From the Department of Molecular Biology and
Pharmacology and Medicine, Washington University School of Medicine,
St. Louis, Missouri 63110, the § Department of Medicine,
Rochester University School of Medicine, Rochester, New York 14642, and
the ¶ Institute of Pathology and Program in Cell Biology, Case
Western Reserve University School of Medicine,
Cleveland, Ohio 44106
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ABSTRACT |
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The mitogen-activated protein kinase (MAPK)
cascade is believed to function as an important regulator of
prostaglandin biosynthesis. Previously we reported that
interleukin-1 induces activation of JNK/SAPK and p38 MAPK with
concomitant up-regulation of cyclooxygenase (Cox)-2 expression and
prostaglandin E2 (PGE2) synthesis. Our experiments demonstrate that overexpression of
MEKK1 (a
constitutively active truncation mutant of MEKK1 containing the
C-terminal 324 amino acids) increases Cox-2 expression and
PGE2 production which is completely blocked by SC68376, a
pharmacologic inhibitor of p38 MAPK.
MEKK1 overexpression results in
activation of both c-Jun N-terminal kinases/extracellular
signal-regulated kinases (JNK/SAPK) and p38 MAPK. Furthermore,
activation of MEKK1 increases SEK1/MKK4 but not MKK3 or MKK6 activity.
These findings suggest that MEKK1
SEK1/MKK4 may function as an
upstream kinase capable of activating both p38 MAPK and JNK/SAPK with
subsequent induction of Cox-2 expression and PGE2
production. We also found that overexpression of the constitutively
active form of SEK1 (SEK1-ED) increases both p38 MAPK and JNK/SAPK
phosphorylation, and increases PGE2 production and Cox-2
expression. By comparison, overexpression of the dominant negative form
of SEK1 (SEK1-AL) decreases the phosphorylation of both p38 MAPK and
JNK/SAPK and reduces Cox-2 expression. Together, this data suggests a
potential role for the MEKK1
SEK1/MKK4
p38 MAPK
Cox-2
cascade linking members of the MAPK pathway with prostaglandin
biosynthesis.
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INTRODUCTION |
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Prostaglandins are ubiquitous compounds involved in various
homeostatic and inflammatory processes throughout the body. They are
formed by the combined action of a phospholipase A2
(PLA2)1 which
liberates arachidonic acid from the sn-2 position of
cellular membrane phospholipids and the cyclooxygenase (Cox) which
converts arachidonic acid to the endoperoxide intermediate
PGH2. PGH2 is subsequently converted to
prostaglandins by the action of cell-specific synthases (1). There are
two cyclooxygenase enzymes which have been identified (60% homology),
Cox-1 and Cox-2. Cox-1 is constitutively expressed in most tissues and
mediates physiologic responses such as regulation of renal and vascular
homeostasis and cytoprotection of the stomach. By comparison Cox-2 is
primarily considered an inducible immediate-early gene product whose
synthesis can be up-regulated by mitogenic or inflammatory stimuli
including: tumor promoters (2), IL-1 (3), endotoxins (4),
platelet-derived growth factor (5), and serum (6).
The physiological role of the cyclooxygenase has been the topic of much interest. Cyclooxygenases are the main therapeutic target for non-steroidal anti-inflammatory drugs which exhibit their antipyretic, analgesic, and anti-inflammatory effects in humans via inhibition of prostaglandin biosynthesis (7). Non-steroidal anti-inflammatory drugs have been effective in the reduction of inflammatory symptoms in carrageenan-induced rat paw inflammation models (8) and in the reduced incidence of colon cancer (9, 10). Moreover, rat intestinal epithelial cells overexpressing Cox-2 demonstrate resistance to apoptosis (11) and Caco-2 human colon cancer cells demonstrate increased metastatic potential when transfected with Cox-2 (12). Perhaps, the most striking evidence for the pervasive role of Cox-2 is obtained from targeted gene disruption models. Mice which are homozygously deficient for Cox-2 develop severe renal pathology and die prematurely of renal failure (13).
