From the Departments of Medicine and
§ Pharmacology, University of Colorado Health Science
Center, Denver, Colorado 80262
Received for publication, April 26, 2000, and in revised form, September 1, 2000
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ABSTRACT |
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Mutations in ras genes have been
detected with high frequency in nonsmall cell lung cancer cells (NSCLC)
and contribute to transformed growth of these cells. It has previously
been shown that expression of oncogenic forms of Ras in these cells is
associated with elevated expression of cytosolic phospholipase
A2 (cPLA2) and cyclooxygenase-2 (COX-2),
resulting in high constitutive levels of prostaglandin production. To
determine whether expression of constitutively active Ras is sufficient
to induce expression of these enzymes in nontransformed cells, normal
lung epithelial cells were transfected with H-Ras. Stable expression of
H-Ras increased expression of cPLA2 and COX-2 protein.
Transient transfection with H-Ras increased promoter activity for both
enzymes. H-Ras expression also activated all three families of MAP
kinase: ERKs, JNKs, and p38 MAP kinase. Expression of constitutively
active Raf did not increase either cPLA2 or COX-2 promoter
activity, but inhibition of the ERK pathway with pharmacological agents or expression of dominant negative ERK partially blocked the
H-Ras-mediated induction of cPLA2 promoter activity.
Expression of dominant negative JNK kinases decreased cPLA2
promoter activity in NSCLC cell lines and inhibited H-Ras-mediated
induction in normal epithelial cells, whereas expression of constructs
encoding constitutively active JNKs increased promoter activity.
Inhibition of p38 MAP kinase or NF- Gain of function mutations in ras genes have been
detected in a variety of human tumors, including colon, prostate, and
lung. Activating mutations in Ras are associated with nonsmall cell lung cancer (NSCLC),1
occurring in ~30% of adenocarcinomas, and just under 10% of other NSCLC types (1). It is presumed that expression of these forms of Ras,
which lack intrinsic GTPase activity, mediate transformation by
constitutively activating downstream effector pathways. We recently
reported that expression of oncogenic forms of Ras was associated with
increased expression of cytosolic phospholipase A2
(cPLA2) and cyclooxygenase-2 (COX-2) in a panel of NSCLC
lines (2). The signaling pathways that mediate induction of
cPLA2 and COX-2 in these cells are undefined.
cPLA2 is the major intracellular form of PLA2,
which selectively hydrolyzes membrane phospholipids at the
sn-2 position and is the rate-limiting enzyme in the
regulated release of arachidonic acid (AA) (3, 4). Free AA is
metabolized through three major pathways to produce eicosanoids.
Cyclooxygenases (COX) convert AA to prostaglandins and thromboxane;
lipoxygenases produce leukotrienes and hydroxyeicosatetraenoic acids
and cytochrome P-450 epoxygenase produces epoxyeicosatrienoic
acids. Two forms of cyclooxygenase have been identified (5). COX-1
appears to be constitutively expressed in most cell types and is
associated with maintenance of vascular tone. COX-2, first identified
as an immediate early response gene (6), is induced in response to
mitogenic stimuli and associated with inflammation. High levels of
cPLA2 and COX-2 expression result in constitutively high
levels of prostaglandin production by NSCLC (7, 8). We (2) and others
(9-11) have demonstrated that nonsteroidal anti-inflammatory agents, which are inhibitors of eicosanoid production, blocked the transformed growth of NSCLC expressing Ras mutations. Because these agents do not
inhibit the growth of most nontransformed cells, it suggests that this
pathway plays a critical role in transformed growth.
Ras has been demonstrated to couple to multiple effector systems,
resulting in activation of distinct physiologic responses (see Ref. 12
for review). These pathways regulate cell proliferation as well as
changes in the cytoskeleton. An important class of Ras effectors is the
mitogen-activated protein kinase (MAP kinase) family. The "classic"
Ras-mediated pathway involves binding and activation of the
serine/threonine kinase Raf-1, which in turn activates the dual
specificity kinase MEK, resulting in activation of the extracellular
signal-regulated kinases (ERKs) (13). ERKs phosphorylate a number of
target proteins, including transcription factors and intracellular
enzymes (14). Expression of constitutively active forms of Ras also can
lead to increased activity of the other members of the MAP kinase
family: the stress-activated protein kinase/c-Jun amino-terminal
kinases (JNKs) and the p38 family of MAP kinase (15, 16). The
physiologic roles of JNKs and p38 MAP kinase appear to be complex and
may depend on the context in which these enzymes are activated.
