From the Department of Molecular Pharmacology and
Biological Chemistry, Northwestern University Medical School,
Chicago, Illinois 60611, the ¶ Division of Environmental and
Occupational Health, School of Public Health, University of Minnesota,
Minneapolis, Minnesota 55455, and the
Program in Pharmacology
and Toxicology, College of Pharmacy, University of New Mexico Health
Sciences Center, Albuquerque, New Mexico 87131
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ABSTRACT |
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Activation of the extracellular signal-regulated
kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is
required for ligand-dependent regulation of numerous
cellular functions by receptor tyrosine kinases. We have shown
previously that although many receptor tyrosine kinase ligands are
mitogens for keratinocytes, cell migration and induction of the
92-kilodalton gelatinase/matrix metalloproteinase (MMP)-9 are
selectively regulated by the epidermal growth factor and scatter
factor/hepatocyte growth factor receptors. In this report we present
evidence of an underlying mechanism to account for these observed
differences in receptor tyrosine kinase-mediated response. Ligands that
are mitogenic, but do not induce MMP-9 or colony dispersion,
transiently activate the p42/p44 ERK/MAP kinases. In contrast, ligands
that stimulate MMP-9 induction and colony dispersion induced sustained
activation of these kinases. The functional significance of sustained
MAPK activation was demonstrated by inhibition of the MAP kinase
kinase MEK1. Disruption of the prolonged signal by addition of the MEK1
inhibitor PD 98059 up to 4 h after growth factor stimulation
substantially impaired ligand-dependent colony dispersion
and MMP-9 induction. These findings support the conclusion that
duration of MAPK activation is an important determinant for certain
growth factor-mediated functions in keratinocytes.
Stimulation of receptor tyrosine kinases by ligand results in the
regulation of multiple signal transduction pathways such as the
mitogen-activated protein kinase
(MAPK)1 cascade (1-3). The
MAPK family of protein kinases includes the extracellular
signal-regulated kinases (ERKs) (4), the c-Jun N-terminal
kinases/stress-activated protein kinases (JNK/SAPKs) (5, 6), and
p38HOG (7). Within the MAPK family, the ERK and JNK
pathways have been reported to be stimulated by receptor tyrosine
kinases in various cell types; however, p38HOG is not
commonly activated by growth factors (3). The MAPK/ERK cascade is best
characterized for its involvement in mitogenic signaling (3, 4), but
additionally, this family of kinases has been implicated in diverse
cellular responses such as chemical or osmotic stress (5-7), cell
differentiation (8), and migration (9, 10).
There is abundant evidence that certain receptor tyrosine kinases are
involved in tumor development or progression (2, 11). In addition to
promoting mitogenic responses in target cells, these receptors are also
capable of regulating cellular functions that are involved in the
acquisition of an invasive phenotype such as modulation of cellular
attachments, proteolysis of extracellular matrix, and directional
migration (2, 11-13). Many growth factors have been reported to
stimulate keratinocyte migration, and it has been shown that receptor
tyrosine kinase and integrin-induced cell migration involve activation
of the Ras/MAPK signal transduction pathway (9, 10). Similarly, receptor tyrosine kinase ligands induce a number of extracellular matrix-degrading proteases including MMP-9 (13-16), and the ERK, JNK,
and p38 MAPK pathways have been reported to contribute to MMP-9 gene expression (15, 17-19).
In the course of previous studies, we have shown that although many
receptor tyrosine kinase ligands are mitogenic for keratinocytes, only
a subset of receptors, the epidermal growth factor (EGF) receptor and
c-Met (scatter factor/hepatocyte growth factor receptor), were also
motogenic (16). Receptor specificity for stimulating migration
coincided with ligand-mediated invasion through a reconstituted basement membrane and induction of MMP-9. An association between MMP-9
expression and keratinocyte migration is suggested by detection of
MMP-9 during wound healing and the correlation between MMP-9 expression
and squamous cell carcinoma (SCC) invasiveness (13, 20-22). In our
studies, we found that MMP-9 plays a functional role in EGF- and
SF/HGF-induced migration, as inhibition of MMP-9 activity impaired
receptor tyrosine kinase-dependent SCC locomotion.