Recently, a novel class of cytokine-suppressive anti-inflammatory drugs have been shown to be inhibitors of endotoxin-stimulated tumor necrosis factor and IL-1 induction (14). These cytokine-suppressive anti-inflammatory drugs have been further shown to inhibit the catalytic activity of p38 MAPK. These and other recent findings serve to potentially link prostaglandin biosynthetic pathways which mediate inflammatory responses, with activation of the mitogen-activated protein kinase (MAPK) signaling cascade.
Components of the MAPK pathway have been implicated as mediators of
phosphorylation of intracellular substrates such as protein kinases and
transcription factors (15) as well as regulators of cell growth and
differentiation (16). In mammalian cells, at least three different
subfamilies of MAPK have been identified each having several isoforms.
They include the extracellular signal-regulated kinases (ERKs), p44
MAPK (ERK1) and p42 MAPK (ERK2); stress-activated protein kinases
(SAPKs), also called c-Jun N-terminal kinases (JNKs): which include
JNK2 (p54 SAPK/
/
) and JNK1 (p45 SAPK
/
) and the p38 MAPKs
(
,
,
, and
). These kinases are in turn activated by
distinct upstream MAPK/ERK kinases (MEKs, MKKs) which recognize and
phosphorylate threonine and tyrosine residues within a tripeptide motif
(Thr-X-Tyr) required for MAPK activation (17). Once
phosphorylated, these MAPKs then activate their specific substrates on
serine and/or threonine residues to produce their effects on downstream
targets. Recent work has demonstrated that both SAPK/JNK and p38 MAPK
cascades are activated preferentially by the inflammatory cytokines
IL-1
and tumor necrosis factor-
, as well as by a wide variety of
cellular stresses such as ultraviolet light, ionizing radiation,
hyperosmolarity, heat shock, oxidative stress, etc. (17). These
findings further implicate the role of these two kinase pathways as
important signaling mechanisms underlying the inflammatory process.
Recent work has linked activation of the prostaglandin generative
pathway with the MAPK pathway. Lin et al. (18) have
demonstrated that the activation of cytosolic phospholipase
A2 (cPLA2) is mediated by MAPK. Furthermore,
Kramer et al. (19) have demonstrated that thrombin activates
both ERK and p38 MAPK in human platelets which may utilize
cPLA2 as a downstream target. We have shown that IL-1 stimulation of renal mesangial cells mediates prostaglandin
E2 production and Cox-2 expression with concomitant
activation of p38 MAPK and SAPK-mediated signaling pathways (20, 21).
Furthermore, we have demonstrated that inhibition of p38 MAPK with a
pyridyl oxazole results in the down-regulation of IL-1 mediated Cox-2 expression and PGE2 production, suggesting a role for p38
in the modulation of prostaglandin synthesis (20). Previous work by Templeton et al. (22) has shown that cells induced to
overexpress a constitutively active form of MEKK1 directly activate
the SAPK pathway. The availability of this model system encouraged
exploration of possible SAPK-mediated effects on prostaglandin
production suggested by our previous cytokine stimulation data. In our
current study, we describe the ability of
MEKK1 expressing cells to
induce Cox-2 expression and PGE2 production and analyze the
intermediate kinases involved in the regulation of prostaglandin
biosynthesis.