Activation in response to cell stresses or growth factors is mediated
through specific upstream kinases: MKK4/7 for JNKs (17, 18) and MKK3/6
for p38 MAP kinase (19, 20). Both JNKs and p38 MAP kinase have been
shown to phosphorylate transcription factors, including c-Jun, ATF-2,
and members of the ets family including Elk-1 (21).
Multiple additional Ras effectors have been described. Ras activation
also leads to stimulation of phosphatidylinositol 3-kinase in some cell
types (22) and subsequent activation of the serine/threonine kinase Akt
(23). Activation of Akt may be critical for cell survival, possibly
through inhibition of apoptotic pathways (24, 25). Expression of Ras
mutants containing mutations in the effector domains suggests that
activation of multiple effector pathways is required for the full
physiologic response to activated Ras (26).
Although studies in NSCLC cell lines have implicated a role for Ras in
induction of cPLA2, these cells contain large number of
mutations and aberrations in signaling pathways, making it difficult to
define the critical molecular pathways regulating cPLA2
expression. We have therefore sought to examine the effects of
constitutively active forms of Ras on cPLA2 expression in
an untransformed lung epithelial cell line. In this study we report that expression of H-Ras is sufficient to mediate induction of cPLA2 expression in RL-65 cells, a neonatal, untransformed,
immortalized rat epithelial cell line (27), and have begun to define
the downstream effector pathways.
Reagents and Constructs--
The cPLA2 reporter
construct contains 2.4 kb of a 5'-region ligated into the promoterless
luciferase vector PA3luc (28). The COX-2 promoter ligated to a
promoterless luciferase vector was a gift of Dr. Harvey Herschman (UCLA
Medical Center, Los Angeles, CA). The Elk-Gal4, Jun-Gal4, and
ATF-2-Gal4 plasmids encode the DNA-binding domain of Gal4 fused to the
activation domain of Elk-1, c-Jun, and ATF-2, respectively. The UAS-luc
plasmid contains five Gal4 DNA-binding sites upstream from a minimal
thymidine kinase promoter with a luciferase reporter. The
The truncation mutant encoding the region from Cell Culture and Transfection--
RL-65 lung epithelial cells
were obtained from American Type Tissue Culture and grown in
Dulbecco's modified Eagle's medium/F-12 supplemented with
NaHCO3 (25 mM), sodium selenite (25 nM), insulin (5 µg/ml), human transferrin (10 µg/ml),
ethanolamine (100 µM), phosphoethanolamine (100 µM), hydrocortisone (0.5 µM), forskolin (5 µM), retinoic acid (50 nM), bovine pituitary
extract (150 µg/ml), penicillin (100 units/ml), and streptomycin (100 units/ml), according to the producer's recommendation. A549 human lung
cancer cells were obtained from the University of Colorado Cancer
Center Tissue Culture Core and grown in RPMI containing 10% fetal calf serum.
For transient transfections, cells were electroporated as described
previously (2). Briefly, two million cells were electroporated in
duplicate dishes using a geneZAPPER electroporator (IBI). Unless otherwise stated cells were transfected with 2 µg of the
cPLA2 promoter, 2 µg of CMV-
For stable transfection, H-Ras cDNA was inserted at the
HindIII site of the retroviral expression vector pMV7 (34)
and packaged into a replication-deficient retrovirus as described
previously (35). Polybrene (8 µg/ml) was added to the
retroviral-containing medium from the 293T packaging cells and filtered
prior to a 24-h incubation with subconfluent monolayers of RL-65 cells.
The infected cells were replated, selected for G418 resistance, and
expanded. Control cell lines (Neo) were selected by infecting cells
with virus lacking a cDNA insert. Clones were selected by
immunoblotting with anti-H-Ras antibodies.
Enzyme Assays and Immunoblotting--
Measurements of
cPLA2 activity were performed as described previously (36).
Briefly, cell were scraped into a homogenization buffer containing 50 mM Hepes, pH 7.5, 1 mM EGTA, 1 mM
EDTA, and a mixture of protease inhibitors using a Dounce homogenizer.