The present study focuses on the signaling requirements for receptor
tyrosine kinase-dependent MMP-9 induction, and we present evidence of an underlying basis for receptor specificity in the regulation of keratinocyte migration and invasion. In particular, we
investigated the apparent paradox that although the MAPK pathway is
essential for receptor tyrosine kinase-induced mitogenesis, there is
demonstrable receptor specificity with regard to regulation of MMP-9
expression and cell migration. We find that although ligand-dependent activation of the ERK and JNK pathways was
readily detected, sustained activation of p42/p44 ERK/MAPK following
growth factor stimulation was associated with induction of MMP-9
expression and keratinocyte motility. Furthermore, attenuation of the
sustained signal by inhibition of MEK1 abrogated ligand-induced MMP-9
expression and migration. These results suggest that
ligand-dependent regulation of cellular responses related
to an invasive phenotype is dictated by the capacity of certain
receptor tyrosine kinases to stimulate sustained activation of the MAPK
signaling cascade.
Cell Lines and Cell Culture--
SCC-12F cells were originally
derived from a tumor of the facial epidermis (23) and were generously
provided by Dr. William A. Toscano, Jr. (Tulane University, New
Orleans, LA). SCC-12F cells were maintained in a 1:1 mixture of
Dulbecco's modified Eagle's medium: Ham's F-12 nutrient mixture
(DMEM:F-12) containing 5% iron-supplemented defined calf serum
(HyClone Laboratories, Inc., Logan, UT). For all experiments involving
growth factor addition, SCC cells were placed into DMEM:F-12 containing
0.1% bovine serum albumin (BSA) for 24-48 h prior to growth factor addition. Murine EGF was obtained from Biomedical Technologies Inc.
(Stoughton, MA); keratinocyte growth factor (KGF) was purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY), and scatter factor/hepatocyte growth factor (SF/HGF) was a generous gift from Genentech. All other growth factors were purchased from Life
Technologies, Inc. PD 98059 and SB202190 were obtained from Calbiochem
and dissolved in dimethyl sulfoxide (Me2SO). The final
concentration of Me2SO did not exceed 0.1% (v/v) in any experiment.
Measurements of Cell Motility--
Evaluation of colony
dispersion (cell scattering) was performed as described previously
(24). Briefly, cells were subcultured and maintained in growth medium
until colonies of greater than 16 cells were established. Cultures were
deprived of growth factors and serum for 24 h prior to treatment
with or without ligand at the concentrations and times indicated in the
figure legends. Colony dispersion was documented by photography.
Photographs of cell cultures were taken at a magnification of × 10 or × 25 using a Nikon N2000 camera mounted upon a Nikon
Diaphot-TMD inverted phase contrast microscope. Results shown are
representative of at least three independent experiments.
Western Blot Analysis of Activated MAPK--
Activated MAPK
species were detected using phosphospecific antibodies directed against
the dually phosphorylated, active forms of the proteins according to
the vendor's instructions. Parallel control blots were obtained using
phosphorylation state-independent pan antibodies for detection of the
MAPK species. SCC-12F cells were serum-deprived for 24 h prior to
stimulation with ligand at the concentrations and for the times
indicated in the figure legends. Control and treated cells were rinsed
with ice-cold phosphate-buffered saline and then lysed in sample buffer
containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10%
glycerol, 50 mM dithiothreitol, and 0.1% w/v bromphenol
blue. Typically, 10 µg of total cell lysate for each sample was
separated on a 10% SDS-polyacrylamide gel and then transferred onto
polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA).
Membranes were blocked with 2% milk in 10 mM Tris-HCl, pH
8.0, 150 mM NaCl, 0.05% Tween 20 (TBST) for 30 min at room
temperature. Membranes were then incubated with primary antibody at
1:1000 dilution in TBST containing 0.25% gelatin (TBSTG) overnight at
4 °C for the phospho-specific phospho-p44/42 MAPK kinase
(Thr202/Tyr204) monoclonal antibody (New
England Biolabs, Beverly, MA). Membranes were then washed in TBSTG for
15 min at room temperature, followed by incubation with a 1:2500
dilution of goat anti-rabbit-conjugated horseradish peroxidase
secondary antibody (Promega, Madison, WI) for 1 h at room
temperature. The membranes were washed with TBST for 1 h at room
temperature and developed using the SuperSignal chemiluminescent
detection system (Pierce). Detection of total ERK was accomplished
using a pan-ERK antibody (Transduction Labs, Lexington, KY). Membranes
were incubated with primary antibody at a 1:5000 dilution in TBSTG for
30 min at 37 °C, washed with TBSTG as above, and then incubated with
a 1:10,000 dilution of goat anti-mouse-conjugated horseradish
peroxidase secondary antibody (Promega, Madison, WI) for 1 h at
room temperature.