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EXPERIMENTAL PROCEDURES |
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Reagents-- Myelin basic protein (MBP) was purchased from Sigma. The p38 MAPK inhibitor SC68376 was kindly provided by Dr. Joe Portnova (G. D. Searle Corp.) and dissolved in dimethyl sulfoxide. Fetal bovine serum was purchased from Life Technologies, Inc. Polyclonal or monoclonal rabbit or mouse IgG antibodies against Cox-2 and Cox-1 were from Cayman Chemical Co. Inc.; cPLA2, MEKK1, MKK3, MKK4, MKK6, JNK, phospho-specific JNK, ERK, and p38 MAPK antibodies were from Santa Cruz Biotechnology Inc. Phospho-specific p38 MAPK, SEK/MKK4, and MKK3/MKK6 were from New England BioLabs. Phospho-specific ERK antibody was from Promega. pET28-c-Jun, a histidine-tagged fusion protein expression plasmid that encodes c-Jun-(1-79) which contains the amino-terminal activation domain of c-Jun and a mutant c-Jun-(1-79, S63A/S73A), in which serine 63 and 73 of c-Jun-(1-79) were mutated to alanine, were generously provided by Dr. Maryann Gruda (Department of Molecular Biology, Bristol Myers Squibb Pharmaceutical Research Institute, Princeton, NJ). Both wild type His-c-Jun-(1-79) and mutant His-c-Jun-(1-79, S63A/S73A) were expressed as a histidine-tagged fusion protein in Escherichia coli NovaBlue (DE3) and purified by His-bind resin (Novagen). pGEX ATF-2-(1-96) was obtained from Dr. J. Silvio Gutkind (Molecular Signaling Unit, Laboratory of Cellular Development and Oncology, NIH). pGEX p38 MAPK was kindly donated by Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical Center). GST-ATF-2-(1-96) and GST-p38 MAPK were expressed as a GST fusion protein in E. coli and purified by GST-binding resin (Pharmacia Biotech Inc.).
Cell Culture and Transfection--
All experiments were
performed with NIH 3T3 cells as well as NIH 3T3 cells stably
transfected with an IPTG inducible EE-epitope-tagged MEKK1 truncation
mutant (containing the C-terminal 324 amino acids, MEKK-1) subcloned
into the lacSwitch promoter construct (Stratagene) (22, 23).
Normal NIH 3T3 cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 25 mM HEPES. MEKK1 3T3 cells were maintained in the above media with 500 mg/liter of
G418 and 200 mg/liter of hygromycin.
MEKK1 expression was induced
using 1 mM IPTG for various time periods.
Western Blot Analysis--
At the time of harvest, cells were
washed with ice-cold phosphate buffer and lysed in whole cell extract
buffer (25 mM HEPES-NaOH (pH 7.7), 0.3 M NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 0.1%
Triton X-100, 0.5 mM dithiothreitol (DTT), 20 mM -glycerophosphate, 100 µM NaVO4, 2 µg/ml leupeptin, and 100 µg/ml
phenylmethylsulfonyl fluoride) to which 6 × Laemmli sample buffer
was added before heating. After boiling for 5 min, equal amounts of
protein were run on 10% SDS-PAGE. Proteins were transferred to
polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp.,
Bedford, MA). The membranes were saturated with 5% fat-free dry milk
in Tris-buffered saline (50 mM Tris, pH 8.0, 150 mM NaCl) with 0.05% Tween 20 (TBS-T) for 1 h at room
temperature. Blots were then incubated overnight with primary
antibodies at 1:1000 dilution in 5% bovine serum albumin TBS-T. After
washing with 5% milk TBS-T solution, blots were further incubated for
1 h at room temperature with goat anti-rabbit or mouse IgG
antibody coupled to horseradish peroxidase (Amersham) at 1:3000
dilution in TBS-T. Blots were then washed five times in TBS-T before
visualization. Enhanced chemiluminescence (ECL) kit (Amersham) was used
for detection.
In-gel Protein Kinase Assay--
Harvested cells were
solubilized in whole cell extract buffer. Protein kinase assays were
performed using our previously described methods (20). Briefly,
SDS-polyacrylamide was polymerized in the presence or absence of 200 µg/ml His-c-Jun-(1-79), His-c-Jun-(1-79, S63A/S73A), or 400 µg/ml
MBP. After electrophoresis, SDS was removed by incubation in 20%
isopropyl alcohol in 50 mM Tris-HCl (pH 8.0) for 1 h.
The gel was then washed for 1 h with 1 mM DTT, 50 mM Tris-HCl (pH 8.0). To denature the proteins, gels were
incubated in 6 M guanidine-HCl, 20 mM DTT, 2 mM EDTA, 50 mM Tris-HCl (pH 8.0) for 1 h.