Extracts were centrifuged at 100,000 × g for 1 h,
and high speed supernatants were matched for protein. Activity was
determined using [14C]arachidonoylphosphatidylcholine in
the presence of 4 mM Ca2+. Results are
expressed as picomoles of arachidonic acid released/mg of protein. For
immunoblotting, cells were lysed in buffer containing 50 mM
For measurement of ERK activity, cells were transiently transfected
with an expression plasmid encoding HA-tagged ERK-1, along with either
H-Ras, BxB-Raf, or empty vector. After 48 h, cells were washed
with ice-cold phosphate-buffered saline, and lysed in lysis buffer (50 mM Based on our studies in NSCLC, we sought to determine whether
expression of constitutively active forms of Ras is
sufficient to induce cPLA2 in normal lung
epithelial cells. We therefore examined the effects of H-Ras expression
in a normal lung epithelial cell line (RL-65). RL-65 cells, derived
from rat lung, display an epithelial morphology (27) and do not form
colonies in soft agar. Stable cell lines expressing H-Ras were obtained
by retroviral infection of RL-65 cells and selection in G-418 as
described previously (35). Control cells (Neo) were infected with
retrovirus lacking a cDNA insert. Expression of cPLA2
and COX-2 were determined by immunoblotting and quantitated by
densitometry. The results of two representative clones of 10 examined
are shown in Fig. 1. Expression of
cPLA2 was increased 3- to 4-fold in these clones (Fig.
1A). Both clones expressing H-Ras also had significantly increased levels of cPLA2 activity, as measured in
cell-free extracts with exogenous substrate (Fig. 1B).
Increased expression of COX-2 was also observed in both clones (2- to
3-fold).
B had no effect on
cPLA2 expression. Truncational analysis revealed that the
region of the cPLA2 promoter from
58 to +12 contained
sufficient elements to mediate H-Ras induction. We conclude that
expression of oncogenic forms of Ras directly increases
cPLA2 expression in normal epithelial cells through
activation of the JNK and ERK pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B-luc
reporter consists of three consensus NF-
B sites upstream from a
promoterless luciferase vector. The dominant negative form of I-
B
(I-
B-(
1-36)) encodes an amino-terminal truncation of the
first 36 amino acids of I-
B, which contain two phosphorylation sites
required for NF-
B activation (29, 30). The dominant negative forms
of JNK kinases (DN-MKK4 and DN-MKK7), containing Lys to Met mutations
in the ATP binding sites, were prepared as described previously (31).
Fusion proteins consisting of MKK7 fused to specific JNK isoforms were
prepared as described by Zheng et al. (32), by fusing the
murine MKK7
1 (18) to the rat p46 JNK3 cDNA (33) or
JNK2.2 The dominant negative
form of ERK-1 contained similar Lys to Met mutations in the ATP-binding
site. Antibodies against cPLA2 and COX-2 were purchased
from Santa Cruz Biotechnologies (Santa Cruz, CA).
[14C]Arachidonoylphosphatidylcholine was from Amersham
Pharmacia Biotech.
102 to +12 (28) was
used to generate additional truncations by polymerase chain reaction.
The following primers were used: for the
58 mutant sense
primer: GGTACCACCTTAACATCCACAGAG; for the
37 mutant sense primer:
GGTACCAGCCCATTTCTTAGCCCCT; for the
12 mutant sense primer: GGTACCAGCGGGAGAAGACTTTCTCA. The antisense primer for all three was the
same and was derived from the PA3-Luc vector: CCACACCCTTAGGTAACCCAGTAGATCC.
-gal, and 2 µg of other
DNA (e.g. H-Ras, BxB-Raf). Total DNA concentration for each
transfection was matched with plasmid lacking an insert. Following
electroporation, cells were incubated in standard media for 48 h.
Cells were then harvested, and luciferase and
-galactosidase
activity was determined as described previously (2). Results are
expressed as luciferase units normalized to
-gal.
-glycerophosphate, pH 7.2, 1 mM EGTA, 2 mM
MgCl2, 100 µM sodium vanadate, 0.5% Triton
X-100. Extracts were centrifuged at 10,000 × g, and
supernatants were matched for protein. Extracts were separated by
SDS-polyacrylamide gel electrophoresis and transferred to an Immobilon
membrane. Blots were probed with either a monoclonal antibody against
cPLA2 or a rabbit polyclonal antibody against COX-2. Blots
were visualized using the ECL system from Amersham Pharmacia Biotech
(Arlington Heights, IL).