Kinase Assays--
Protein A-agarose was purchased from Life
Technologies, Inc. [
The immunocomplexes were washed and incubated in kinase buffer as
follows. JNK was incubated at 30 °C for 20 min in kinase buffer
(12.5 mM MOPS, pH 7.5, 20 mM
Zymogram Analysis--
SCC-12F cells were serum-deprived for
24 h prior to growth factor treatment in fresh serum-free medium.
Conditioned medium (CM) collected from control (untreated) and growth
factor-treated cell cultures was analyzed for proteinase activity by
substrate-gel zymography as described previously (16). Equal total
protein from experimental samples was fractionated on 10%
SDS-polyacrylamide gels containing 0.1% gelatin. Following
electrophoresis, gels were washed with 2.5% Triton X-100 for 30 min at
room temperature and then incubated with substrate buffer (50 mM Tris, 0.2 M NaCl, 5 mM
CaCl2, 0.02% Brij, pH 7.6) 24-48 h at 37 °C.
Proteinase activity is visualized as clear areas in a Coomassie
Blue-stained gel. Results shown are representative of a minimum of
three independent experiments.
Inhibition of MEK1 Activity Impairs Receptor Tyrosine
Kinase-dependent Colony Dispersion and MMP-9
Induction--
Receptor tyrosine kinase activation promotes MMP-9
expression in multiple cell types, including keratinocytes (13, 15, 16). We have shown previously that not all mitogenic ligands for
keratinocytes are capable of inducing MMP-9 expression and colony
dispersion; rather, only activation of a small subset of receptors (the
EGF receptor and c-Met) regulate these cellular processes (16). Other
studies have shown that MAPK activity is essential for MMP-9 expression
in oncogenic transformed rat embryo cells and in tumorigenic SCC cells
displaying constitutive activation of both ERK and JNK/SAPK (17, 18).
As stimulation of the Ras/MAPK pathway is shared by receptor tyrosine
kinases, but only a limited number of receptors induced MMP-9
expression in keratinocytes, we investigated whether MAPK activity was
essential for growth factor-mediated MMP-9 induction in a human
keratinocyte cell line.
Both ERK1 (p42-kDa MAPK) and ERK2 (p44-kDa MAPK) are activated by the
MAP kinase kinase MEK1 (28). The inhibitor PD 98059 prevents activation
of MEK1 by upstream kinases (29) and has been reported to selectively
inhibit the ERK/MAP kinase pathway. MAPK activity has been shown to be
required for cell migration (9, 10), so we determined the
concentrations of PD 98059 that effectively inhibited
ligand-dependent colony dispersion. SCC-12F cells were
stimulated with epidermal growth factor (EGF) or scatter
factor/hepatocyte growth factor (SF/HGF) in the presence or absence of
the MEK1 inhibitor PD 98059, and ligand-dependent colony
dispersion was evaluated (Fig. 1,
A-F). When SCC-12F cells were incubated with increasing
concentrations of PD 98059 for 15 min prior to growth factor
stimulation, partial abatement of EGF-mediated colony dispersion was
detected with 10 µM PD 98059, and complete inhibition of
the response was detected at 30 µM PD 98059 (Fig. 1,
D and E). These concentrations are consistent with those reported for half-maximal (IC50 = 2 µM) and complete (50 µM) inhibition of MEK1
by PD 98059 in vitro (29). A similar PD
98059-dependent impairment of migration was noted when
cells were stimulated with SF/HGF (data not shown).
Our previous studies demonstrated that MMP-9 plays a functional role in
ligand-dependent colony dispersion (16); therefore, we
investigated whether MAPK activation was essential for EGF or
SF/HGF-dependent induction of MMP-9. Gelatin zymography was performed on conditioned media collected 24 h after ligand
stimulation in the presence and absence of PD 98059 (Fig.
2). The MEK1 inhibitor PD 98059 had no
effect on basal MMP-9 levels (Fig. 2); however, preincubation of cells
with concentrations of PD 98059 that inhibited colony dispersion (Fig.
1, E and F) reduced EGF- and SF/HGF-induced MMP-9
expression (Fig. 2). Together, these results demonstrate that
inhibition of MEK1 impairs receptor tyrosine
kinase-dependent colony dispersion and MMP-9 induction,
suggesting that ERK1 and/or ERK2 activation is essential for these
responses in a human keratinocyte cell line.
