From the Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262
Received for publication, December 4, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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Urokinase-type plasminogen activator (uPA)
regulates the remodeling of extracellular matrix and controls
reparative processes such as wound healing and liver regeneration. Here
we show inducible uPA expression is controlled by MEKK1, a MAPK kinase
kinase that regulates the ERK1/2 and JNK pathways. MEKK1 is
activated in response to growth factors and cytoskeletal changes. We
have found MEKK1 to be necessary for uPA up-regulation in response to
treatment with phorbol 12-myristate 13-acetate or basic fibroblast
growth factor. We demonstrate that growth factor-treated
MEKK1-deficient fibroblasts display greatly reduced uPA expression and
activity compared with control fibroblasts. Further, we show that
growth factor-induced uPA expression requires
MEKK1-dependent MKK1 and JNK activity and that transfection
of MEKK1 into knockout cells restores inducible uPA expression and
activity. Importantly, disrupted expression of MEKK2, a related MAPK
kinase kinase, had no effect on uPA activity. Therefore, we
conclude that MEKK1 expression is required for PMA- or FGF-2-induced
signals to control uPA expression and function.
Tissue remodeling in response to stress is a critical homeostatic
function in multiple organ systems. One physiological process involving
tissue remodeling is that of wound healing. Wound healing is a complex
process involving multiple cell types and regulated proteolytic
degradation of extracellular matrix components (1). The loss of
activity of some key proteases results in an impaired ability to repair
wounds (2-4). Among the important enzymes expressed and secreted
during the wound healing response is the serine protease uPA1 (2, 5, 6). Known uPA
substrates are diverse, and include the zymogen plasminogen (7). uPA
activity promotes a proteolytic cascade by converting plasminogen to
its active form, plasmin, which can then cleave and activate matrix
metalloproteinases (8), thereby enhancing tissue remodeling processes
by degrading extracellular matrix components. Beyond wound healing, uPA
is required for liver regeneration (9), whereas disregulation of uPA
activity plays a major role in human diseases including cancer
metastasis (7, 10). As uPA can have a profound impact on homeostasis,
both activity and expression are subject to tight regulation. uPA
activity is controlled post-translationally through cleavage to produce an activated form (7) as well as through localization by binding to its
cognate receptor (uPAR) (7). uPA is also regulated at the
transcriptional level. AP-1 transcription factor duplexes play a major
role in control of inducible uPA expression through binding of enhancer
regions in the promoter of the uPA gene (11). AP-1 complex components
and assembly are, in turn, subject to regulation by MAPK activity.
Transcription factor and AP-1 component c-Jun may be
phosphorylated and consequently activated by the MAPK c-Jun
NH2-terminal kinase (JNK) (12). JNK may also phosphorylate the CREB (cAMP-responsive element-binding protein) family member ATF-2.
ATF-2/c-Jun heterodimers have been shown to be a predominant form of
AP-1 that promotes uPA expression (11), and therefore JNK activity is
an integral part of uPA regulation. Another MAPK, extracellular
signal-regulated kinase (ERK1/2), is also key to AP-1 regulation, as
its activation leads to the induction of c-Fos, which may join with
c-Jun to form a second AP-1 heterodimer complex for the regulation of
uPA expression (12).
MEKK1 is a 196-kDa serine-threonine kinase that functions as an MAPKKK
in the JNK pathway and can modulate the ERK1/2 pathway (13). Although
regulation of these MAPK pathways has been attributed to other MAPKKKs,
genetic studies have begun to define a specific role for MEKK1 in
tissue homeostasis. Targeted disruption of the MEKK1 gene in mice
results in a failure to activate JNK in response to aortic banding, and
MEKK1 In this work, we demonstrate that MEKK1 regulates uPA expression
in response to growth factor receptor ligation and by phorbol ester stimulation. Our data are consistent with MEKK1 being a junction
that integrates different signals to common pathways required for uPA expression.
Antibodies and Reagents--
The anti-phospho JNK and
anti-phospho-ERK monoclonal antibodies and the rabbit polyclonal
antibody against ERK2 were purchased from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). The rabbit polyclonal antibody against uPA was a
generous gift of the Finsen Laboratory (Copenhagen, Denmark). HRP-sheep
anti-mouse IgG was purchased from Amersham Biosciences. Protein
A-HRP conjugate was purchased from Zymed Laboratories (San Francisco,
CA). The constructs for full-length uPA and the uPA promoter were
purchased from American Type Culture Collection (Manassas, VA).