-glycerophosphate, pH 7.2, 0.5% Triton X-100, 5 mM EGTA, 100 µM sodium orthovanadate, 1 mM dithiothreitol, 2 mM MgCl2, 0.06 unit of aprotinin, 0.1 mM phenylmethylsulfonyl fluoride,
and 20 µM leupeptin). Insoluble material was pelleted by
centrifugation (10 min, 14,000 × g), and supernatants
were matched for protein. ERKs were immunoprecipitated by incubating for 2 h with anti-HA antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and protein G-Sepharose beads. The beads were washed three
times in lysis buffer, and kinase activity was determined using
epidermal growth factor-receptor peptide as described previously (35).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Induction of cPLA2 and COX-2
expression in RL-65 cells by H-Ras. RL-65 cells were stably
transfected with H-Ras or with empty vector (Neo), and
stable transfectants were selected for G418 resistance. Results are
shown for two representative clones (H-Ras-2 and H-Ras-31). A, cell
lysates were prepared, and equal amounts of lysate protein were
immunoblotted for cPLA2 (top panel) or COX-2
(bottom panel). B, cell lysates from Neo and two
clones were assayed for cPLA2 activity using
[14C]PC as substrate. Results represent the mean of
triplicate determinations ± S.E. *, p < 0.01, **, p < 0.05 versus Neo.
To examine mechanisms leading to increased cPLA2
expression, RL-65 cells were transiently cotransfected with a construct
encoding 2.4 kb of the rat cPLA2 promoter ligated to a
promoterless luciferase reporter (PA3-Luc) along with an expression
plasmid for H-Ras. Transient cotransfection of H-Ras increased
cPLA2 promoter activity 5- to 8-fold (Fig.
2A). Treatment of the cells
with BZA-5B, a farnesyl transferase inhibitor, which has been shown to
block Ras function (37), inhibited the induction of promoter activity, suggesting that this represents a direct effect of Ras. These data are
consistent with our previous finding demonstrating inhibition of
cPLA2 promoter activity and protein expression in two NSCLC lines (2). Transient expression of H-Ras also increased COX-2 promoter
activity (Fig. 2B), consistent with increased levels of
expression of both enzymes detected in stable transfectants.
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Ras activation results in activation of multiple MAP kinase pathways in other cell types. Activation of MAP kinase family members was determined by cotransfecting RL-65 cells with plasmids encoding the activation domain of specific transcription factors fused to the DNA-binding domain of Gal4, along with a plasmid containing five Gal-4 binding domains upstream of a promoterless luciferase reporter (UAS-luc). Separate experiments were performed with Elk-Gal4, Jun-Gal4, and ATF-2-Gal4. Elk-Gal4-mediated increases in luciferase are a measure of ERK activity, because ERKs are the major kinase phosphorylating Elk. Jun-Gal4 activation is a measure of JNK activity, and ATF-2-Gal4 activation is a measure of JNK and p38 MAP kinase activity. Expression of H-Ras resulted in increased luciferase activity with all three Gal-4 fusions (Table I), indicating that H-Ras activates all three MAP kinase pathways in these cells. We therefore sought to examine the role of these pathways in regulation of the cPLA2 promoter.
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Activation of ERKs via Ras is mediated through activation of the
serine-threonine kinase Raf. Expression of constitutively active Raf
(BxB-Raf) increased ERK activity as assessed with Elk-Gal4 fusions to
approximately the same extent as expression of H-Ras (Table I), but did
not significantly increase cPLA2 promoter activity (Fig.
2A) or COX-2 promoter activity (Fig. 2B). These results were confirmed by transient transfection of cells with epitope-tagged ERK-1, followed by immunoprecipitation and assay of
kinase activity in the immunoprecipitate (data not shown). To further
assess the role of the ERK pathway in regulation of the
cPLA2 promoter, transfected cells were treated with the MEK inhibitor PD98059 (38). Treatment with 50 µM
PD98059, a concentration that blocks ERK activation in these cells,
decreased the Ras-mediated induction of the cPLA2 promoter
by ~50%(Fig. 3A). In
separate studies, RL-65 cells were cotransfected with a construct
encoding a dominant negative form of ERK-1 or (DN-ERK). This construct has been used in studying the COX-2 promoter (39). Expression of DN-ERK
inhibited the Ras-mediated induction of the cPLA2 to a
similar extent as seen with PD98059 (Fig. 3B). Based on
these findings we would propose that activation of ERKs is necessary but not sufficient for induction of cPLA2 promoter
activity.