Duration of p42/p44 ERK Activation as a Consequence of Receptor
Tyrosine Kinase Activation--
MEK1 activates both the p42 and p44
ERKs, and the ERK/MAPK pathway is involved in receptor tyrosine
kinase-regulated mitogenic responses, yet many mitogenic ligands were
unable to promote keratinocyte colony dispersion or MMP-9 induction (9,
10, 16). Our findings indicate that the ERK/MAPK pathway is required
for EGF- and SF/HGF-dependent colony dispersion and MMP-9
induction (Figs. 1 and 2, data not shown). Therefore, we tested
selected motogenic and non-motogenic ligands to determine whether
growth factor-dependent activation of specific MAPK
isoforms was associated with the observed differences in response. The
concentration of growth factors (typically 10 nM) used to
stimulate cells corresponded to concentrations previously reported to
induce a mitogenic response in this cell line (16). Activated p42 and
p44 ERKs were detected using a phospho-specific antibody that only
recognizes the dually phosphorylated and active form of the MAP kinase
(30).
As shown in Fig. 3, serum-deprived
SCC-12F cells displayed low basal MAPK activation in the absence of
growth factor and stimulation of cells with EGF rapidly induced ERK1
and ERK2 activation (Fig. 3A). When the activities of
different growth factors were compared, EGF, SF/HGF, insulin-like
growth factor (IGF) 1, and keratinocyte growth factor (KGF) all induced
ERK1 and ERK2 activation (Fig. 3, A
In addition to activation of specific MAPK isoforms, variations in
duration of MAPK activation has been associated with differences in
functional outcome in response to receptor tyrosine kinase ligands (8,
31). We have shown that ERK activation is required for EGF- and
SF/HGF-dependent MMP-9 induction and colony dispersion (Figs. 1 and 2, data not shown); however, ERK1 and ERK2 were activated by ligands (KGF and IGF-1) that do not induce either of these responses
(Ref. 16, Fig. 3). Therefore, we evaluated the duration of MAPK
activation following stimulation with motogenic and non-motogenic growth factors. Stimulation of serum-deprived SCC-12F cells by EGF or
SF/HGF resulted in a rapid activation of p42 and p44 ERK that was
sustained for up to 6 h (Figs. 3 and 4, data not shown). This is
in contrast to the response observed after stimulation with IGF-1 or
KGF, where the typical duration of MAPK activation did not exceed
1 h before returning to base-line levels (Fig. 3). Thus, ligands
that promote keratinocyte migration and MMP-9 induction also stimulate
a prolonged duration of ERK1 and ERK2 activation.
Growth Factor Activation of JNK/SAPK and
p38HOG--
As shown in Figs. 3 and
4, there is a correspondence between
growth factors that promote sustained ERK1/2 activation and those that
induce colony dispersion and MMP-9 activity. There is evidence that the
JNK/SAPK and p38 pathways are also involved in expression of MMP-9 (13,
17-19), and constitutive activation of JNK/SAPK is detected in
UM-SCC-1 cells displaying high basal MMP-9 expression (18). Therefore,
we wanted to determine if specific receptor tyrosine kinase ligands
differed in their ability to activate stress-activated members of the
MAPK family.
We monitored activation of JNK and p38 by EGF, IGF-1, SF/HGF, and KGF
by measurement of catalytic activity in immunocomplex assays (26, 27).
The motogenic ligands EGF and SF/HGF stimulated robust activation of
JNK (~10-fold); KGF was a more modest JNK activator (~5-fold), and
IGF-1 did not stimulate JNK activation (Fig.
5). The time courses of EGF and
SF-HGF-stimulated JNK activation were similar with JNK activity
detected within 5 min, and peak activity was observed between 15 and 30 min, and activity returned to background levels within 2 h (Fig.
5). KGF also stimulated JNK activation, with maximal activity apparent
at the 15-min time point.
It has been reported that the p38HOG pathway is
not typically activated by receptor tyrosine
kinase-dependent signals (3, 7); however,
EGF-dependent activation of p38 has been reported (27).
Modest activation (3-5-fold) of p38 was detected in response to EGF,
SF/HGF, and KGF, but not IGF-1, in SCC-12F cells (Fig. 6, data not shown). As with JNK, the
activation was transient with a return to base-line activity within
2 h for EGF and KGF. In contrast, SF/HGF appeared to stimulate a
low (2-3-fold) but sustained activation of p38. Although phorbol
ester-induced MMP-9 expression is eliminated by the p38 inhibitor SB
203580 (19), EGF-mediated migration and MMP-9 induction were
essentially unimpaired by the p38 inhibitor SB202190 (data not shown).