Proteins were then renatured by overnight incubation in 1 mM DTT, 2 mM EDTA, 0.04% Tween 20, 50 mM Tris-HCl (pH 8.0). For the protein kinase assays, gels
were equilibrated for 1 h in kinase buffer containing 1 mM DTT, 0.1 mM EGTA, 20 mM
MgCl2, 40 mM HEPES-NaOH (pH 8.0), 100 µM NaVO4. The kinase reaction was carried out
for 1 h in kinase buffer with 30 µM ATP and 5 µCi/ml [-32P]ATP. Finally, the gels were washed
extensively in 5% trichloroacetic acid and 1% sodium pyrophosphate
until washes were free of radioactivity. Autoradiography of dried gel
was performed at
80 °C.
Immunecomplex p38 MAPK Activity Assay--
The cell extracts
were immunoprecipitated by incubation overnight with anti-p38 MAPK
antibody and then with protein A-Sepharose beads for 3 h at
4 °C. The beads were washed 3 times with 1 ml of ice-cold whole cell
extract buffer. The immunecomplex p38 MAPK activity assay using MBP or
GST-ATF-2-(1-96) as the substrate was performed at 30 °C for 20 min
in 30 µl of kinase reaction buffer (5 µg of MBP or
GST-ATF-2-(1-96), 20 µM ATP, 10 µCi of [-32P]ATP, 25 mM HEPES, and 20 mM MgCl2). The reaction was terminated with
Laemmli sample buffer and the products were resolved by 10% SDS-PAGE.
The phosphorylated MBP or GST-ATF-2 was visualized by autoradiography.
Immunecomplex MKK3, MKK4, and MKK6 Activity Assay-- The cell extracts were immunoprecipitated by incubation overnight with anti-MKK3, MKK4, or MKK6 antibodies and then incubated with protein A-Sepharose beads for 3 h at 4 °C. The beads were washed 3 times with 1 ml of ice-cold whole cell extract buffer. The immunecomplex MKK3, MKK4, or MKK6 activity assay using GST-p38 MAPK (10 µg/reaction) as the substrate was performed at 30 °C for 20 min in 30 µl of kinase reaction buffer (10 µg of GST-p38 MAPK, 100 µM ATP, 25 mM HEPES, and 20 mM MgCl2). The reaction was terminated with Laemmli sample buffer and the products were resolved by 10% SDS-PAGE. The phosphorylated p38 MAPK was detected by anti-phospho-specific p38 MAPK antibody by Western blot analysis and detected by enhanced chemiluminescence.
PGE2 Determination-- PGE2 in the overlying culture media was measured by a PGE2 ELISA Kit (Cayman Chemical).
Statistical Analysis-- All the experiments were performed at least three times. Data were expressed as the mean ± S.E. Statistical analysis was performed by using a paired or unpaired Student's t test. A difference with a p value of 0.05 was considered statistically significant.
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RESULTS |
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MEKK1 Activation Induces PGE2 Production and Cox-2
Expression--
A MEKK1 inducible model system was utilized in which
NIH 3T3 cells were stably transfected with a constitutively active
truncated fragment of MEKK1 (the C-terminal 324 amino acids,
MEKK-1)
under the control of an IPTG inducible promoter. Confluent normal NIH 3T3 cells as well as
MEKK1 3T3 cells were incubated for varying periods of time with 1 mM IPTG to confirm overexpression of
MEKK1 protein by Western blot assay using an anti-MEKK1 antibody.
Normal NIH 3T3 cells were virtually unaffected by IPTG stimulation. By comparison, in MEKK1 3T3 cells,
MEKK1 expression was minimally detected under basal conditions and was maximally induced at 12-36 h
following IPTG stimulation (Fig. 1).
Notably, the MEKK1 antibody detected duplicate bands of equal intensity
following IPTG stimulation. Previous data suggests that the lower band
represents untagged
MEKK1 (resulting from an internal initiation
product), whereas the upper band represents the EE epitope-tagged
MEKK1 (24). In this study, we examined whether activation of
MEKK1 expression induces prostaglandin biosynthesis. Comparison of
untreated
MEKK1 3T3 cells with cells induced by IPTG demonstrates an
approximately 4-fold enhancement in PGE2 production (Fig.