Cell Lines--
Both primary and immortalized mouse embryo
fibroblasts (MEFs) were obtained as described previously (17).
Cell Culture, Stimulation, and Lysis--
MEFs were cultured in
IMDM medium (Invitrogen) containing penicillin/streptomycin (1%,
Invitrogen), L-glutamine (2 mM, Invitrogen), monothioglycerol (0.0012%, Sigma), and 10% (v/v) fetal calf serum (Gemini Bioproducts, Woodland, CA) at 37 °C in a humidified
atmosphere. Human FGF-2 was purchased from Upstate Biotechnology Inc.
(Lake Placid, NY). PMA was purchased from Sigma. After stimulation, the
cells were lysed in 0.5 ml of sample buffer (125 mM
Tris-Cl, pH 6.8, 20% glycerol, 4.6% SDS, 0.1% bromphenol blue, and
10% 2-mercaptoethanol) and incubated at room temperature for 20 min. After centrifugation at 14,000 × g for 5 min,
post-nuclear detergent cell lysates were collected.
Northern blot Analysis--
Stimulated cells were lysed, and RNA
was extracted with RNeasy (Qiagen, Valencia, CA) according to
manufacturer's recommendations. RNA analysis by Northern hybridization
was performed as described previously (33). The membrane was hybridized
with a 460-bp uPA cDNA fragment (EcoRI digest).
Transient Transfection--
Adherent cells were transfected by
lipofection. Briefly, 4 × 105 cells were
seeded on 100-mm cell culture plates and incubated in complete medium
overnight. cDNA expression constructs were incubated in serum-free
medium with LipofectAMINE (Invitrogen) at room temperature for 30 min
and then diluted with serum-free medium and incubated with cells at
37 °C for 2 h, after which time the LipofectAMINE mixture was
replaced with complete medium and the cells returned to 37 °C for
24 h. Complete medium was then removed, the cells were rinsed, and
incubation was continued with serum-free medium for an additional
24 h. For production of stable transfectants, cells were subjected
to selection and maintained in the presence of G418.
Immunoblotting--
Proteins were separated by SDS-PAGE and then
transferred to nitrocellulose (Schleicher & Schuell). Membranes were
blocked in 5% milk (diluted in Tris-buffered saline and 0.1% Tween
20) and incubated with the appropriate antibody at 4 °C overnight. HRP-protein A or HRP-sheep anti-mouse IgG was used as secondary reagent. After extensive washing, the targeted proteins were detected by enhanced chemiluminescence (ECL). Where indicated, blots were stripped by treatment with 2% SDS and 100 mM
2-mercaptoethanol in Tris-buffered saline and then reprobed with the
desired antibodies and detected by ECL.
Luciferase Assay--
The Luciferase assay kit was purchased
from Promega (Madison, WI), and assays were carried out according to
manufacturer's recommendations.
uPA Colorimetric Assay--
Urokinase-specific chromogen S2444
was purchased from DiaPharma Group Inc. (West Chester, OH). Tissue
culture medium was removed from the stimulated MEFs, diluted with S2444
in Tris buffer (50 mM Tris and 38 mM NaCl), and
incubated at 37 °C for 24 h. uPA activity cleaves S2444 to
produce p-nitroaniline, which was assessed by color
change at 405 nm.
Fluorescent Microscopy--
Immortalized, serum-starved MEFs
were attached to coverslips and stimulated with 100 nM PMA
for the indicated times. Cells were fixed in 3% paraformaldehyde, 3%
sucrose pH 7.4, washed, permeabilized with 0.2% Triton, and blocked in
4% bovine serum albumin. Cells were bound by anti-uPA and
Cy3-conjugated anti-rabbit.
