From the Department of Life Science, Kwangju
Institute of Science and Technology, Kwang-Ju 500-712, § College of Pharmacy, Pusan National University, Pusan
609-735, and ¶ College of Pharmacy, Chonnam National University,
Kwang-Ju 500-757, Korea
Received for publication, March 5, 2001, and in revised form, April 24, 2001
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ABSTRACT |
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Activation of Ras signaling by growth factors has
been associated with gene regulation and cell proliferation. Here we
characterize the contributory role of cytosolic phospholipase
A2 in the oncogenic Ha-RasV12 signaling
pathway leading to activation of c-fos serum
response element (SRE) and transformation in Rat-2 fibroblasts. Using a c-fos SRE-luciferase reporter gene, we showed that
the transactivation of SRE by Ha-RasV12 is mainly via a
Rac-linked cascade, although the Raf-mitogen-activated protein
kinase cascade is required for full activation. In addition, Ha-RasV12-induced DNA synthesis was significantly
attenuated by microinjection of recombinant RacN17, a
dominant negative mutant of Rac1. To identify the mediators downstream
of Rac in the Ha-RasV12 signaling, we investigated the
involvement of cytosolic phospholipase A2. Oncogenic
Ha-RasV12-induced SRE activation was significantly
inhibited by either pretreatment with mepacrine, a phospholipase
A2 inhibitor, or cotransfection with the antisense
oligonucleotide of cytosolic phospholipase A2. We also
found cytosolic phospholipase A2 to be situated downstream
of Ha-RasV12 in a signal pathway leading to transformation.
Together, these results are indicative of mediatory roles of Rac and
cytosolic phospholipase A2 in the signaling pathway by
which Ha-RasV12 transactivates c-fos SRE and
transformation. Our findings point to cytosolic phospholipase
A2 as a novel potential target for suppressing oncogenic
Ha-RasV12 signaling in the cell.
Ras is a 21-kDa guanine nucleotide-binding protein that functions
as a molecular switch linking upstream activators, such as growth
factor receptors and nonreceptor tyrosine kinases, to several
downstream effectors (1, 2). The best characterized Ras-activated
pathway involves a Raf-MAPK1
cascade that includes Raf-1, MAPK kinase, and the mitogen-activated kinases extracellular signal-regulated kinases 1 and 2 (3-5), activation of which stimulates the transcriptional activity of p62TCF/Elk-1 (6-9). In addition, regulation of
c-fos transcription by serum response element (SRE) is
itself regulated by several proteins, including serum response factor
(SRF) and p62TCF/Elk-1 (10-13). In that regard, activation
of the MAPK cascade is known to stimulate interaction between
p62TCF/Elk-1 and SRF at SRE, thus providing a direct link
between MAPK activity and induction of c-fos (10, 11).
In addition to the Raf-MAPK cascade, an essential role of Rac, a member
of Rho family GTPases, in the Ras signaling pathway has been
demonstrated by several groups (14, 15). Rho family GTPases were once
thought to be involved primarily in organizing the actin cytoskeleton
(16). However, over the past several years, it has become evident that
Rho GTPases also carry out critical functions in the control of cell
proliferation and SRE activation (14, 15, 17-19). Unlike the Raf-MAPK
cascade, which activates SRE in a
p62TCF/Elk-1-dependent manner, Rac and other
Rho family GTPases were shown to stimulate SRE largely via a
p62TCF/Elk-1-independent pathway, which probably involves
direct activation of SRF (6-10, 19). Thus, the Rac-linked pathway is
suggested to act as another effector pathway of Ras in the cell (14,
15). Consistent with this, cooperation between Rac and Raf-MAPK
cascades was shown to cause transformation synergistically (15). In
addition, Rat-1 fibroblasts expressing RacV12, a constitutively
activated mutant of Rac1, displayed all the features of malignant
transformation (14), again supporting the role of Rac as a downstream
mediator of Ras in a signal pathway leading to transformation. However, the downstream elements of the Rac signaling cascade that mediates transformation remain to be identified. Although c-Jun N-terminal kinase (JNK) could be speculated as a downstream mediator, Rac mutants
defective in activating JNK were still shown to induce transformation
(20), suggesting that activation of JNK is probably not involved in
Rac-mediated cell transformation. It has been reported that the
p21-activated serine/threonine kinases might be involved in Rac
transformation, because expression of a kinase-deficient p21-activated
serine/threonine kinase 1 mutant inhibited Ras-transformation (21).
However, other groups reported that p21-activated serine/threonine kinase binding was dispensable for Rac-induced transformation, and thus
the role of p21-activated serine/threonine kinases in transformation is
still unclear (15).
It was recently demonstrated that when activated, Rac in turn activates
cytosolic phospholipase A2 (cPLA2), and there
is a resultant release of arachidonic acid (AA), a principal product of
cPLA2 activity (22-24). This makes it likely that
cPLA2 is a downstream mediator of Rac signaling. Consistent
with this, cPLA2 has been shown to be necessary for Rac in
mediating actin remodeling or c-fos SRE activation (23). For
instance, the inhibition of cPLA2 by either pretreatment
with mepacrine, a potent inhibitor of phospholipase A2, or
cotransfection with antisense cPLA2 oligonucleotide dramatically repressed Rac-induced SRE activation (23). In addition, in
actin remodeling, Rac was shown to stimulate growth
factor-dependent actin stress fiber formation via
cPLA2 and subsequent metabolism of AA metabolism by
5-lipoxygenase (25). Together, these observations place
cPLA2 downstream of Rac in a pathway leading to SRE
activation or actin remodeling. Thus, activated Ras may stimulate the
Rac-cPLA2-dependent pathway as well as the
Raf-MAPK-linked cascade to activate SRE and transformation.