2B). Since cyclooxygenase is a
key rate-limiting enzyme which mediates prostaglandin biosynthesis, we
further examined whether the enhancement of PGE2 production was correlated with changes in the expression of these enzymes. As
shown in Fig. 2A, induction of
MEKK1 by IPTG resulted in
a significant increase in Cox-2 expression. However, induction of
MEKK1 did not increase either Cox-1 or cPLA2 protein
expression (data not shown). To demonstrate whether MEKK1-induced
PGE2 synthesis is mediated by the activation of Cox-2, a
Cox-2 specific inhibitor, NS-398, was utilized. As shown in Table
I, NS-398, at a concentration of 0.1 µM, completely blocked PGE2 production
induced by the activation of MEKK1. These findings suggest that Cox-2
plays an important role in MEKK1-induced PGE2
biosynthesis.
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MEKK1 induces p46 SAPK and p54 SAPK Activity--
To
investigate the signal transduction pathway responsible for mediating
induction of COX-2 expression in MEKK1 cells, in-gel kinase assays
using His-c-Jun-(1-79) as a substrate were performed to measure SAPK
activity following
MEKK1 induction by IPTG. Fig. 3A demonstrates that two bands
corresponding to the molecular weights of p46 SAPK and p54/55 SAPK were
maximally induced in MEKK1 3T3 cells by 24 h after IPTG induction.
Similar experiments utilizing mutated His-c-Jun-(1-79, S63A/S73A) as a
substrate demonstrated no phosphorylation of this substrate (Fig.
3B). Similarly, Western blot analysis using an
anti-phospho-specific JNK which recognizes both phosphorylated (and
presumably active) p46 SAPK and p54/55 SAPK further illustrated that
MEKK-1 induction could increase phosphorylation of both p46 SAPK and
p54/55 SAPK, respectively (Fig. 3C). Western blot analysis
employing a pan-JNK antibody (which also recognizes murine SAPK)
indicated that
MEKK1 induction did not influence total protein
expression of p46 SAPK or p54/55 SAPK (Fig. 3D).
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MEKK1 Induces p38 MAPK Activity--
Recent data generated in
our laboratory has suggested that the p38 MAPK activated pathway may
contribute to the signaling mechanism for IL-1
-induced Cox-2
expression and prostaglandin synthesis in renal mesangial cells (20).
With this data in mind and the above observations linking Cox-2
expression with MEKK-1 activation, we assessed IPTG-induced
MEKK1
3T3 cells for p38 MAPK activity. As shown in Fig.
4, p38 MAPK activity increased within
24-36 h after
MEKK1 induction which was examined by immunocomplex kinase assay using MBP (Fig. 4A) or GST-ATF-2-(1-96) (Fig.
4B) as the substrate. This increase in activity was further
correlated with Western blot analysis illustrating increased
phosphorylation of p38 MAPK at 24 h following IPTG stimulation
(Fig. 4D). Western blot analysis using anti-p38 MAPK
indicated that total protein expression was unaffected by
MEKK1
induction (Fig. 4C). Together, these results demonstrate a
role for MEKK1, a kinase heretofore considered upstream of the SAPK
pathway, as an activator of p38 MAPK in an intact cell system.
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p38 MAPK Mediates MEKK1-induced COX-2 Expression--
To ascertain
whether p38 MAPK signaling pathways mediate PGE2 production
and cyclooxygenase expression induced by MEKK1 induction, we studied
the effects of SC68376, an inhibitor of p38 MAPK. SC68376 directly
inhibits p38 MAPK catalytic activity (IC50 of 2-5
µM) without influencing ERK and JNK activity. When
MEKK1 3T3 cells were treated with 1 mM IPTG and 10 µM SC68376 for 24 h, the inhibitor completely
eliminated Cox-2 expression and concomitant PGE2 production normally induced by
MEKK1 induction. As shown in Fig.