Zymogram--
To analyze uPA activity in MEF cell culture
supernatants 1.2 × 106 MEFs were plated into a 10-cm
tissue culture dish and allowed to adhere overnight. Medium was then
removed, and the cells were rinsed with 5 ml of serum-free IMDM to
remove residual serum. Cells were incubated for 24 h in 4 ml of
serum-free IMDM with either vehicle or stimuli. Cell-free supernatant
(150 µl) samples were mixed with 50 µl of nondenaturing sample
buffer (40% glycerol, 8% SDS, 250 mM Tris-HCl, pH 6.8, 0.05% bromphenol blue), and proteins were separated within 10%
SDS-PAGE co-polymerized with 0.025 unit of plasminogen (Calbiochem) and
2 µg/ml MEKK1 Regulates PMA-induced uPA Expression--
Wound healing
requires coordinated proteolysis of various wound matrix components
(1). We observed that MEKK1-deficient mice developed wounds, through
over-grooming or fighting, that were persistent and healed poorly (not
shown). Importantly, these persistent wounds were not observed in mice
deficient for MEKK2, a related MAPKKK. This apparent healing defect is
very similar to one reported for mice deficient in the protease uPA.
Carmeliet and colleagues (18) observed facial wound healing defects in uPA-deficient mice. uPA activity regulates the activation of other proteases necessary for the healing process and has been linked to
activation of immunoregulatory cytokine TGF- MEKK1-deficient Fibroblasts Lose uPA Activity--
Because our
data indicated that MEKK1 plays a role in regulation of uPA expression,
we wanted to determine whether the apparent loss of uPA message
observed in MEKK1 FGF-2-induced uPA Expression Requires MEKK1--
Because MEKK1 is
required for PMA-induced uPA expression, we wanted to determine whether
MEKK1-deficient fibroblasts were similarly impaired for uPA induction
in response to growth factors that activate ERK1/2 and JNK signaling.
When the FGF receptor tyrosine kinase is bound by its cognate
ligand fibroblast growth factor 2 (FGF-2), it initiates a potent ERK1/2
signal (22). Further, FGF-2 stimulation induces uPA expression in
NIH3T3 fibroblasts (22). Northern blot analysis revealed that FGF-2 was
capable of up-regulating uPA expression in wild-type fibroblasts (Fig. 3A). However, as with PMA
stimulation, MEKK1-deficient fibroblasts were unable to up-regulate uPA
expression to FGF-2 (Fig. 3A). Importantly, ligand-induced
uPA induction was restored in MEKK1-deficient cells that had been
transfected with MEKK1 (Fig. 3A, add back), demonstrating that FGF-2-induced uPA expression is
MEKK1-dependent and not caused by epigenetic
differences between cell lines. This loss of uPA expression resulted in
a functional loss of uPA activity (Fig. 3B). Consistent with
the Northern blot analysis, FGF-2-inducible secreted uPA
activity is reduced to background levels in MEKK1-deficient cells (Fig.
3B). Furthermore, expression of wild-type MEKK1 in MEKK1 MEKK1 Regulates MAPK Signaling Downstream of FGF-2 Receptor
Ligation--
Control of uPA expression is largely dependent on the
binding of the consensus AP-1-PEA3 sequence within the uPA promoter (21, 23). Both ERK1/2 and JNK signaling impact AP-1 activity (12), and
we have previously shown that MEKK1 activity plays a role in both
pathways (13). To determine whether MEKK1 expression is necessary for
FGF-2-induced ERK1/2/JNK signaling, we stimulated either wild-type or
MEKK1-deficient fibroblasts and assessed MAPK activity by immunoblot
with antibodies specific for activated ERK or JNK (Fig.
4A). We clearly show that
ligand-induced ERK and JNK phosphorylation is dramatically reduced in
the MEKK1-deficient samples (Fig. 4A). Importantly, other
FGF-2-induced pathways, such as phosphatidylinositol 3-kinase
activation and consequent AKT phosphorylation, remain intact in
MEKK1-deficient cells (Fig. 4B). This finding demonstrates
that disruption of MEKK1 expression specifically impacts MAPK
signaling.
We wanted to determine whether ERK1/2, JNK, or both of these signaling
pathways were responsible for FGF-2-induced uPA activity. We therefore
repeated the zymography assay using wild-type fibroblasts treated with
the MEK inhibitor UO126 or the JNK inhibitor SP600125. Analysis of uPA
activity revealed it to be highly sensitive to MEK inhibition, as U0126
treatment blocked FGF-2-induced uPA expression (Fig.