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The role of JNKs in regulation of cPLA2 expression was
examined by cotransfection with dominant negative JNK kinases
(DN-MKK4/7). Coexpression of either DN-MKK4 or DN-MKK7 inhibited the
H-Ras-mediated induction of cPLA2 promoter activity by
~60-70% (Fig. 4A).
Expression of both dominant negative constructs did not result in any
further inhibition. Blocking both the ERK and JNK pathways resulted in somewhat greater inhibition but failed to completely suppress in the
induction of the promoter by H-Ras (DN-MKK4, 40% inhibition; PD98059, 50% inhibition; DN-MKK4+PD98059, 61% inhibition).
Expression of wild-type MKK4 or MKK7 had no effect (data not shown).
Correspondingly, expression of DN-MKK4 or DN-MKK7 also inhibited
steady-state cPLA2 promoter activity in A549 cells (Fig.
4B), a human NSCLC line expressing oncogenic forms of Ras
(2). It has recently been reported that chimeric proteins of JNK
kinases fused to JNKs act as constitutively active JNKs (32). We
therefore constructed expression plasmids encoding MKK7 fused to
different JNK isoforms.2 Cells were cotransfected with
these constructs along with the cPLA2 promoter vector. As
shown in Fig. 5, expression of MKK7/JNK fusions increased promoter activity to ~50% of the level achieved with expression of H-Ras. Combined, the data in Figs. 4 and 5 indicate
that JNKs are involved in the induction of the cPLA2 promoter. The ability of ERKs to synergize with JNKs was examined by
cotransfection of cells with BxB-Raf along with the MKK7/JNK constructs. However, no further increase in promoter activity was found
with the combination of enzymes above that obtained with the MKK7/JNK
constructs alone (data not shown). To further test a cooperative role
for JNKs and ERKs in driving the promoter, we performed transient
transfection experiments with a constitutively active form of MEKK1.
Expression of MEKK1 in transient transfections increased both ERK and
JNK activity, as assessed by cotransfection with either Elk-Gal4 and
Jun-Gal4 (Table I). Expression of constitutively active MEKK1 also
markedly increased cPLA2 promoter activity (Fig. 6). Because expression of MEKK1 appeared
to systematically increase expression of -gal, presumably by
activating the CMV promoter, we chose to use expression of Renilla
luciferase driven by the thymidine kinase promoter to normalize for
transfection efficiency in these experiments.
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The role of p38 MAP kinase was examined by treating transfected cells with SB203580, a specific p38 MAP kinase inhibitor (40). At a concentration of 10 µM, which blocked H-Ras-mediated p38 MAP kinase activation (data not shown), no inhibition of the H-Ras-mediated induction of the cPLA2 promoter was detected (Fig. 3A). Transient expression of constitutively active MKK6, which specifically activates p38 MAP kinase (20), also failed to increase promoter activity or affect the induction observed with expression of H-Ras (data not shown).
The cPLA2 promoter contains a putative NF-B-site at
760 to
751(28), which has been suggested to be critical for
induction of the enzyme. Transient expression of H-Ras in RL-65 cells
increased NF-
B activity, as determined with a plasmid containing
three consensus NF-
B sites (
B-luc). This induction was blocked by expression of a dominant negative form of I-
B lacking the regulatory phosphorylation sites required for dissociation from NF-
B
(I-
B-(
1-36)) (Fig. 7A).
However, expression of I-
B-(
1-36) failed to block the increase
in cPLA2 promoter activity observed with H-Ras (Fig. 7B), indicating that NF-
B does not contribute to Ras
regulation of the cPLA2 promoter. Induction of the COX-2
promoter was also not inhibited by expression of I-
B-(
1-36),
and, in fact, induction of promoter activity by H-Ras was somewhat
increased (Fig. 7C).
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To begin to define the regions of the promoter required for induction
by H-Ras, we prepared a series of truncations of the original 2.4-kb
fragment (28). Removal of all but the last 58 bp of the promoter did
not significantly change either basal promoter activity or
H-Ras-mediated induction (Fig.