5, SC68376 exhibits selectivity for
inhibition of Cox-2 protein expression. There was negligible influence
on MEKK-1, SAPK, and cPLA2 expression (data not shown).
Similarly, PGE2 production induced by
MEKK1 was
completely inhibited by the presence of 10 µM SC68376
(Fig. 5). The above observations confirm that the activation of p38 MAPK provides a crucial signaling mechanism which promotes Cox-2 expression and prostaglandin biosynthesis.
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SEK1/MKK4 Mediates MEKK1-induced COX-2 Expression--
Previous
data has suggested that SEK1/MKK4 is the immediate upstream kinase of
SAPK/JNK (26, 27) whereas MKK3 and MKK6 are immediately upstream of p38
MAPK (26, 28-30). Therefore, we decided to investigate whether this
proposed signaling cascade was responsible for expression of Cox-2 and
activation of p38 MAPK in MEKK1 inducible cells. In accordance with
previous findings, IPTG induction of
MEKK1 produced phosphorylation
of SEK1/MKK4 (Fig. 6A).
Furthermore, our experiments also demonstrated that the active
MEKK1
resulted in an increase of SEK1/MKK4 activity which was demonstrated by
its ability to enhance p38 MAPK phosphorylation (Fig. 6B).
However, phosphorylation of both MKK3 and MKK6 (as evidenced using an
anti-phospho-MKK3/MKK6 antibody) was not induced by IPTG stimulation
(Fig. 7A). Furthermore, these
findings were reinforced by immunocomplex kinase assays using p38 MAPK
as the substrate for either MKK3 (Fig. 7B) or MKK6
activities (Fig. 7C). As a positive control, anisomycin (31)
activated MKK3/MKK6 phosphorylation and activation in NIH 3T3 cells
(Fig. 7A, right panel). Thus in both circumstances, either
via kinase activity assay or direct phosphorylation analysis, MKK3 and
MKK6 were not activated with IPTG induction of p38 MAPK activity. These
findings suggest a signaling mechanism for activation of p38 MAPK
involving activation of SEK1/MKK4 induced by MEKK1.
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DISCUSSION |
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The MAPK family consists of at least three different subgroups which include: ERKs, JNKs (SAPKs), and p38 MAPK kinases. Among these groups, the JNK/SAPK and p38 MAPK pathways share activation by inflammatory cytokines, bacterial endotoxins, and environmental stress (17, 32-34), whereas the ERKs are preferentially activated by mitogens of the receptor tyrosine kinase family. Once activated, these MAPKs can phosphorylate and activate transcription factors which regulate gene expression. However, the physiological consequences for the cell following activation of these kinases remain unclear. Investigators have implicated the role of the JNK/SAPK and p38 MAPK pathway in the regulation of inflammatory mediators, cytokine production (14), platelet aggregation (35), and neuronal apoptosis (36). These kinases are in turn activated by upstream mitogen-activated protein/Erk kinases (MEKs) such as SAPK/ERK kinase-1 (SEK1, also known as MKK4) which is upstream of JNK/SAPK and MKK3/MKK6 which are upstream of p38 MAPK. These MEKs are in turn activated by MEK kinases of which MEKK 1, 2, 3, and 5 have been characterized to date (25, 37). Previous data has implicated MEKK1 and MEKK2 as preferential activators of the JNK/SAPK pathway. MEKK 3 exhibits preference for activation of the ERK pathway in vivo (37). Currently, the identity of the MEKK (or MEKKs) upstream of the p38 MAPK pathway is unknown. Although SEK1/MKK4 can activate p38 in vitro (26), previous studies failed to demonstrate that inducible expression of MEKK1 can activate p38 MAPK in vivo.
The data presented in this paper provides evidence for the role of
MEKK1 as an upstream activator of both p46 and p54 SAPK as well as p38
MAPK. More importantly, we provide evidence that cells overexpressing
MEKK1 can result in phosphorylation and activation of p38 MAPK. The
activation and phosphorylation of both p46 and p54 SAPK as well as p38
MAPK peak within 24 h of IPTG exposure.