The aim of the present study, therefore, was to characterize the
contribution made by cPLA2 to SRE activation and
transformation induced by oncogenic Ras. With the aid of a
c-fos SRE reporter plasmid, we found that transactivation of
SRE by Ha-RasV12 is mainly mediated via the
cPLA2-linked cascade. In addition, we present evidence
suggesting the role of cPLA2 as a downstream mediator of
Ha-RasV12 in a signaling to transformation.
Together, our findings point to cPLA2 as a novel target for
suppressing oncogenic Ha-RasV12 signaling in the cell.
Chemicals and Reagents--
Antisense cPLA2
oligonucleotide (GsTsGCTGGTAA GGATCTsAsT) is directed against codons
4-9 of the human cytosolic, Ca2+-dependent
PLA2 gene; two linkages are phosphothiolated at both the 5'
and 3' ends. Antisense and control (GsTsGCTCCTAAGTTTCTsAsT) cPLA2 oligonucleotides were purchased from Biomol (Plymouth
Meeting, PA). Mepacrine and wortmannin were from Sigma;
nordihydroguaretic acid, indomethacin, and AACOCF3 were
from Biomol; PD 098059 was from Research Biochemical
International (Natick, MA). Bromodeoxyuridine (BrdUrd) and
monoclonal anti-BrdUrd antibody were purchased from Amersham Pharmacia
Biotech. All other chemicals were from standard sources and were
molecular biology grade or higher.
Plasmids and DNA Manipulations--
Reporter genes pSRE-Luc and
pSREmt-Luc contain positions Cell Culture, DNA Transfection, and Luciferase
Assay--
Rat-2 fibroblasts were obtained from the American Type
Culture Collection (CRL 1764) and grown in DMEM supplemented with 0.1 mM nonessential amino acids (Life Technologies, Inc.), 10%
fetal bovine serum (FBS), and penicillin (50 units/ml)-streptomycin (50 mg/ml) (Life Technologies, Inc.) at 37 °C under a humidified atmosphere of 95% air, 5% CO2 (v/v). The stable
Rat2-HO6 clone expressing Ha-RasV12, a constitutively
activated Ha-Ras mutant, has been described previously (28). Transient
transfection was carried out by plating ~5 × 105
cells in 100-mm dishes for 24 h, after which calcium phosphate:DNA precipitates prepared with a total of 20 µg of DNA, including 3 µg
of pSRE-Luc and 5 µg of a GTPase expression vector (e.g. pEXV-RacN17), were added to each dish (29). To control for
variations in cell number and transfection efficiency, all clones were
cotransfected with 1 µg of pCMV-
Luciferase activity was assayed in 10-µl samples of extract using a
luciferase assay system (Promega) according to the manufacturer's protocol; luciferase luminescence was counted in a luminometer (Turner
Design, TD-20/20) and normalized to cotransfected [3H]AA Release--
Rat-2 cells were plated to a
density of 1 × 105 cells/well in 6-well plates and
maintained in DMEM supplemented with 10% FBS. After 4 h, 2 µCi/ml [3H]AA (Amersham Pharmacia Biotech) was added to
each well and incubated for an additional 36 h, after which the
cells were washed at least three times with medium. The cells were then
transfected with pSPORT-Ha-RasWT or
pSPORT-Ha-RasV12 using the calcium phosphate:DNA
precipitation method. After incubating the cells for 6 h at
37 °C, the medium was exchanged for fresh DMEM supplemented with
0.5% FBS and incubated for another 6 h; [3H]AA
released into the medium during that period was assayed by scintillation counting. At the end of each experiment, the cells were
solubilized in 0.5 ml of EtOH, and total intracellular incorporation was determined so that counts could be corrected to intracellular pools
of AA.
Microinjection and Immunofluorescence Microscopy--
The
procedure for microinjection of purified fusion protein has been
described. Briefly, Rat-2 cells were plated on scored 12-mm coverslips,
incubated for 24 h, and then rendered quiescent by starvation for
48 h in serum-free DMEM. On the day of injection, the coverslips
were transferred to 35-mm culture dishes, and Ha-Ras or
Ha-RasV12, along with 2 mg/ml Rat IgG or
RacN17 protein, was microinjected using glass capillary
needles, yielding about 150-200 microinjected cells/coverslip. Two h
after microinjection, BrdUrd was added, and the cells were incubated
for an additional 16 h at 37 °C. The cells were then fixed for
20 min at 22 °C in acid alcohol (90% EtOH, 5% acetic acid, 5%
H2O), after which they were incubated for 60 min at
22 °C with mouse anti-BrdUrd antibody, followed by 60 min with
rhodamine-conjugated anti-mouse IgG antibody and then 60 min with
fluorescein isothiocyanate-labeled anti-rat IgG. The coverslips were
then washed intensively and mounted. DNA synthesis by individual cells
was assessed as a function of BrdUrd incorporation, which was
photographed and analyzed using an Axioskop fluorescence microscope
(Carl Zeiss). The immunofluorescent staining of the injected cells
indicated that about 80% of the cells were successfully microinjected.
