration by the MEK1
inhibitor PD98059 may reflect relative differences in the dependence of
the EGFR on ERK1/2 versus ERK5 in fibroblasts as compared
with epithelial cells or may reflect the ability of PD98059 to
significantly inhibit ERK5 activation in their cells at the doses utilized.
The requirement of these fairly rapid cell morphologic changes for ERK
activation suggests that there may be ERK phosphorylation targets
involved other than the classically described nuclear transcriptional
regulatory proteins. We have recently found that, following HGF or EGF
stimulation of mIMCD-3 cells, activated ERK1/2 associates with and
phosphorylates the receptor docking protein GAB1 (36). This protein is
of particular interest, because it has been found to associate with
both the activated EGFR (4) and c-met (3) and mediate signaling
interactions with the PI3K, PLC In conclusion, HGF-mediated cell migration and branching process
formation requires the coordinate activation of the PI3K, PLC
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and epidermal growth factor (EGF), are capable of mediating
branching morphogenesis (1, 2). In contrast, despite the presence of
the EGFR in both cell types, only HGF is capable of mediating branching
morphogenesis in MDCK cells, suggesting that these two receptors
initiate morphogenesis utilizing similar, but not identical, signaling
pathways. Upon activation, both the c-met receptor and the EGFR are
tyrosine-phosphorylated and subsequently interact with GRB-2 and
GAB1 (GRB-2-associated binding protein) (3, 4). These adapter
molecules allow the activated receptor to initiate signaling through
phosphoinositide 3-kinase (PI3K), phospholipase C-
(PLC
), SHPTP
and mitogen-activated protein kinase (MAPK) (3, 5-10). We have found
that activation of the PI3K results in an increase in
receptor-mediated activation of PLC
and that these signaling
pathways then mediate PKC activation as an important component of the
cell migratory response (11, 12). However, the role of MAPK signaling
in epithelial cell motility and tubulogenesis, independent of its role
in cell proliferation, is not as well defined.
for a
normal motility response, whereas the c-met receptor appears to utilize
the ERK1/2 signaling pathway during cell migration and branching
morphogenesis and EGF-dependent cell morphogenesis is
primarily dependent on ERK5 activation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 1% Triton X-100, 2 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin A, and 25 mM
sodium fluoride), vortexed, and incubated on ice for 10 min and
centrifuged at 14,000 rpm for 10 min. Protein assay was
performed on the supernatant, and 1 mg of protein was incubated with 40 µl of protein G-Sepharose beads (VWR Scientific Products) and
3 µg of goat-anti ERK5 antibody (Santa Cruz Biotechnology) for 3 h at 4 °C. The immune complex was then washed twice with the lysis
buffer followed by two washes in reaction buffer (20 mM
Tris-Cl, pH 7.5, 2 mM EGTA, 2 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 0.5 µg/ml pepstatin A, and 25 mM
sodium fluoride) and incubated for 30 min at 30 °C with 20 µg of
myelin basic protein (Sigma) in a buffer containing 20 mM
Tris-Cl (pH 7.5), 10 mM MgCl2, 100 µM ATP, and 10 µCi of [
-32P]ATP. The
supernatant was separated by SDS-PAGE, and phosphorylation of MBP was
analyzed via autoradiography. The bands were cut out of the membrane
and counted in a scintillation counter to quantify the kinase activity.
To compare values from separate experiments, counts from each
experiments were normalized to their respective control.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Inhibition of MAPK by PD98059 and
U0126. mIMCD-3 cells were pretreated with appropriate doses of
either PD98059 (A) or U0126 (B) for 20 min prior
to stimulation for 10 min with either HGF (40 ng/ml) or EGF (20 ng/ml).
The activation state of ERK1/2 was determined by SDS-PAGE separation of
40 µg of whole cell lysates followed by immunoblotting with
anti-phospho-MAPK. Quantitation of inhibition was performed using IMAGE
1.62 (National Institutes of Health).
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Fig. 2.
Inhibition of HGF- and
EGF-dependent mIMCD-3 cell migration. Directed cell
migration was performed using a modified Boyden chamber. A,
pretreatment with 50 µM PD98059 results in 75%
inhibition of HGF-directed cell migration and 37% inhibition of
EGF-dependent cell migration (p < 0.001 for HGF versus HGF + PD98059, p < 0.001 for
EGF versus EGF + PD98059). B, pretreatment with
the PLC inhibitor U73122 (1 µM) caused complete
inhibition of both HGF- and EGF-dependent cell migration
(p < 0.01 for HGF versus HGF + U73122 and
for EGF versus EGF + U73122). C, pretreatment
with the PI3K inhibitor wortmannin (100 nM) results in
~60% inhibition of both HGF- and EGF-mediated cell migration
(p < 0.001 for HGF versus HGF + wortmannin
and for EGF versus EGF + wortmannin).