MEKK1 induction does
not affect the phosphorylation and activation of ERK. These results
provide an important indication that intact mammalian cells induced to
overexpress
MEKK1 can phosphorylate and activate p38 MAPK,
suggesting that MEKK1 is one of the upstream kinases regulating the p38
MAPK pathway.
Interestingly, our experiments also demonstrate that MEKK1 induction
by IPTG results in increased activity of SEK1/MKK4, but not MKK3 or
MKK6. Co-transfection of 293 cells with MKK4 and p38 MAPK demonstrated
activation of the p38 MAPK (38). Similarly, our results indicate that
overexpression of the constitutively active form of SEK1 not only
increases JNK/SAPK activity but also enhances p38 MAPK activity.
However, overexpression of the kinase dead form of SEK1 decreases both
JNK/SAPK and p38 MAPK activity. The above results clearly suggest that
the MEKK1
SEK1/MKK4 kinase cascade not only activates JNK/SAPK, but
may also function as an upstream activator p38 MAPK pathway in
vivo. Since several reports suggest that MKK3 and MKK6 are the
immediate upstream kinases which activate p38 MAPK, it remains a
possibility that MKK3/MKK6 utilizes an alternative pathway for
activation of p38 MAPK.
Prostaglandins are formed by the dual action of a phospholipase A2 which releases arachidonic acid from membrane phospholipids and cyclooxygenase which converts its substrate to prostaglandin H2 which is subsequently converted to prostaglandins by cell-specific synthases. Cyclooxygenases (Cox-1 and Cox-2) function as rate-limiting enzymes in the biosynthesis of prostaglandins. Cox-2 has been identified as an inducible enzyme stimulated by cytokines, tumor promoters, and hormones (39). Previous work from our laboratory (21) has suggested that Cox-2 can be activated by IL-1 stimulation of renal mesangial cells. Cytokine treatment induces concomitant activation of JNK/SAPK and p38 MAPK (20, 21). Further evidence has demonstrated that pharmacologic inhibition of p38 MAPK by SC68376 inhibited cytokine-induced stimulation of Cox-2 and PGE2 synthesis, suggesting that p38 MAPK pathway is one of the important signaling mechanisms modulating cytokine-induced Cox-2 gene expression and prostaglandin biosynthesis (20).
In this study we show that 3T3 cells activated to overexpress MEKK1
exhibit up-regulated expression of Cox-2 and increased PGE2
synthesis. The Cox-2 specific inhibitor, NS-398 (0.1 µM), completely inhibited PGE2 production induced by the
activation of MEKK1. Thus, we believe that the activation of Cox-2
plays a critical role in MEKK1-induced PGE2 biosynthesis.
However, overexpression of
MEKK1 does not result in up-regulation of
Cox-1 or cPLA2 protein expression. In order to further
determine whether Cox-2 expression was mediated by either the SAPK/JNK
or the p38 MAPK pathway, we utilized SC68376, an oxazole compound which
inhibits p38 MAPK (IC50 of 2-5 µM). This
compound does not affect p38 MAPK phosphorylation, however, it inhibits
the ability of p38 MAPK to activate downstream targets. Furthermore,
the selectivity of this inhibitor has been shown by its inability to
affect either ERK or SAPK/JNK activity or phosphorylation. Using
SC68376, we demonstrated that inhibition of p38 MAPK completely blocked
the effects of
MEKK1 overexpression on Cox-2 expression and
prostaglandin biosynthesis. These observations clearly implicate the
role of p38 MAPK as an important mediator of MEKK1-induced Cox-2
expression and PGE2 production. Previous data from
Herschman's (40) laboratory demonstrated that the JNK pathway is
involved in platelet-derived growth factor-induced transcriptional
regulation of Cox-2. Therefore, their results in addition to our own,
suggest that the activation of both p38 MAPK and JNK pathways are
involved in MEKK1-induced Cox-2 expression and PGE2
synthesis.