Western Blot Analysis--
Protein samples were heated at
95 °C for 5 min and subjected to SDS-polyacrylamide gel
electrophoresis on 8% acrylamide gels, followed by transfer to
polyvinylidine difluoride membranes for 2 h at 100 V using a Novex
wet transfer unit. Membranes were then blocked overnight in
Tris-buffered saline with 0.01% (v/v) Tween 20 and 5% (w/v) nonfat
dried milk, after which they were incubated for 2 h with the
primary antibody (anti-cPLA2 or anti-tubulin) in
Tris-buffered saline and then for 1 h with horseradish
peroxidase-conjugated secondary antibody. The blots were developed
using enhanced chemiluminescence kits (ECL, Amersham Pharmacia
Biotech). Bands on XAR-5 film (Eastman Kodak Co.) corresponding to
cPLA2 were measured by densitometry.
Soft Agar Analysis and Cell Growth Experiments--
For the soft
agar clonability assays, 103 or 104 cells
suspended in 4 ml of agar (Noble, Difco; 0.3% in growth medium with
10% FBS) were poured onto a 6-ml basal layer (0.6% agar in
DMEM) in 100-mm plates. The plates were incubated at 37 °C for 10 days, and the colonies were counted by staining them with
p-iodonitro tetrazolium violet dye as described previously
(30). For the cell growth experiments, Rat-2 or Rat2-HO6 cells were
plated onto a 6-well plate (105 cells/plate) in 1 ml of
DMEM containing 10% FBS. On the next day, the medium was replaced with
serum-free medium or serum-free medium containing mepacrine. The viable
cell number was counted at 36 h later.
Leukotriene LTC4/D4/E4
Assays--
Rat-2 and Rat2-HO6 cells (3 × 105) were
plated on 60-mm dishes and incubated in DMEM supplemented with 10% FBS
for 24 h. Then, the culture medium was replaced with DMEM
containing 0.5% FBS for an additional 24 h. For the measurements
of the level of LTC4/D4/E4, the
plates were rinsed twice with cold phosphate-buffered saline and mixed
with 4 times their volume of absolute ethanol and left at 4 °C for
30 min. The resulting precipitate was removed by centrifugation at
10,000 rpm for 30 min at 4 °C. The ethanolic supernatant and culture
medium containing the leukotrienes were collected through a C2
reverse phase column (Amersham Pharmacia Biotech, RPN 1903). The methyl
formate in the eluted samples was then removed by evaporation under
vacuum, and the samples reconstituted in assay buffer were stored under
argon at p62TCF/Elk-1-independent Activation of c-fos SRE by
Ha-RasV12--
As a first step in characterizing the
downstream signaling cascades elicited by constitutively activated,
oncogenic Ha-RasV12, we investigated the mechanisms by
which they stimulate c-fos SRE. Because activation of
c-fos by normal Ha-RasWT was previously shown to
be dependent upon p62TCF/Elk-1 binding to SRF (3-5), we
initially used a luciferase reporter gene under the control of a human
c-fos minimal promoter fused to SRE oligonucleotide to
assess the extent to which Ha-RasV12 requires
p62TCF/Elk-1 binding to stimulate SRE (Fig.
1A). Cotransfection with either pSPORT-Ha-RasWT or pSPORT-Ha-RasV12
caused dose-dependent activation of c-fos SRE
(Fig. 1B). To assess the role of Elk-1/p62TCF,
pSREmt-Luc, containing a mutant oligonucleotide
(AGG to TGT), with an
intact SRF interaction site but lacking a p62TCF/Elk-1
binding site (13, 31), was used as a reporter gene (Fig.
1A). Unlike transfection of pSPORT-Ha-RasWT,
which activated c-fos SRE in a
p62TCF/Elk-1-dependent manner, transfection
with pSPORT-Ha-RasV12 stimulated both pSRE-Luc and
pSREmt-Luc to similar degrees (~12-fold increase over a pSPORT
control vector), indicating that Ha-RasV12 acts
independently of p62TCF/Elk-1 (Fig. 1C).
Preferential Sensitivity of Ha-RasV12 to Inhibition of
Rac--
Ras activates the MAPK and Rac pathways via interactions with
Raf-1 and PI 3-kinase, respectively, and proper function of both
pathways is required for efficient mitogenesis or transformation by Ras
(14, 15, 26). To obtain further insight into the signaling mechanism by
which Ha-RasV12 mediates c-fos SRE activation,
therefore, we examined the effect of cotransfecting vectors encoding
dominant negative mutants of either Rac1 (RacN17) (17, 18)
or Raf-1 (craf301) (27). As shown in Fig.
2A, both RacN17
and craf301 significantly inhibited Ha-RasWT-induced SRE
activation. On the other hand, although strongly inhibited by
RacN17, transactivation of SRE by Ha-RasV12 was
only partially affected (~20% inhibition) by craf301 (Fig. 2A). Activation of SRE by RhoAV14, a
constitutively activated RhoA mutant transfected as a control, was
unaffected by either RacN17 or craf301 (Fig.
2A). SRE activation by Ha-RasWT thus appears to
be via a pathway dependent on both Raf-MAPK and Rac, although the
contribution of the latter was relatively small. Activation by
Ha-RasV12, by contrast, appears to be largely via the
Rac-linked pathway.