are
required for PDGF-induced cell motility (8, 9). To determine whether
these pathways were also differentially required in HGF- and
EGF-dependent cell migration, we utilized the PI3K inhibitor wortmannin and the PLC inhibitor U73122. As compared with
inhibition of MEK1, inhibition of phospholipase C equally inhibited
HGF- and EGF-induced cell motility (control, 6.33 ± 0.84; U73122,
10.6 ± 3.3; HGF, 211.8; HGF + U73122, 13.5 ± 1.88; EGF,
180.4 ± 20.1; EGF + U73122, 9.67 ± 1.63)(Fig. 2B). Similarly, inhibition of the PI3K equally inhibited
both HGF- and EGF-induced motility by ~60% (control, 10.2 ± 1.3 cells/mm2; wortmannin, 9.17 ± 0.91; HGF, 309 ± 24; HGF + wortmannin, 127 ± 12.6; EGF, 327 ± 26.9; EGF + wortmannin, 128.7 ± 6.12) (Fig. 2C). This partial
inhibition of cell motility with wortmannin is comparable to that seen
in PDGF-dependent migration (8). These results indicate
that, although activation of the PI3K and PLC
are equally important
for HGF- and EGF-mediated cell migration, ERK1/2 activation appears to
be differentially required for HGF- and EGF-dependent motility.
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Fig. 3.
Phosphorylation of ERK5 following EGF
stimulation of mIMCD-3 cells. A, immunoprecipitation of
ERK5 with the anti-ERK5 antibody followed by immunodetection with
anti-ERK5 reveals a size shift in ~30% of the immunoprecipitated
ERK5 following EGF stimulation (lane 7). A prolonged
exposure reveals a similar size shift of less than 5% of ERK5
following HGF stimulation (not shown). Although 50 µM
PD98059 had little effect on the EGF-dependent shift of
ERK5 (lane 8), 20 µM U0126 completely
prevented the ERK5 gel retardation. B, immunoprecipitation
of ERK5 with anti-ERK5 followed by immunodetection with an antibody
specific for MEK5-activated ERK5 (anti-phospho-ERK5) reveals activated
ERK5 following EGF stimulation (lane 5) but not HGF
stimulation (lane 2). Again, U0126 fully inhibits
EGF-dependent phosphorylation of ERK5 (lane 7),
whereas PD98059 inhibits the phosphorylation by ~50% (lane
6). C, immunoprecipitation of ERK5 followed by an MBP
substrate kinase assay reveals activation of ERK5 by EGF and not HGF.
D, quantitation of ERK5 kinase activity by -scintillation
counting of the MBP substrate from three independent experiments
reveals a 3-fold increase in ERK5 kinase activity following EGF
stimulation, with no effect of HGF. PD98059 inhibits
EGF-dependent ERK5 activation by 62%, whereas U0126
inhibits ERK5 kinase activity by 97%.
View larger version (15K):
[in a new window]
Fig. 4.
Inhibition of ERK5 activation prevents
EGF-dependent morphogenesis. A, mIMCD-3
cell migration performed following pretreatment with 20 µM U0126 results in nearly complete inhibition of both
HGF- and EGF-dependent cell migration (p < 0.001 for HGF versus HGF + U0126 and p < 0.001 for EGF versus EGF + U0126). B, mIMCD-3
branching process formation was examined following 24-h incubation in
type 1 collagen gels. Incubation in the presence of 50 µM
PD98059 resulted in 76% inhibition of HGF-dependent
branching process formation and 23% inhibition of
EGF-dependent branching morphogenesis. In contrast, 20 µM U0126 caused complete inhibition of branching process
formation by both growth factors (n = 3 for each
condition; p < 0.003 for HGF versus HGF + PD98059 and <0.001 for HGF versus HGF + U0126,
p = not significant for EGF versus
EGF+PD98059 and p < 0.001 for EGF versus
EGF + U0126).
exhibit elongated
branching process formation as an early precursor to multicellular
tubulogenesis (1, 28). To determine if MAPK activation is necessary for
this process, mIMCD3 cells were suspended in a type 1 collagen and
treated with HGF or EGF in the presence of the MEK inhibitors PD98059
or U0126 and the number of processes/cell was then determined 24 h
following matrix polymerization. Similar to the results observed with
cell migration, HGF-induced branching process formation was inhibited
76% by the MEK1 inhibitor PD98059, whereas EGF receptor-induced
morphogenesis was inhibited only 23% (Fig. 4B). In
contrast, U0126 entirely inhibited both HGF- and EGF-induced branching
process formation (control, 0.72 ± 0.13 processes/cell; PD98059,
0.91 ± 0.08; U0126, 0.12 ± 0.14; HGF, 3.63 ± 0.25;
HGF + PD98059, 1.41 ± 0.27; HGF + U0126, 0.25 ± 0.06; EGF,
2.59 ± 0.13; EGF + PD98059, 2.15 ± 0.49; EGF + U0126,
0.35 ± 0.05). Again, this result suggests that
HGF-dependent morphogenesis is primarily dependent on
ERK1/2 activation whereas EGF-induced branching process formation is
ERK5-dependent.