Taken together, these findings suggest that the
p38HOG pathway does not play a central role in
growth factor-induced keratinocyte migration or MMP-9 expression or
account for receptor specificity in the regulation of these cellular
responses.
Sustained ERK Activation Is Required for Receptor Tyrosine
Kinase-dependent Migration and MMP-9 Induction--
As
shown in Figs. 3 and 4, prolonged activation of the p42/p44 ERKs
corresponded with the capacity of specific receptor tyrosine kinases to
promote keratinocyte migration and MMP-9 induction. In order to
determine the potential significance of sustained ERK activation for
these two cellular responses, MAPK activation following EGF or SF/HGF
stimulation was disrupted by inhibiting MEK1 with PD 98059 at various
times after growth factor addition. MMP-9 activity was analyzed in
conditioned medium collected from SCC-12F cells treated with EGF in the
absence or presence of PD 98059 added at various time points after
growth factor addition (Fig.
7A).
Ligand-dependent induction of MMP-9 was inhibited when PD
98059 was added 1 h before EGF addition, when both were
administered concurrently and when PD 98059 was added at time points up
to 4 h after EGF stimulation (Fig. 7A). However,
addition of the MEK1 inhibitor at time points beyond 4 h after EGF
stimulation no longer effectively blocked EGF-dependent
MMP-9 induction (Fig. 7A). This suggests that the
MEK1-dependent signal required for MMP-9 induction was
fully transmitted by this point. In addition, EGF- and SF/HGF-mediated
SCC-12F colony dispersion was inhibited by addition of PD 98059 at time
points up to 6 h after growth factor addition (data not shown). To
confirm that MAPK activation was inhibited by the MEK1 inhibitor at the
extended time points, cell lysates were collected 30 min after
treatment with PD 98059, and activated ERKs were detected by immunoblot
analysis. As shown in Fig. 7B, PD 98059 treatment after EGF
stimulation effectively blocked the sustained MAPK activation. Thus,
inhibition of MEK1 transformed the pattern of EGF-dependent
MAPK activation to resemble that observed for IGF-1 and KGF (Fig. 3),
and importantly, interruption of sustained MAPK activation resulted in
loss of the EGF-dependent cellular responses (Fig. 7, data
not shown). Together, these findings indicate that sustained activation
of the MAPK signaling cascade is required for receptor tyrosine
kinase-dependent MMP-9 induction and cell migration.
We have demonstrated previously that growth factor-regulated
keratinocyte migration and MMP-9 induction is selectively mediated by
the EGF receptor and c-Met in SCC lines and normal human keratinocytes (16). The mechanisms responsible for specificity of response to
different receptor tyrosine kinases remain unclear; however, it has
been proposed that the duration of ERK activation may determine cellular responses to ligand. In one well characterized example, nerve
growth factor causes sustained ERK activation and promotes PC12 cell
differentiation, whereas differentiation is not induced by EGF which
only transiently activates ERKs within this cell type (31, 33, 34).
In this report we present evidence that sustained versus
transient activation of the ERK/MAPK signaling cascade represents an
underlying mechanism to account for receptor tyrosine kinase specificity in ligand-induced keratinocyte migration and MMP-9 induction. Both IGF-1 and KGF transiently activate the ERK/MAPK pathway
and stimulate keratinocyte proliferation but do not induce colony
dispersion or MMP-9 expression (Ref. 16, Figs. 3 and 4). In contrast,
EGF- and SF/HGF-mediated motility and MMP-9 induction are associated
with sustained duration of MAPK activation (Figs. 3, 4, and 7). In
examining the signaling consequences of receptor tyrosine kinase
activation, we found that ligand stimulation enhances activity of
similar MAPK constituents (p42/p44 ERK) regardless of the functional
outcome (Figs. 3 and 4). The distinguishing characteristic of receptors
that regulate MMP-9 induction and colony dispersion is their ability to
promote prolonged ERK activation (Figs. 3, 4, and 7B).
Importantly, interruption of the sustained signal using the MEK1
inhibitor PD 98059 rendered EGF inactive for migration and MMP-9
induction (Fig. 7, data not shown), indicating that prolonged duration
of MAPK activation is required for both of these cellular responses.