Consistent with the aforementioned results, PD 098059, a specific MAPK
kinase inhibitor (32), markedly inhibited SRE activation by
Ha-RasWT (e.g. ~75% inhibition at 10 µM) but had a substantially smaller effect on
Ha-RasV12-induced activation (Fig. 2B). The
levels of expression of Ha-RasWT and Ha-RasV12
were similar (data not shown), meaning that the reduced sensitivity to
inhibition of Raf-MAPK on the part of Ha-RasV12 was not due
to the differential expression of Ras isoforms. Together with the
p62TCF/Elk-1-independent nature (Fig. 1C),
therefore, Ha-RasV12 signaling to SRE seems to be largely
via the Rac-linked pathway, although the Raf-MAPK cascade seems to
still be required for efficient signaling.
Role of PI 3-Kinase in Ha-RasV12 Signaling--
It has
been reported that PI 3-kinase is situated downstream of Ras in the
pathway leading to Rac activation (26). Therefore, to further
investigate the contributing role of Rac-linked signaling to
Ha-RasV12-induced SRE activation, we tested the effect of
wortmannin (33), a specific PI 3-kinase inhibitor, and observed that
wortmannin selectively and dose-dependently inhibited SRE
activation by Ha-RasV12 but had minimal effects on
activation by Ha-RasWT (Fig.
3A). As an example,
pretreatment with 0.1 µM wortmannin inhibited
Ha-RasV12-induced SRE activation by ~70% but had little
effect on Ha-RasWT-induced SRE activation. Similarly,
transient transfection with a dominant negative PI 3-kinase mutant,
pSG5- Preferential Inhibition of Ha-RasV12-induced DNA
Synthesis by Microinjection of RacN17--
In another
approach aimed at evaluating the role of Rac in Ha-RasV12
signaling, recombinant RacN17 protein was microinjected
into cells, and Ha-RasV12-stimulated DNA synthesis was
assessed by indirect immunofluorescence. Groups of 150-200 quiescent
cells on coverslips were microinjected with Ha-RasWT,
Ha-RasV12, or RacN17 plus
Ha-RasV12 or Ha-RasWT along with control rat
IgG and then labeled with BrdUrd. Ha-RasWT stimulated DNA
synthesis in ~40% of the microinjected cells, as indicated by their
BrdUrd-labeled nuclei, whereas Ha-RasV12 stimulated 70% of
cells to incorporate BrdUrd (Fig. 4).
Coinjection of RacN17 reduced the fraction of cells
stimulated to initiate DNA synthesis by Ha-RasV12 from 70 to 40% but had little effect on Ha-RasWT-induced DNA
synthesis. The results of three independent experiments are graphically
summarized in Fig. 4B; they provide direct evidence that Rac
is a critical link in the signal transduction pathway by which
Ha-RasV12 stimulates DNA synthesis and, presumably, cell
proliferation.
cPLA2 as a Downstream Mediator of
Ha-RasV12 Signaling--
In fibroblasts, Rac stimulates
growth factor-dependent actin stress fiber formation via
PLA2 activation and subsequent metabolism of AA by
lipoxygenase (22). In addition, we observed that cPLA2 is a
principal downstream mediator of Rac-induced activation of c-fos SRE, JNK, and reactive oxygen species (23, 24, 34). It
seems probable, therefore, that cPLA2 is situated
downstream of Ha-RasV12 and mediates Rac-linked signals. To
test this likelihood, the contributing role of cPLA2 in
Ha-RasWT or Ha-RasV12-induced SRE activation
was examined using an antisense oligonucleotide against
cPLA2. As shown in Fig.
5A, cotransfection of the
antisense cPLA2 oligonucleotide, but not the control
oligonucleotide, dose-dependently inhibited
Ha-RasV12-induced SRE activation (e.g. ~70%
inhibition by 0.5 µM antisense cPLA2).
The antisense oligonucleotide inhibited
Ha-RasWT-induced SRE activation to a smaller degree.
Separately, the expression level of cPLA2 was evaluated on
Western blot analysis using cPLA2-specific rabbit
polyclonal antibodies (Fig. 5A). The expression level of cPLA2 is clearly diminished by cotransfection with 0.5 µM antisense, but not control, oligonucleotides, whereas
no change was observed in the level of tubulin, which was used as a
control. These results suggest that cPLA2 is clearly
involved in Ha-RasV12-induced signaling to SRE activation.
Similarly, pretreating cells with mepacrine (22), a specific inhibitor
of PLA2, dose-dependently inhibited
Ha-RasV12-induced SRE activation but had a smaller effect
on Ha-RasWT-induced SRE activation (Fig. 5B). As
an example, 2.5 µM mepacrine reduced
Ha-RasV12-induced SRE activation by ~70% but reduced
Ha-RasWT-induced SRE activation by only 25-30%,
indicating that PLA2 activity is preferentially
involved in the Ha-RasV12-signaling pathway.
Encouraged by the above results, we tested whether the level of AA, a
principal product of cPLA2, is indeed enhanced by
Ha-RasV12 in the cells. Consistent with the proposed role
of cPLA2 as a downstream mediator of Ha-RasV12,
transient transfection with Ha-RasV12 expression plasmid
significantly elevated levels of AA in a dose-dependent manner, an effect that was selectively inhibited by mepacrine (Fig.
6). Together, our results strongly
suggest the mediatory role of cPLA2 in
Ha-RasV12 signaling in the cell.
Mepacrine, a PLA2 Inhibitor, Suppresses
Ha-RasV12 Transformation--
Considering the reported
activity of Ha-RasV12 as a transforming oncogene,
the cPLA2-linked cascade may also play a critical role in
the transforming activity of Ha-RasV12. To test this
possibility, we examined whether cPLA2 inhibition shows any
transformation suppression activity to Rat2-HO6, a transformed Rat-2
cell line stably expressing Ha-RasV12 (28). By
dose-dependent analysis as shown in Fig.