How does MEKK1 activate p38 MAPK which, in turn, induces Cox-2 expression? Since our results indicate that MEKK1 activation increases SEK1/MKK4 activity without affecting MKK3 or MKK6 activity, our hypothesis is that SEK1/MKK4 may mediate this signaling mechanism. Overexpression of the constitutively active form of SEK1 results in increased phosphorylation of JNK/SAPK and p38 MAPK. The kinase negative mutation, SEK1-AL, inhibits phosphorylation of both aforementioned kinases. This evidence suggests that SEK1/MKK4 is the upstream kinase of both JNK/SAPK and p38 MAPK pathway. Furthermore, our experiments also demonstrate that activated SEK1 induces Cox-2 expression and PGE2 production whereas dominant negative SEK1 inhibits this response. SEK1-induced Cox-2 expression and PGE2 production are completely inhibited by p38 MAPK inhibitor. Together, these results illustrate the ability of SEK1/MKK4 to mediate MEKK1-induced Cox-2 expression and PGE2 production and serve as an upstream kinase modulating p38 MAPK activation.
The implications of the data presented link the MAPK pathway and the
prostaglandin generative pathway. Previous work has identified ERK as
an activator of cPLA2 in Chinese hamster ovary cells (18). Furthermore, recent data generated in thrombin-stimulated platelets indicate the possible role of p38 MAPK as an activator of
cPLA2 (19, 35). In contrast, our data demonstrates that NIH
3T3 cells stimulated to overexpress MEKK1 and activate p38 MAPK do not exhibit any perceptible changes for cPLA2 total
protein. We cannot exclude the possibility that increased
cPLA2 activity may be involved in the regulation of
MEKK1-induced prostaglandin biosynthesis. However, our data demonstrate
that the Cox-2 inhibitor can completely block MEKK1-induced
PGE2 production. Therefore, we believe that even if
cPLA2 is involved in this regulation, it would have a minimal role in PGE2 production when Cox-2 is inhibited.
These results suggest that cPLA2 is not the only target for
activation by the MAPK pathway, but Cox-2 is a key mediator.
Our experiments demonstrate that overexpression of MEKK1 increases
Cox-2 expression and PGE2 production which is completely blocked by SC68376, a pharmacologic inhibitor of p38 MAPK.
MEKK1 overexpression results in activation of both JNK/SAPK and p38 MAPK.
Induction of
MEKK1 increases SEK1/MKK4 but not MKK3 or MKK6
activity. Overexpression of the constitutively active form of SEK1
increases both p38 MAPK and JNK/SAPK phosphorylation, and enhances
PGE2 production and Cox-2 expression which can be completely inhibited by a p38 MAPK inhibitor. Overexpression of the
dominant negative SEK1-AL decreases the phosphorylation of both p38
MAPK and JNK/SAPK and reduces Cox-2 expression. Collectively, this data
suggests that MEKK1
SEK1/MKK4 may function as an upstream kinase
pathway which activates both p38 MAPK and JNK/SAPK and, in turn,
induces Cox-2 expression and PGE2 production. Together, our
current findings suggest a novel signaling pathway by which MEKK1
SEK/MKK4
p38 MAPK
Cox-2. This pathway provides further evidence for the role of the MAPK pathway in the mediation of prostaglandin biosynthesis and as a potential target for modulation of
the inflammatory response.
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FOOTNOTES |
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* This work was supported by United States Public Health Awards DK 38111 and DK 50606.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Professor of
Medicine and Molecular Biology and Pharmacology, Barnes Jewish
Hospital, 216 South Kingshighway, Renal Div., Box 8305, St. Louis, MO
63110. Tel.: 314-454-8495; Fax: 314-454-8430; E-mail:
morrison{at}pharmdec.wustl.edu.
1
The abbreviations used are: PLA2,
phospholipase A2; Cox, cyclooxygenase; PGE2,
prostaglandin E2; IL, interleukin; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; ERK,
extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase;
MBP, myelin basic protein; GST, glutathione S-transferase;
IPTG, isopropyl-1-thio--D-galactopyranoside; DTT,
dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MEK,
mitogen-activated protein/Erk kinases.
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