Several lines of evidence indicate that MMP-9 gene
expression is regulated by activation of various MAP kinase signaling
pathways. Oncogenic transformation with v-Src or v-Ras up-regulate
MMP-9 expression, as does diverse stimuli such as EGF, tumor necrosis factor- These studies, together with our findings, suggest that prolonged
activation of MAPK activity may be a general requirement for induction
of MMP-9 expression. Constitutive up-regulation of MMP-9 through
oncogenic transformation by v-Ras has been reported to be through a
MEK1-independent activation of MAPK (36). In contrast, our results
demonstrate that PD 98059 interferes with growth factor-induced ERK
activation and MMP-9 expression, suggesting that receptor tyrosine
kinase-mediated MMP-9 induction requires a MEK1-dependent
pathway. The requirement for MEK1 activity in the sustained MAPK
activation as detected in this system suggests that input from upstream
signaling effectors, such as MEK1, is involved in the duration of MAPK activation.
Interestingly, there is also evidence that prolonged MAPK activation
may play a role in cell migration. Sustained ERK2 activation has been
correlated with Ret- and fibroblast growth factor-mediated scattering
in SK-N-MC cells (37). This finding, in conjunction with our results,
suggests that sustained ERK2 activation is a common requirement for
receptor tyrosine kinase-regulated colony dispersion. One possible
consequence of sustained ERK activation might be the induction of gene
products specifically required for migration. We have shown that MMP-9
activity is involved in growth factor-induced colony dispersion (16),
and therefore, MMP-9 may represent one such gene product.
Analysis of the MMP-9 promotor has identified potential cis acting
elements such as the
12-O-tetradecanoylphorbol-13-acetate-response element, Ets,
and NF-kB that are likely to confer induction of MMP-9 in response to
growth factors or oncogenic transformation (17, 35, 36). Interestingly,
in one model system where cells displayed constitutive activation of
MAPK as part of the transformed phenotype, the accompanying
up-regulation of MMP-9 expression was dependent upon
AP-1/12-O-tetradecanoylphorbol-13-acetate response element
sites within the MMP-9 promotor region and one of the essential
12-O-tetradecanoylphorbol-13-acetate response elements bound
a Jun D/c-Fos heterodimer (18). A possible mechanism by which sustained
MAPK activation could result in MMP-9 induction is through regulation
of essential transcription factors such as c-Fos. Expression of this
immediate early gene is dependent on MAPK activation, and furthermore,
phosphorylation of c-Fos by MAPK enhances its activity (38). Thus, in
one possible signaling scenario, initial activation of MAPK may be
required to induce c-Fos, and sustained activation may serve to enhance
c-Fos transcriptional activity and AP1-dependent expression
of the MMP-9 gene (18, 38). It is possible that a sequence
of MAPK-dependent induction followed by
MAPK-dependent phosphorylation of specific transcription factors may represent a mechanism whereby duration of MAPK activation distinguishes between a mitogenic or motogenic response in target cells. Analysis of the contributions of sustained MAPK activation to
differential gene expression will require further study.
The ERK/MAPK signal transduction cascade plays an important role in
mediating cellular responses to growth factor receptor activation. In a
limited number of cellular systems including PC12 cells and K562
megakaryocytes, sustained activation of MAP kinases is a requirement
for cell differentiation (8, 31, 34, 39). The findings presented in
this paper support the conclusion that sustained activation of MAP
kinase signaling cascades may play an important role in dictating other
cellular responses such as migration, metalloproteinase expression, and
invasive capacity that require de novo protein and RNA
expression. Additionally, the ability of a particular receptor tyrosine
kinase to promote sustained MAPK activation may serve as an underlying
biochemical mechanism to account for receptor specificity in cellular
responses to growth factors.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-32P]ATP was purchased from NEN
Life Science Products. Anti-JNK and anti-p38 antibodies were purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
pGEX-2T-c-Jun-(1-232) was the gift of Dr. Daniel Mueller (Department
of Medicine, University of Minnesota). pGEX-3X-ATF-2 was the gift of
Dr. Benoit Dérijard (Center de Biochimie, Nice, France).
Glutathione S-transferase fusion proteins were expressed and
purified as described previously (25). The immunoprecipitation of JNK
(26) and p38 (27) was conducted according to published methods.