5). Interestingly, treatment with p38
inhibitor SB203586 enhanced FGF-2-induced uPA activity (Fig. 5). We
conclude that regulation of both the ERK1/2 and JNK signaling pathways,
a property of MEKK1 signaling, is necessary for FGF-2-induced uPA
expression. Altogether, our data are consistent with JNK and ERK1/2
activities, modulated by MEKK1, functioning in concert to regulate uPA
expression.
Protease-dependent tissue remodeling plays an
important role in normal homeostasis and wound healing and is a factor
in significant human pathologies including myocardial infarction (24)
and cancer metastasis (7). Further, uPA has been linked to arterial
neointima formation and vascular wound healing in mouse models
(25, 26). The protease plasmin, a vital component in these
processes, itself is capable of degrading multiple matrix components.
Further, plasmin regulates the activity of matrix metalloproteinases
(27) by cleaving and consequently activating inactive precursors. As
unrestricted proteolysis would be deleterious to the organism, plasmin
activity is tightly regulated. Proteins upstream of plasmin that
activate this protease cascade are therefore an integral part of the
remodeling process. uPA can activate plasmin by cleaving plasminogen,
the precursor zymogen form of plasmin. Understanding the regulation of
uPA expression is therefore critical in defining the molecular basis of
normal reparative processes and the pathophysiology of multiple
diseases. Indeed, the capacity to remodel the mix of components that
comprise the extracellular matrix is of paramount importance for
maintaining homeostasis.
As uPA activates plasmin, the loss of uPA expression observed in
MEKK1 As uPA protein was detectable by immunoblot in all of the basal
fibroblasts tested, we conclude that MEKK1 expression is not absolutely required for basal uPA expression. Thus, other regulatory pathways independent of MEKK1 regulate uPA expression. In contrast to
inducible uPA expression, basal uPA transcription has been shown to be
driven by SP1 binding to the proximal promoter (30). SP1-dependent transcription is regulated differently than
that reliant upon AP-1 (12, 31) and may in part be regulated through ERK1/2 signaling (32). Indeed, our results showing basal uPA activity
to be sensitive to MEK but not JNK inhibition is consistent with
SP1-dependent constitutive transcription being controlled by the ERK1/2 pathway, independently of JNK activity (Fig. 4). There
are additional MAPKKKs capable of activating the JNK and ERK1/2
pathways that are regulated by upstream stimuli different from those
that activate MEKK1. The MEKK1 and MEKK2 knockouts have defined their
selective roles in regulating different physiological functions. The
pathological consequences observed with MEKK1 or MEKK2 deletion are
different, even though the downstream MAPK signaling pathways remain
intact. For this reason, MAPKKK knockouts, like those of MEKK1 and
MEKK2, can be more subtle than total loss of a signaling pathway,
such as found for the JNKs, but can be much more telling in
regards to physiological regulation in adult animals. The MEKK1
knockout defines MEKK1 as a critical MAPKKK controlling the expression
of uPA from multiple stimuli. MEKK1 expression is required for uPA
induction downstream of both receptor tyrosine kinase (FGF
receptor) and protein kinase C activation (PMA), and thus these
pathways converge at or upstream of MEKK1 (Fig.
6). Our data place MEKK1 at the nexus of
the signaling pathways that control uPA expression and, as such, reveal
the utility of modulating MEKK1 as a means of regulating
uPA-dependent cell functions. The control of proteolytic
degradation of extracellular matrix required for tumor metastasis is
just one example of such a function. The importance of MEKK1 in these
processes validates the use of MAPKKKs as targets for inhibition
by small molecule inhibitors in specific diseases including cancer
metastasis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice display reduced tolerance to hypertensive cardiac
insult (14). Indeed, MEKK1-deficient mice show an increased propensity
to cardiac rupture and congestive heart failure consistent with
defective tissue remodeling
capability.2 We have
previously shown MEKK1 to be activated in response to an array of
stimuli, including epidermal growth factor receptor ligation (15) and
cytoskeletal alteration by treatment with nocodazole and taxol (13,
16). Further, we have found serum and lysophosphatidic acid-induced JNK
activation to be reduced in MEKK1-deficient embryonic stem cells (13).