7A, mepacrine (1 µM) was shown to cause a significantly reduced cell
growth in Rat2-HO6, with little effect on the growth of Rat-2 normal cells. In addition, morphological reversion of Rat2-HO6 by mepacrine (1 µM) was observed, but there was no effect on the
morphology of Rat-2 cells (Fig. 7B). Clearly, the
morphology of the oncogenic Ras-transformed Rat2-HO6 cells was reverted
to that of Rat-2 parental cells, showing a flat and dispersed
phenotype. In accordance with this result, mepacrine clearly diminished
the colony formation in soft agar plates of Rat-HO6 cells (Fig.
7C), suggesting that cPLA2 is critical for the
transforming activity of Ha-RasV12. Thus, the
cPLA2-linked cascade by Ha-RasV12 appears to be
commonly essential for the signaling cascades induced by
Ha-RasV12 leading to c-fos SRE expression and
transformation. Importantly, the resulted preferential sensitivity of
Ha-RasV12-transformed cells to cPLA2 inhibition
led us to suggest that cPLA2 could be an ideal target
against Ha-RasV12-induced transformation.
We showed that the Rac-linked cascade apparently plays a crucial
role in Ha-RasV12 signaling leading to transactivation of
c-fos SRE and transformation. Several approaches were taken
to show that the Rac-linked cascade is required for
Ha-RasV12-induced signaling. First, cotransfection of
RacN17 dramatically inhibited SRE stimulation by
Ha-RasV12 but had only minor effects on
Ha-RasWT-induced signaling (Fig. 2A). Besides
c-fos SRE activation, Ha-RasV12-induced DNA
synthesis is also preferentially mediated by the Rac-linked pathway, as
shown in the microinjection experiment (Fig. 4). The aforementioned
findings provide direct evidence that Rac is a crucial link in the
signal transduction pathway mediating Ha-RasV12-induced DNA
synthesis and, presumably, cell proliferation.
In addition, our findings suggest that cPLA2 is situated
downstream of Ha-RasV12, mediating Ha-RasV12
signaling to transformation. For example, cotransfection of antisense oligonucleotide against cPLA2 or pretreatment with
mepacrine markedly inhibited Ha-RasV12-induced SRE
activation but inhibited Ha-RasWT-induced activation to a
much smaller degree (Fig. 5), suggesting that cPLA2 is
preferentially involved in the signaling by Ha-RasV12. The
preferential involvement of cPLA2 in oncogenic
Ha-RasV12 signaling points to cPLA2 as a
possible target for suppressing the transforming activity of
Ha-RasV12. Consistent with this idea, treatment of Rat2-HO6
cells with mepacrine significantly reduced the growth and colony
formation in soft agar plates of Rat2-HO6 (Fig. 7). Furthermore, we
observed that by transient cotransfection with plasmids expressing
Ha-RasV12 and annexin-1, which was shown to specifically
inhibit cPLA2 by direct interaction (35, 36), the number of
transformed foci formations was significantly reduced compared with
that by Ha-RasV12 alone (data not shown), thus again
suggesting the mediatory role of cPLA2 in oncogenic
Ha-RasV12 signaling. In support of the suggested role of
cPLA2 as a downstream mediator of
Ha-RasV12, transient expression of Ha-RasV12
induced a dose-dependent generation of AA, a principal
product of cPLA2, an effect that was selectively inhibited
by mepacrine (Fig. 6A). Interestingly, we also observed that
the expression level of cPLA2 protein is elevated in
Rat2-HO6 cells (data not shown). Thus, longer-term exposure of
Ha-RasV12 is suggested to induce the regulation of
cPLA2 at the level of gene expression as well as activity.
Similar to our result, it has been reported that microinjection of Ras
oncogene protein results in the stimulation of PLA2
activity and that the effects of Ras protein on the activity of
PLA2 reflect a critical aspect of the mitogenic activity of
Ras proteins (37). In addition, the increased expression of
cPLA2 protein was reported in human cancer cell lines
harboring oncogenic Ras mutations (38).
From the results of the present study, we speculate that the
Ha-RasV12-evoked cascade leading to SRE activation or
transformation may be somewhat different from that evoked by wild-type
Ha-Ras, although the exact mechanism by which the differential effects
are accomplished is not clear. In addition, the details of the
Ha-RasV12-mediated signaling pathway to cPLA2
stimulation remains obscure. Indeed, it has been well characterized
that a Raf-MAPK-linked cascade, in addition to the Rac-linked
cascade, contributes to cPLA2 stimulation (39, 40).
For example, according to a report from Leslie and co-workers (39),
extracellular signal-regulated kinases phosphorylate cPLA2
on Ser-505, which modestly increases its catalytic activity.
Recent reports also show that p38 kinase is the MAPK responsible for
cPLA2 phosphorylation in thrombin- and collagen-activated
platelets and in tumor necrosis factor- On the other hand, a number of reports have suggested that
cPLA2 is stimulated via Rac (22, 23, 34, 44, 45). As reported previously, cPLA2 mediates a variety of cellular
activities (e.g. stimulation of c-fos SRE or JNK
and generation of reactive oxygen species, among others) that are
induced by Rac activation, thus suggesting stimulation of
cPLA2 by Rac1. This means that cPLA2
stimulation may be either Rac-dependent or Raf-MAPK
kinase-MAPK-dependent (Rac-independent), and our earlier
findings indicate that in certain cases, the former predominates. For
example, C2-ceramide stimulates cPLA2 activity in Rat-2
fibroblasts (about a 4.2-fold increase, as measured by AA release), and
the effect is dramatically inhibited by RacN17 expression (46).