Briefly, cells were incubated in a 5% CO2 humidified
incubator at 37 °C in DMEM:F-12, 0.1% BSA in the presence or
absence of the indicated agent. Cells were then washed with ice-cold
phosphate-buffered saline and harvested in lysis buffer. JNK lysis
buffer contained 25 mM HEPES, pH 7.5, 300 mM
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, pH
8.0, 0.1% Triton X-100, 0.5 mM dithiothreitol, 20 mM
-glycerophosphate, 1 mM
Na3VO4, 20 µg/ml aprotinin, 50 µg/ml
leupeptin, 1 mM phenylmethylsulfonyl fluoride. p38 lysis
buffer contained 20 mM Tris-Cl, pH 7.4, 1% Triton X-100,
10% glycerol, 137 mM NaCl, 2 mM EDTA, pH 8.0, 25 mM
-glycerophosphate, 1 mM
Na3VO4, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin.
Cell lysates were centrifuged at 10,000 × g, 4 °C
for 10-20 min. The protein concentration of the supernatant was
quantitated using the Bradford Assay (Bio-Rad, Cambridge, MA). 200 µg
of protein was incubated with 5 µl of the appropriate antibody and 25 µl of protein A-agarose at 4 °C for 1-2 h.
-glycerophosphate, 7.5 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3VO4,
0.5 mM NaF) in the presence of 1 µg of glutathione
S-transferase-c-Jun and 5 µCi of
[
-32P]ATP. p38 was incubated at 30 °C for 20 min in
kinase buffer (25 mM HEPES, pH 7.4, 25 mM
-glycerophosphate, 25 mM MgCl2, 2 mM dithiothreitol, 0.1 mM
Na3VO4) in the presence of 2 µg of
glutathione S-transferase-ATF-2 and 7.5 µCi of
[
-32P]ATP. The kinase reactions were terminated by the
addition of Laemmli sample buffer. The proteins were resolved using
10% SDS-PAGE mini-gels. Substrate phosphorylation was detected by
autoradiography and quantified using a Bio-Rad model GS-700 Imaging Densitometer.
RESULTS
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Fig. 1.
Inhibition of MEK1 by PD 98059 impairs
EGF-mediated colony dispersion. SCC-12F cells were maintained in
DMEM:F-12 containing 5% calf serum and were serum-starved 24 h
prior to growth factor stimulation. Cell were either unstimulated
(A) or stimulated with 10 nM EGF in the presence
of (B) 0, (C) 3 µM, (D)
10 µM, (E) 30 µM, or
(F) 100 µM PD 98059 which was added 15 min
prior to EGF addition. Colony dispersion was analyzed 18 h
following growth factor addition.
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Fig. 2.
Inhibition of MEK1 by PD 98059 impairs
EGF-dependent MMP-9 induction. SCC-12F cells were
grown to near-confluency in DMEM:F-12 containing 5% calf serum and
were serum-starved 24 h prior to growth factor stimulation.
Gelatin zymography was performed on serum-free conditioned media from
SCC-12F cells stimulated with EGF (10 nM) 24 h in the
presence or absence of increasing concentrations of PD 98059 as
indicated. The upper band represents the 92-kDa
gelatinase/MMP-9, and the lower band represents the 72-kDa
gelatinase/MMP-2.
D). Therefore,
selective activation of a particular MAPK isoform (p42 or p44 ERK) did
not account for the functional differences between motogenic and
non-motogenic receptor tyrosine kinases. These findings were confirmed
in an in-gel kinase assay using myelin basic protein as a substrate
(data not shown).
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Fig. 3.
Time course of MAPK activation following
exposure to various RTK ligands. SCC-12F cells were grown to
near-confluency in DMEM:F-12 containing 5% calf serum and were
serum-starved 24 h prior to growth factor stimulation. SCC-12F
cells were stimulated in serum-free DMEM:F-12 containing 0.1% BSA with
10 nM EGF (A), 20 ng/ml SF/HGF (B),
10 nM IGF-1 (C), or 10 nM KGF
(D) for 5, 15, or 30 min, or 1 or 2 h as indicated.
Whole cell lysates were collected, fractionated on 10% SDS-PAGE, and
active phosphorylated proteins detected by immunoblot analysis with the
phospho-MAPK antibody (New England Biolabs) as described under
"Experimental Procedures."
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Fig. 4.
Extended time course of
SF-dependent MAPK activation. SCC-12F cells were grown
as described in the legend to Fig. 3 and stimulated with 20 ng/ml
SF/HGF in serum-free DMEM:F-12 containing 0.1% BSA for 5 or 15 min or
1, 2, 4, or 6 h as indicated. Whole cell lysates were collected,
fractionated on 10% SDS-PAGE, and active phosphorylated proteins
detected by immunoblot analysis with the phospho-ERK antibodies (New
England Biolabs). Similar findings were obtained following treatment
with EGF.