MEKK1 is thus a MAPKKK that regulates MAPK signaling in response to
cytoskeletal changes and a diverse group of growth factors.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-casein (Sigma). The gel was washed for 1 h in 300 ml
of 2.5% Triton X-100. The gel was rinsed three times with water to
remove the Triton X-100 and rocked for an additional 2 h in
water. For casein proteolysis, the gel was immersed in zymography
enzyme buffer (25 mM Tris, pH 7.4, 100 mM NaCl,
and 10 mM CaCl2) and incubated for 20-24 h at
37 °C. Following this incubation the gel was stained for 2 h in
Coomassie Blue stain (40% methanol, 10% acetic acid and 2.5 g/liter
R250 Coomassie Blue dye (Sigma)) and destained with multiple changes of
destain solution (30% methanol, 10% acetic acid) until light bands
appeared on a blue background.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(19). MEKK1 signaling
through ERK1/2 and JNK pathways controls AP-1 activity (12), and uPA
expression is transcriptionally regulated through the binding of
AP-1-PEA3 complexes to sequences in the uPA promoter (11), which
suggested to us that MEKK1 might regulate uPA expression. Thus, we
investigated the role of MEKK1 in the regulation of uPA expression. We
tested fibroblasts derived from both parental strain and MEKK1
/
embryos (MEFs) for uPA expression. PMA induces a dynamic reorganization
of the actin cytoskeleton and is a well characterized, potent
stimulator of uPA expression (20, 21). As we have demonstrated
previously that MEKK1 regulates both ERK1/2 and JNK signaling
(13), we predicted that MEKK1-deficient cells would show impaired
PMA-induced uPA induction. We observed a dramatic reduction in
PMA-induced uPA mRNA in MEKK1-deficient fibroblasts as measured by
Northern blot analysis (Fig.
1A). This loss of uPA
induction observed in Northern blot analysis was mirrored in reporter
assays using luciferase-based uPA promoter constructs (Fig. 1,
B-D), which indicated that MEKK1 is required for PMA to
stimulate uPA promoter activity. Activation of the uPA promoter requires the kinase activity of MEKK1, as luciferase activity of
MEKK1
/
fibroblasts transfected with a kinase-inactive MEKK1 mutant
mimicked that of control cells (Fig 1C). Loss of uPA
induction in response to PMA is a function of MEKK1 deficiency because
the stable expression of MEKK1 in knockout cells restored regulation of
the uPA promoter (Fig. 1, C and D). Thus, our
data indicate that MEKK1 is essential for PMA-induced uPA
expression.
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Fig. 1.
MEKK1 is required for uPA
expression. PMA-induced uPA expression was assessed by Northern
blot (A) and luciferase reporter assays (B-D).
A, wild-type and MEKK1 /
fibroblasts were treated with
100 nM PMA for 18 h. RNA (5 µg) was probed for uPA
and
-actin. B, wild-type and MEKK1
/
immortalized
fibroblasts were transfected with a uPA-promoter/luciferase (uPA/Luc)
construct and treated with 100 nM PMA for 18 h. Fold
increase in uPA induction was assessed by luciferase assay.
C, MEKK1
/
fibroblasts were transiently transfected with
empty vector, wild-type MEKK1, or kinase-inactive MEKK1 K1253M together
with uPA/Luc and treated with PMA. D, MEKK1
/
fibroblasts
clones that stably express wild-type or kinase-inactive MEKK1 were
treated with PMA. Both C and D were assayed for
uPA induction as described in B. The results shown are
the mean ± S.E. of at least three independent experiments.
/
cells would result in a corresponding decrease
in uPA activity. Consistent with decreased uPA message in MEKK1
/
cells, a colorimetric assay for measuring uPA activity confirmed that
MEKK1 deficiency is also associated with a reduction in secreted
urokinase activity (Fig. 2A).