View larger version (14K):
[in a new window]
Fig. 5.
Expression of dominant negative ERK5 prevents
EGF-dependent branching morphogenesis. Wild-type
ERK5 (BMK1), dominant negative ERK5
(dnBMK1), or empty pcDNA3 vector was cotransfected into
mIMCD-3 cells with pEGFP 24 h prior to incubation in type 1 collagen gels in the presence of either HGF or EGF. Cells expressing
dnBMK1 or wild-type BMK1 were identified by EGFP fluorescence, and
processes were scored following 24 h of incubation in a matrix.
A, mIMCD-3 cells expressing either the pCDNA3 vector or
pCDNA3 + dnBMK1 were identified by coexpression of EGFP and
photographed at magnification × 60 using confocal microscopy.
Representative cells are shown demonstrating inhibition of EGF-induced
process formation in dnBMK1-expressing cells. B,
quantitation of processes from cells expressing ERK5 reveals that
expression of the empty pcDNA3 vector or BMK1 had no effect on HGF
or EGF-dependent branching process formation. In contrast,
expression of dnBMK1 resulted in complete inhibition of EGF-mediated
morphogenesis with no effect on HGF-dependent branching
process formation (30 cells/well were scored in triplicate wells from
two separate experiments for each condition; p = not
significant for HGF + BMK1 versus HGF + dnBMK1,
p < 0.01 for EGF + BMK1 versus EGF + dnBMK1).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
100
µM PD98059 (data not shown).
, and SHPTP. Overexpression of either
the GAB1 c-met receptor binding domain (3) or a truncated GAB1
construct missing the PH1 domain that mediates membrane localization
(37) results in a dominant negative phenotype for HGF-induced
epithelial tubulogenesis. Thus, this docking protein appears to be a
critical signaling intermediate in induction of the tubulogenic
phenotype. Examination of the GAB1 sequence reveals multiple potential
ERK phosphorylation sites (PX(S/T)P), including sites
immediately adjacent to the potential SH2 interaction sites of the
PI3K, suggesting that ERK phosphorylation of GAB1 might regulate the
degree or duration of signaling activation following receptor
stimulation. In addition, activated ERK may play a direct role in
regulating the cellular machinery required for morphogenesis. For
example, Xie et al. (16) found that EGF-mediated disassembly
of focal adhesions was dependent on ERK activation and Klemke et
al. (38) have found that activated ERK can associate with and
phosphorylate myosin light chain kinase, thereby increasing its kinase
activity and enhancing cell migration. More recently, Chen et
al. (39) have found that the assembly of the tight junctional
proteins claudin-1 and ZO-1 was regulated by MAPK activation. Whether
ERK5 can mediate these functions downstream of EGFR activation remains to be explored.
, and
ERK1/2. Although EGF-dependent morphogenesis appears to
have similar requirements for PI3K and PLC
activation, this receptor
pathway utilizes the alternative MEK5/ERK5 MAPK pathway for cell
morphogenesis. This differential signaling in growth factor-induced
morphogenesis may help to further explain how cell types expressing
identical growth factor receptors can demonstrate divergent morphogenic responses.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. J. D. Lee for his kind gift of the BMK1 constructs, and we thank Fiona Watson and Eisuke Nishida for their helpful comments.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK54911 (to L. G. 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.
§ Both authors contributed equally to this work.
¶ To whom correspondence should be addressed: Yale University School of Medicine, 333 Cedar St., LMP 2093, New Haven, CT 06520. Tel.: 203-785-7111; Fax: 203-785-7068; E-mail: anil.karihaloo@yale.edu.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M009963200
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ABBREVIATIONS |
---|
The abbreviations used are:
HGF, hepatocyte
growth factor;
EGF, epidermal growth factor;
EGFR, EGF receptor;
MDCK
cells, Madin-Darby canine kidney cells;
PI3K, phosphoinositide
3-kinase;
PLC, phospholipase C
;
PDGF, platelet-derived growth
factor;
MAPK, mitogen-activated protein kinase;
ERK1/2, extracellular
signal-regulated kinases 1 and 2;
MEK, MAPK/ERK kinase;
DMEM/F12, Dulbecco's modified Eagle's medium/Ham's F12 medium;
PAGE, polyacrylamide gel electrophoresis;
GFP, green fluorescence protein;
EGFP, enhanced GFP;
BMK1, ERK5 (also called Big MAPK);
dnBMK1, dominant
negative BMK1;
SHPTP, SH phosphotyrosine phosphatases.
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