Similarly, we observed that epidermal growth factor-evoked
cPLA2 activity in Rat-2 fibroblasts is largely Rac-dependent (31), that RacN17 inhibits cPLA2
activation induced by hydrogen peroxide (47), and that phorbol
12-myristate 13-acetate stimulation of SRE is selectively suppressed by
inhibiting cPLA2 (35). More recently, we observed that
phorbol 12-myristate 13-acetate induces SRE activation primarily via a
Rac-cPLA2-dependent cascade, because phorbol
12-myristate 13-acetate-induced cPLA2 activation was shown
to be dramatically inhibited by RacN17
expression.2
The aforementioned findings strongly indicate that Rac is a
principal mediator of cPLA2 stimulation in some cases,
although Raf-MAPK may contribute to full activation. We would predict a similar scenario for cPLA2 stimulation by oncogenic
Ha-RasV12. Interestingly, Goldschmidt-Clermont (48) and
co-workers reported that reactive oxygen species generated by
Ha-RasV12 somehow mediate oncogenic signaling in
fibroblasts, and they proposed that Rac, not Raf-MAPK kinase-MAPK, is
involved in the signaling to reactive oxygen species generation, thus
mediating Ha-RasV12 signaling to transformation. Our recent
results suggest that Rac signaling to reactive oxygen species
generation is through cPLA2 activation in Rat-2 fibroblasts
(44), thus suggesting a mediatory role of a Rac-cPLA2
cascade for the efficient transformation by oncogenic
Ha-RasV12. In any event, there is an apparent signaling
link between Ha-RasV12 and cPLA2 stimulation.
In support of the signaling link between Ras and cPLA2,
Warner et al. (49) reported that Ras is essential for
epidermal growth factor-induced AA release in Rat-1 fibroblasts.
We do not yet know in detail the downstream molecule(s) by
which cPLA2 mediates oncogenic H-RasV12
signaling. Nonetheless, because nordihydroguaretic acid, a general lipoxygenase inhibitor, markedly inhibited the colony formation in soft
agar plates of Rat2-HO6 (Fig.
8A), we predict that
leukotriene synthesis by lipoxygenase is probably involved. In
contrast, no detectable inhibition was observed by treatment with
indomethacin, a cyclooxygenase inhibitor (Fig. 8A).
Therefore, leukotriene synthesis by lipoxygenase is possibly situated
downstream of cPLA2, mediating Ha-RasV12
signaling to transformation. Consistent with the proposed role of
leukotriene as downstream mediator, Rat2-HO6 cells show a significantly enhanced level of leukotriene
C4/D4/E4 compared with Rat-2 cells, an effect that was selectively inhibited by mepacrine (Fig.
8B).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
53 to +45 of the c-fos
promoter situated upstream of the luciferase gene, with wild-type or
mutant SRE oligonucleotides (23-mers) inserted at the
53 position
(23). pSPORT-Ha-Ras and pSPORT-Ha-RasV12 were from
Dr. P. Kirshmeier (Schering-Plough Research Institute). pEXV,
pEXV-RacN17, and pEXV-RhoV14
(RhoAval14) expression vectors were from Dr. A. Hall
(University College London, London, UK). Dominant negative mutants of
PI 3-kinase (pSG5-
p85
) and Raf-1 (craf301, a kinase-defective
form of Raf-1) were from Dr. J. Downward (Imperial Cancer Research
Center) and Dr. U. R. Rapp (University of Wurzburg),
respectively (26, 27).
GAL, a eukaryotic expression
vector in which the Escherichia coli
-galactosidase (lac
Z) structural gene is under the transcriptional control of the
cytomegalovirus promoter. The total quantity of DNA in each
transfection was kept constant at 20 µg by adding appropriate
quantities of sonicated calf thymus DNA (Sigma). After incubating
6 h with the calcium phosphate:DNA precipitates, the cells were
rinsed twice with phosphate-buffered saline before incubating in fresh
DMEM supplemented with 0.5% FBS for an additional 36 h.
Thereafter, cell extracts were prepared by rinsing each plate twice
with phosphate-buffered saline and lysing the cells in 0.2 ml of lysis
solution (0.2 M Tris, pH 7.6, 0.1% Triton X-100). The
lysed cells were scraped and spun for 1 min, and the supernatants were
assayed for protein concentration and luciferase and
-galactosidase activities.
-galactosidase activity.
-galactosidase assays were carried out using 50-µl aliquots of extract diluted with 100 µl of H2O and 150 µl of 2× reaction buffer (3 mg/ml
O-nitrophenyl-
-galactopyranoside, 2 mM
MgCl2, 61 mM Na2HPO4,
39 mM NaH2PO4, 100 mM
2-mercaptoethanol). When a faint yellow color appeared, the reactions
were stopped by the addition of 350 µl of 1 M
Na2CO3, and the optical density at 410 nm was
measured in a spectrophotometer. The results were then used to
normalize luciferase activity to transfection efficiency. Protein
concentrations were routinely measured using the Bradford procedure
with Bio-Rad dye reagent (Bio-Rad) and using bovine serum albumin as a
standard. Transfection experiments were performed in duplicate with two
independently isolated sets, and the results were averaged.