In addition, zymographic analysis demonstrated the requirement of MEKK1
for regulation of uPA activity. We co-polymerized plasminogen and
casein in SDS-PAGE and then used this gel to separate proteins of
conditioned medium from wild-type and MEKK1
/
fibroblast cultures. In a subsequent incubation in reaction buffer, in-gel uPA activated plasminogen to form active plasmin, which then cleaved casein. Upon
staining of the gel with Coomassie Blue, areas of casein proteolysis
could then be observed as clear bands. Our results demonstrate that PMA
stimulates uPA protein expression and secretion from wild type
fibroblasts (Fig. 2B). In contrast, uPA activity is markedly
inhibited in MEKK1
/
fibroblasts in response to PMA. Immunoblotting
confirmed that induction of secreted uPA protein expression in both
primary and immortalized fibroblasts is MEKK1-dependent (Fig. 2B). We further verified the requirement of MEKK1 in
uPA expression by immunofluorescence analysis. Stimulation of wild-type fibroblasts with PMA resulted in an increased accumulation of uPA in
the Golgi complex (Fig. 2C). Although MEKK1
/
fibroblasts exhibit a basal level of uPA expression, the presence of
Golgi-associated uPA was not observed in PMA-stimulated MEKK1
/
fibroblasts (Fig. 2C). To confirm the specificity of MEKK1
in regulating fibroblast uPA expression, we repeated the zymography
assay with MEKK2
/
fibroblasts. Our zymography assays clearly showed
that PMA-induced uPA activity in MEKK2
/
fibroblasts was not reduced
when compared with activity observed in wild-type fibroblasts (Fig.
2D). Altogether, these findings demonstrate that MEKK1 is an
MAPKKK that controls uPA expression in fibroblasts.
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Fig. 2.
uPA protease activity is reduced in
MEKK1 /
but not
MEKK2
/
fibroblasts. Secreted uPA protease activity specifically is
regulated by MEKK1. A, concentrated tissue culture medium
conditioned with wild-type or MEKK1
/
fibroblasts (untreated or
treated with 100 nM PMA for 24 h) was incubated with
urokinase-specific chromogen S2444 for 24 h. PMA-induced uPA
activity was determined by measuring the change in absorbance at 405 nm. Results shown are the mean ± S.E. of at least three
independent experiments. B and D, proteins of
media from wild-type (WT), MEKK1
/
, or MEKK2
/
fibroblast cultures were separated by SDS-PAGE co-polymerized with
plasminogen and casein (see "Experimental Procedures"), and uPA
activity was assessed by casein proteolysis. NS, no
stimulus. B, secreted uPA protein levels were assessed by
immunoblot with anti-uPA antibodies. The displayed zymograms are
representative of at least three independent experiments. C,
wild-type and MEKK1
/
fibroblasts were serum-starved overnight and
then treated with PMA as described previously. Fibroblast uPA was bound
by anti-uPA antibodies and anti-rabbit Cy3.
/
fibroblasts, either through transient transfection (Fig. 3C) or in stably transfected cell lines (Fig. 3B,
add back), restored uPA activity. Furthermore, this
restoration of uPA activity absolutely requires MEKK1 activity, as
expression of a kinase-inactive MEKK1 mutant did not rescue uPA
activity (Fig. 3C). Our results indicate that MEKK1 is
absolutely required for FGF-2-induced uPA up-regulation in
fibroblasts.
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Fig. 3.
MEKK1 activity required for uPA induction by
FGF-2. FGF-2-induced uPA expression was assessed by Northern blot.
Wild-type, MEKK1 /
, and MEKK1
/
add-back (stable MEKK1
transfectants) fibroblasts were treated with FGF-2 (10 ng/ml, 10 h). A, RNA (5 µg) was probed for uPA and
-actin.
B, zymogram analysis of uPA activity from tissue culture
media of treated or untreated fibroblasts. C, zymogram of
tissue culture media from fibroblasts transiently transfected with
empty vector, wild-type MEKK1, or kinase-inactive MEKK1 K1253M.
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Fig. 4.
MEKK1 regulates MAPK signaling downstream of
stimulation with FGF-2. Wild-type (WT) KK1 /
fibroblasts were serum-starved overnight, stimulated with FGF-2 (10 ng/ml) for the indicated times, and then lysed. A, equal
amounts of lysate were separated by 10% SDS-PAGE, and MAPK signaling
assessed by immunoblot with antibodies to phosphorylated forms of ERK
and JNK. An anti-ERK2 immunoblot was performed to confirm equal
loading. B, FGF-2-induced activation of the AKT signaling
pathway was assessed by anti-phospho-AKT immunoblot with equal loading
confirmed by total AKT immunoblot.