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Fig. 5.
Activation of JNK by various RTK
ligands. SCC-12F cells were incubated for the indicated times with
10 nM EGF, 20 ng/ml SF/HGF, 10 nM IGF-1, or 10 nM KGF. The cells were then lysed and assayed for JNK
activity as described under "Experimental Procedures." JNK activity
is indicated by the phosphorylation of glutathione
S-transferase-c-Jun. The data shown are representative of
two independent experiments.
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Fig. 6.
Activation of p38 by various RTK
ligands. SCC-12F cells were incubated for the indicated times with
10 nM EGF, 20 ng/ml SF/HGF, 10 nM IGF-1, or 10 nM KGF. The cells were then lysed and assayed for p38
activity as described under "Experimental Procedures." p38 activity
is indicated by the phosphorylation of glutathione
S-transferase-ATF-2. The data shown are representative of
three independent experiments. No stimulation of p38 was detected
following treatment with IGF-1. NS, nonspecific
phosphorylation; A, anisomycin that serves as a positive
control.
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Fig. 7.
Inhibition of MEK by PD 98059 post-EGF
stimulation impairs RTK-dependent MMP-9 induction and MAPK
activation. A, gelatin zymography (0.1%) analysis was
performed on serum-free conditioned media from unstimulated cells and
cells stimulated with EGF (10 nM) for 24 h in the
presence or absence of 50 µM PD 98059 added at increasing
times following growth factor addition. B, SCC-12F cells
were grown to sub-confluency in DMEM:F-12 containing 5% calf serum and
were serum-starved 24 h prior to growth factor stimulation.
SCC-12F cells were stimulated with EGF (10 nM) in
serum-free DMEM:F-12 containing 0.1% BSA for 0 or 5 min or 4 or 6 h, and then PD 98059 (30 µM) was added as indicated
followed by an additional 30 min of incubation. Whole cell lysates were
collected, fractionated on 10% SDS-PAGE, and active phosphorylated
proteins detected by immunoblot analysis with the phospho-specific ERK
antibody (New England Biolabs) as described under "Experimental
Procedures."
DISCUSSION
, and phorbol ester tumor promoters (35, 36). Phorbol ester
stimulation of MMP-9 expression was reportedly through the p38HOG pathway as use of the p38 inhibitor SB203580 blocked
phorbol ester-dependent MMP-9 induction and cell invasion
but not mitogenesis (19). In addition, disruption of the ERK- or
JNK-dependent signaling decreases endogenous MMP-9
expression in UM-SCC-1 cells (18). Although the JNK and p38 MAPK
cascades may be necessary for induction of MMP-9 in response to various
stimuli, our data suggest that neither pathway is sufficient for growth
factor-stimulated MMP-9 expression. JNK and p38 activities were
induced by KGF (Figs. 5 and 6) even though KGF does not induce
MMP-9 in these cells (16). Duration of ERK activation corresponds to
this receptor-mediated response, and it is intriguing that
MAPK-dependent up-regulation of MMP-9 expression is
apparent in several systems where MAPK is constitutively activated
either through oncogenic transformation or endogenously as part of the
transformed phenotype (17, 18, 35, 36).
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant RO1AR42989 (to L. G. H.), by National Institutes of Health Grant CA72498 (to E. V. W.), and by a Pharmaceutical Research and Manufacturers of America Foundation Research Starter Grant (to E. V. W.).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.
§ Supported by National Institutes of Health Training Grant T32GM08061.
** To whom correspondence should be addressed: Program in Pharmacology and Toxicology, UNM Health Sciences Center, 2502 Marble NE, Albuquerque, NM 87131. Tel.: 505-272-2482; Fax: 505-272-6749; E-mail: lghudson{at}unm.edu.
The abbreviations used are: MAPK, mitogen-activated protein kinase; MAP, mitogen-activated protein; MEK1, MAP kinase kinase; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; IGF-1, insulin-like growth factor 1; KGF, keratinocyte growth factor; SF/HGF, scatter factor/hepatocyte growth factor; SCC squamous cell carcinoma, JNK/SAPKs, c-Jun N-terminal kinases/stress-activated protein kinases; MMP, matrix metalloproteinase; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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