50 °C until assayed for
LTC4/D4/E4 using a specific
enzyme-linked immunosorbent assay (Amersham Pharmacia Biotech, RPN 224)
as instructed by the manufacturer. The enzyme immunoassay was
calibrated with standard LTC4/D4/E4
from 0.75 to 48 pg/well. The sensitivity, defined as the amount of
LTC4/D4/E4 needed to reduce zero
dose binding, was 0.5 pg/well, which is equivalent to 10 pg/ml. The
statistical significance of
LTC4/D4/E4 assays was assessed with
analysis of variance (ANOVA) (p < 0.01).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
p62TCF/Elk-1-independent
activation of SRE by oncogenic Ha-RasV12.
A, schematic diagram illustrating the pSRE-luciferase
reporter gene vectors used in the study. The structures of constructs
containing either wild-type or mutant SRE oligonucleotide sequences
(23-mer) inserted at the 53 position of a truncated c-fos
promoter fused to the luciferase gene are shown. The filled
circles denote the methylation interference pattern for the SRF
ternary complex with p62TCF/Elk-1. B,
dose-dependent activation of SRE by transient
cotransfection of pSPORT, pSPORT-Ha-Ras, or
pSPORT-Ha-RasV12. Transfectants were serum-deprived in DMEM
with 0.5% (v/v) FBS for 36 h before luciferase assay.
C, p62TCF/Elk1-independent SRE activation by
Ha-RasV12. Reporter gene vectors, pSRE-Luc (3 µg) or
pSREmt-Luc (3 µg), were transiently cotransfected with 5 µg of
pSPORT, pSPORT-Ha-Ras, or pSPORT-Ha-RasV12. Relative
activation of pSRE-Luc was calculated as described under
"Experimental Procedures."
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Fig. 2.
Preferential sensitivity of normal
Ha-RasWT to inhibition of the Raf-MAPK cascade.
A, pSRE-Luc (3 µg) and expression vectors encoding Ha-Ras,
Ha-RasV12, or RhoAV14 (RhoAval14)
(5 µg) were transiently cotransfected with 5 µg of dominant
negative mutants, RacN17, or craf301. Total amounts of DNA
were kept constant at 20 µg with calf thymus carrier DNA.
Transfectants were serum-deprived for 36 h prior to luciferase
assay. B, effect of PD 090859 on Ha-Ras or
Ha-RasV12-mediated SRE activation. pSRE-Luc (3 µg) was
transiently cotransfected with pSPORT, pSPORT-Ha-Ras, or
pSPORT-Ha-RasV12 (5 µg), after which the transfectants
were exposed to the indicated concentrations of PD 098059 for 24 h
before harvest for assay.
p85
, dose-dependently inhibited the effects of
Ha-RasV12 but attenuated the effects of wild-type Ha-Ras to
a much smaller degree (Fig. 3B).
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Fig. 3.
Preferential sensitivity of
Ha-RasV12 to inhibition of PI 3-kinase. A,
effect of wortmannin on Ha-Ras- and Ha-RasV12-induced SRE
activation. Rat-2 cells transiently cotransfected with pSRE-Luc (3 µg) and pSPORT, pSPORT-Ha-Ras, or pSPORT-Ha-RasV12 (5 µg) were exposed to the indicated concentrations of wortmannin for
24 h prior to harvest for assay. B, pSRE-Luc and
pSPORT, pSPORT-Ha-Ras, or pSPORT-Ha-RasV12 were transiently
cotransfected with selected amounts (0, 1, 3, and 5 µg) of
pSG5-del.p85 , an expression vector encoding a dominant negative PI
3-kinase mutant. Transfectants were serum-deprived in DMEM containing
0.5% (v/v) FBS for 36 h prior to luciferase assay.
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Fig. 4.
Effect of microinjected RacN17
protein on Ha-RasV12-induced DNA synthesis.
A, immunofluorescence of microinjected Rat-2 cells. Phase
contrast (A-C) and fluorescence (D-I) images of
serum-starved cells injected with 7.25 mg/ml RacN17 protein
(A, D, G), Ha-RasV12
protein (B, E, H), or both
(C, F, I), along with Rat IgG (2 mg/ml). Injected cells were identified by cytoplasmic fluorescein
isothiocyanate-labeling (D-F), and BrdUrd
(BrdU) incorporation was identified by nuclear
rhodamine labeling (G-I), as described under
"Experimental Procedures." B, shaded and
open bars represent injected and uninjected cells on the
same coverslips, respectively. The data are the means ± S.E. of
three independent experiments.
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Fig. 5.
Preferential sensitivity of
Ha-RasV12-induced SRE activation to cPLA2
inhibition. A, relative luciferase activity after
pSRE-Luc (3 µg) and pSPORT, pSPORT-Ha-Ras, or
pSPORT-Ha-RasV12 (5 µg) were transiently cotransfected
with the indicated quantities of antisense or control cPLA2
oligonucleotides. Data are expressed as percents of each control
(transfection without oligonucleotides). Levels of protein expression
of cPLA2 and tubulin (control) are shown by immunoblots.
Data are representative of three independent experiments. B,
effect of mepacrine (1, 2.5 µM) on
Ha-RasV12-mediated SRE activation. pSRE-Luc (3 µg) was
transiently cotransfected with pSPORT, pSPORT-Ha-Ras, or
pSPORT-Ha-RasV12 (5 µg), after which the transfectants
were exposed to the indicated concentrations of mepacrine for 12 h
before harvest for assay.