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Fig. 5.
uPA up-regulation requires both ERK1/2 and
JNK activity. Wild-type MEFs were treated with 20 µM
UO126, SP600125, SB203580, or Me2SO vehicle for 60 min. Cells were then stimulated for 24 h with 1 ng/ml FGF-2 or
left untreated. The activity of secreted uPA in the media was assessed
by casein/plasminogen zymography. The displayed zymograms are
representative of at least two independent experiments.
Inh., inhibitor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
fibroblasts would be predicted to result in a reduced capacity to remodel matrix components. This defect could be manifested by an impaired ability to heal wounds, and in fact, this very defect
has been observed in uPA-deficient mice (18). Our observation that
facial wounds persisted in our MEKK1-deficient mouse colony suggests
that MEKK1 is involved in the wound healing process. Mice lacking
MEKK1
/
have an inhibited wound healing response that mimics uPA
deficiency and that is not observed in MEKK2 knockout mice. What
properties of MEKK1 make it an important MAPKKK for tissue remodeling?
First, MEKK1 is activated in response to changes in cytoskeletal
structure (13, 16), cell shape, and adherence (13, 16) and in
response to epidermal growth factor and lysophosphatidic acid (13, 15).
In contrast, other MAPKKKs, like MEKK2, appear to be involved primarily
in responses to specific growth factor and cytokine receptors
but not in changes to the cytoskeleton, adherence, or cell shape
(28). Second, MEKK1 interacts with Rac and Cdc42, important GTPases in
the control of the cytoskeleton, cell shape, and migration. Finally,
MEKK1 regulates the JNK and ERK1/2 pathways, MAPKs that have been shown
clearly to be involved in the regulation of uPA expression. We have
demonstrated that MEKK1-deficient fibroblasts do not have the ability
to up-regulate uPA in response to FGF-2 and PMA. Further, we provide
evidence that FGF-2-induced uPA expression specifically requires
MEKK1-dependent JNK and ERK1/2 activities. It is intriguing
that, whereas other MAPKKKs are known to regulate ERK1/2 or JNK in
response to specific stimuli, MEKK1 remains indispensable for uPA
up-regulation in response to these growth factors. Growth
factor-dependent uPA expression is blocked in
MEKK1-deficient fibroblasts, demonstrating that other MAPKKKs such as
Raf (which regulates ERK1/2 signaling) and MEKK2 (which controls JNK)
are unable to compensate for the loss of MEKK1 activity. One reason for
this requirement may be that, by its ability to coordinately regulate
both ERK1/2 and JNK, MEKK1 is uniquely suited to control uPA induction.
Alternately, MEKK1 may be needed for effective MAPK signaling in
response to these ligands. MEKK1 has been found to associate with
cytoskeletal (29) and focal adhesion proteins,2 suggesting
that MEKK1 localization may figure prominently in the organization of
signaling complexes.
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Fig. 6.
Model depicting MEKK1 pathway controlling uPA
expression. MEKK1 /
fibroblasts are defective in MAPK
activation-dependent uPA expression (see "Discussion"
for details).
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FOOTNOTES |
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* 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 may be addressed: Dept. of Pharmacology,
Rm. 2809, School of Medicine, University of Colorado Health Sciences
Center, 4200 East Ninth Ave., Denver, CO 80262. Tel.: 303-315-1008;
Fax: 303-315-1022; E-mail: bruce.cuevas@uchsc.edu or
gary.johnson{at}uchsc.edu.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M212363200
2 J. Witowsky, A. Abell, N. L. Johnson, G. L. Johnson, and B. D. Cuevas, our unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: uPA, urokinase-type plasminogen activator; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase kinase; ERK, extracellular signal-regulated kinase; MEKK, MAPK/ERK kinase kinase; JNK, c-Jun NH2-terminal kinase; HRP, horseradish peroxidase; MEF, mouse embryo fibroblast; IMDM, Iscove's modified Dulbecco's medium; FGF, fibroblast growth factor; PMA, phorbol 12-myristate 13-acetate; AKT, protein kinase B.
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1. |
Singer, A. J.,
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