8A). Removal of an additional
region down to 37 decreased basal and H-Ras-mediated induction but
did not affect the -fold induction caused by Ras. Finally, truncation
down to
12 abolished both basal and H-Ras-induced promoter activity.
Parallel studies were also performed in A549 cells. Promoter
activity slightly increased in going from
2.4 kb to
58 bp
(Fig. 8B). A further decrease to
37 decreased
promoter activity by ~60%, and a further truncation to
12
abolished promoter activity completely. It thus appears that
removing all but the last 58 bp of the promoter does not
impair activity in either RL-65 cells or NSCLC, and the
construct containing the region from
37 still retains some
basal activity and can be induced by expression of H-Ras.
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DISCUSSION |
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Expression of oncogenic forms of Ras is observed in a variety of cancer cells and is believed to play a critical role in the dysregulated growth of these cells. However, the downstream signaling pathways mediating these effects are less well defined. This is complicated by the identification of a large number of potential Ras effectors (see Ref. 12).
Previous studies from our laboratory have demonstrated that expression of oncogenic Ras in NSCLC is associated with increased expression of cPLA2 and COX-2, resulting in constitutively high levels of eicosanoid production in these cells. The ability of inhibitors of eicosanoid production to block anchorage-independent growth of NSCLC (2), as well as the growth of xenografts of NSCLC in athymic mice3 indicates that this pathway is critical for transformed growth.
Because NSCLCs are likely to manifest mutations in multiple signaling pathways, we sought to determine whether expression of constitutively active Ras is sufficient to induce enzymes in the eicosanoid pathway in normal lung epithelial cells. From our studies we conclude that expression of H-Ras is sufficient to increase expression of both cPLA2 and COX-2 in a nontransformed cell line. This induction is mediated through transcriptional activation of both promoters and is a direct effect of Ras.
As in other cell types, activation of Ras leads to activation of all three members of the MAP kinase family. By expression of Gal4 fusion proteins that encode for the activation domains of known substrates of these kinases, we were able to demonstrate activation of ERKs, JNKs, and p38 MAP kinase. These results were confirmed by direct measurements of kinase activities in cells stably expressing H-Ras (data not shown). Activation of the JNK pathway appears to be both necessary and sufficient for induction of cPLA2. Dominant negative forms of JNK kinases inhibited the Ras-mediated induction. Expression of these constructs also inhibited steady-state cPLA2 promoter activity in NSCLC, suggesting that similar signaling pathways mediate induction of cPLA2 in these cells. Expression of constitutively active JNK constructs, obtained by expressing fusion proteins of JNK fused to MKK7 increased cPLA2 promoter activity. A critical requirement for JNK activation in cPLA2 induction is consistent with a preferential role for this pathway in the mitogenesis of NSCLC (41).
Because activation of JNK activity resulted in only half of the induction seen with H-Ras, it is likely that additional pathways are required for full activation. The ERK pathway appears to be necessary but not sufficient to induce cPLA2 expression. Treatment of cells with a specific pharmacological MEK inhibitor, or expression of a dominant negative form of ERK, both decreased the Ras-mediated induction of the promoter by half, but expression of constitutively active Raf failed to significantly increase promoter activity and did not increase the induction seen with the constitutively active JNK constructs alone.
Expression of MEKK1, which like Ras, stimulated the activity of all the MAP kinase families, resulted in increases in promoter activity similar to that seen with H-Ras. These findings suggest that Ras, and probably MEKK1, engage additional signaling pathways critical for induction of the cPLA2 promoter. This finding was confirmed by the observation that, whereas inhibition of both the ERK and the JNK pathway resulted in further inhibition of promoter activity, residual induction of the promoter by Ras was still observed.
Activation of p38 MAP kinase does not appear to represent this additional pathway. Neither inhibition of p38 MAP kinase with a specific inhibitor nor expression of MKK6, which specifically activates p38 MAP kinase, had any effect on promoter activity. We have also observed that the p38 MAP kinase inhibitor did not alter expression of cPLA2 or COX-2 in several NSCLCs (data not shown). Further studies are required to identify these pathways.