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Fig. 6.
Preferential stimulation of cPLA2
by Ha-RasV12. A, [3H]AA
released from Rat-2 cells prelabeled for 36 h with
[3H]AA (0.5 µCi/ml) and transiently transfected with
pSPORT, pSPORT-Ha-Ras, or pSPORT-Ha-RasV12 (0, 1, or 5 µg). [3H]AA released into the medium was quantified as
described under "Experimental Procedures." B,
mepacrine-sensitive release of [3H]AA. Transfectants were
incubated with 1 µM mepacrine for 6 h prior to
collecting the medium for assay.
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Fig. 7.
cPLA2 inhibition by mepacrine
causes a selective inhibition to the transforming activity by
Ha-RasV12. A, suppression of cancerous cell
growth of Rat2-HO6 by mepacrine in a dose-dependent manner.
Rat-2 and Rat2-HO6 cells were placed to a density of 1 × 105 cells per plate and added with mepacrine (1 µM) or control buffer (phosphate-buffered saline) after
6 h of cell splitting, and the cell number was counted every day.
This experiment was performed in duplicate with two independently
isolated sets, and the results were averaged. B,
morphological reversion of Rat2-HO6 cells by mepacrine. Rat-2 and
Rat2-HO6 cells growing with and without mepacrine (1 µM)
for 24 h were photographed with a phase contrast microscope.
C, inhibition of soft agar growth of Rat2-HO6 in the
presence of mepacrine (1 µM). For the soft agar
clonability assays, 103 or 104 cells
(last panel) suspended in 4 ml of agar (Noble, Difco; 0.3%
in growth medium with 10% FBS) were poured onto a 6-ml base layer
(0.6% agar in DMEM) in 100-mm plates. The plates were incubated at
37 °C for 10 days, and the colonies were counted by staining them
with p-iodonitro tetrazolium violet dye. Data are
representative of two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-stimulated neutrophils
(41-43). However, there is increasing evidence that in some cell
types, phosphorylation of cPLA2 by MAPK is not sufficient to induce AA release. For example, phosphorylation of cPLA2
on Ser-505 is not required for AA release from thrombin-stimulated platelets, but it may be involved in the platelet response to collagen
(41, 42). Thus phosphorylation does not provide definitive proof of a
role for Raf-MAPK in cPLA2 activation, although
Raf-MAPK is generally assumed to contribute at least somewhat.
More information will be required to clarify the role of MAPK pathways
in the regulation of evoked cPLA2 activity.
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Fig. 8.
Leukotriene synthesis by lipoxygenase is
possibly involved in the transforming activity by
Ha-RasV12. A, inhibition of soft agar
growth of Rat2-HO6 in the presence of nordihydroguaretic acid (1 µM). For the soft agar clonability assays,
104 cells of Rat2-HO6 suspended in 4 ml of agar (Noble,
Difco; 0.3% in growth medium with 10% FBS), added with
nordihydroguaretic acid (0.1 or 1 µM) and indomethacin (1 or 10 µM), were poured onto a 6-ml base layer (0.6% agar
in DMEM) in 100-mm plates. The plates were incubated at 37 °C for 10 days, and the colonies were counted by staining them with
p-iodonitro tetrazolium violet dye. Data are representative
of two independent experiments. B, enhanced generation of
leukotriene C4/D4/E4 by Rat2-HO6
and its suppression by mepacrine. Rat-2 and Rat2-HO6 cells were grown
for 24 h in DMEM containing 10% FBS and then harvested for the
quantitation of levels of the leukotriene
C4/D4/E4 mixture as described under
"Experimental Procedures." Values represent the average of three
independent experiments.
In summary, our results clearly indicate that Ha-RasV12 is
selectively sensitive to cPLA2 inhibition, and thus it may
be appropriate to evaluate cPLA2 as a novel target for
suppressing Ha-RasV12 transformation. Given that
cPLA2 is probably a downstream mediator of
Ha-RasV12-induced transformation, further characterization
of the cPLA2 signaling cascade would appear to be a pivotal
step toward a better understanding of oncogenic,
Ha-RasV12-mediated signal transduction.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. A. Hall (University College,
London, UK) and Dr. J. Downward (Imperial Cancer Research
Center, London, UK) for providing us with the RacN17 expression
plasmid and a dominant negative mutant of PI 3-kinase,
pSG5-p85
, respectively.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the National Research Laboratory, Molecular Medical Science Research (02-03-A-05), the Interdisciplinary Research program of the KOSEF (1999-2-20700-004-5), Life Phenomena and Function Research Group Program-2000 from the Ministry of Science & Technology, and the Brain Korea 21 program.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. Tel.: 62-970-2495;
Fax: 62-970-2484; E-mail: jkim@eunhasu.kjist.ac.kr.
Published, JBC Papers in Press, April 25, 2001, DOI 10.1074/jbc.M101975200
2 M.-H. Yoo, C.-H. Woo, H.-J. You, S.-H. Cho, B.-C. Kim, J.-E. Choi, J.-S. Chun, B. H. Jhun, T.-S. Kim, and J.-H. Kim, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; SRE, serum response element; SRF, serum response factor; JNK, c-Jun N-terminal kinase; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; AA, arachidonic acid; PI 3-kinase, phosphoinositide 3-kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; LT, leukotriene; WT, wild-type; Luc, luciferase; mt, mutant.
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