A number of studies have examined induction of cPLA2
expression in nontransformed cells in response to circulating factors. The most potent agents mediating this induction appear to be cytokines. Interleukin-1 induced cPLA2 expression in human synovial
fibroblasts (42). Tumor necrosis factor-
induced expression in Hep-2
cells (43), and HeLa cells (44), and interferon
induced
cPLA2 expression in BEAS-2B epithelial cells (45). A common
effector pathway activated by these agents is NF-
B, and the
observation that the cPLA2 contains a putative NF-
B
caused us to examine this pathway. Although expression of H-Ras
resulted in a marked activation of NF-
B, as assessed by a
NF-
B-sensitive reporter, dominant negative I-
B had no effect on
induction of the cPLA2 promoter, and somewhat potentiated
induction of the COX-2 promoter. It therefore appears that NF-
B is
not involved in the Ras-mediated induction of these enzymes. Analogous
studies are required to directly test whether this element of the
promoter plays a role in cytokine-mediated induction. A number of
studies have shown that expression of constitutively active forms of
Ras leads to the production of specific cytokines. In fibroblasts,
expression of H-Ras led to expression of granulocyte-colony stimulating
factor (CSF), granulocyte-macophage-CSF, and interleukin-1
(46).
Interestingly, elevated cytokine production has been demonstrated in a
number of nonsmall cell lung cancer lines (47-49). However, the
ability of H-Ras to induce the cPLA2 promoter in transient
transfection experiments, where only a small percentage of the cells
are expressing the oncogene, makes it unlikely that the induction
observed in RL-65 cells is mediated through cytokine production.
Induction of COX-2 has been shown to be mediated by both
NF-
B-dependent and -independent pathways. Induction in
response appears to be require activation of NF-
B (50), whereas
induction by platelet-derived growth factor or src signals through the
JNK pathway (51, 52).
The lack of a role for NF-B in regulating the cPLA2
promoter is consistent with our truncational analysis, which
demonstrated that removal of the NF-
B site did not affect
inducibility of the promoter by H-Ras in RL-65 cells, or steady-state
promoter activity in NSCLC. In fact, the minimal region of the promoter that we have defined from
58 to +12 does not contain any of the putative regulatory elements previously identified, suggesting that
novel transcription factors may be involved. Induction of COX-2 through
pathways involving H-Ras has been shown to also be mediated by JNKs, as
well as ERKs (53), and involves binding to a cAMP-response element in
the COX-2 promoter (52). It thus appears that expression of H-Ras leads
to induction of both COX-2 and cPLA2 through similar
downstream signaling pathways. However, these pathways diverge at the
level of the specific transcription factors, and cis-acting
regulatory factors regulating each promoter.
Increased expression of cPLA2 has also been reported in
Ras-transformed fibroblasts (54), resulting in constitutively elevated eicosanoid production, which presumably contributes to the transformed growth of those cells. Studies from our laboratory have demonstrated that, in vascular smooth muscle cells, activation of Ras also leads to
increased cPLA2 expression and eicosanoid production (35).
In these cells increased cPLA2 expression is not associated with increased proliferation but rather appears to be involved in
suppression of muscle-specific gene expression, leading to a less
differentiated phenotype. In conclusion, induction of cPLA2 represents a novel downstream effector of Ras, which is likely to play
an important role in the physiologic responses following Ras activation
in both normal and transformed cells.
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FOOTNOTES |
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* This work was supported by National Cancer Institute Grant SPORE CA-58187 and National Institutes of Health Grants DK-19928 and DK-39902.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: Division of Renal Diseases and Hypertension, Box C-281, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-315-6733; Fax: 303-315-4852; E-mail: Raphael.Nemenoff§UCHSC.edu.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M003581200
2 S.-Y. Han and L. E. Heasley, submitted for publication.
3 L. E. Heasley, D. Chan, and R. A. Nemenoff, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
NSCLC, nonsmall cell
lung cancer;
cPLA2, cytosolic phospholipase A2;
COX-2, cyclooxygenase-2, JNK, c-Jun amino-terminal kinase;
ERK, extracellular signal-regulated kinase;
NF-B, nuclear factor
B;
I-
B, inhibitor of NF-
B;
AA, arachidonic acid;
MAP, mitogen-activated protein;
MEK, MAP kinase/ERK kinase;
kb, kilobase(s);
bp, base pair(s);
DN, dominant negative;
CSF, colony-stimulating
factor.
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