From the Department of Chemistry and Biochemistry, School of Medicine and Revelle College, University of California at San Diego, La Jolla, California 92093-0601
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ABSTRACT |
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Prostaglandins are known to play a central role
in the initiation of labor in humans, and amnionic cells constitute a
major source of these compounds. Prostaglandin synthesis and release by
amnion cells in response to hormones and ligands takes place after a
characteristic 4-5 h lag. However, we report herein that free
arachidonic acid (AA), the metabolic precursor of prostaglandins, can
be induced at much shorter times (1 h) in human amnionic WISH cells by
phorbol 12-myristate 13-acetate (PMA) through activation of protein
kinase C (PKC
). WISH cells were found to possess both cytosolic
group IV phospholipase A2 (cPLA2) and
Group VI Ca2+-independent phospholipase A2
(iPLA2). Of these, the cPLA2 was found to be
the likely mediator of AA mobilization in PMA-activated WISH cells. PMA
also activates phospholipase D (PLD) in these cells and ethanol, a
compound that inhibits PLD-mediated phosphatidic acid (PA) formation,
blocked AA release. Moreover, prevention of PA dephosphorylation by the
PA phosphohydrolase inhibitors propranolol and bromoenol lactone,
resulted in inhibition of AA release by PMA-treated WISH cells.
Collectively, these data suggest that activation of cPLA2
and attendant AA release by phorbol esters in WISH cells requires prior
generation of DAG by phosphatidate phosphohydrolase.
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INTRODUCTION |
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Phospholipase A2 (PLA2)1 constitutes a key regulatory step in the production of prostaglandins (PGs) because it catalyzes the release of arachidonic acid (AA) from the sn-2 position of phospholipids, making the fatty acid accessible to PG synthases. At present, ten different PLA2 groups have been identified (1-3). Those include five groups of small secreted PLA2s, which show millimolar requirements for Ca2+ (Groups I, II, III, V, and X), and two groups of intracellular, high molecular weight enzymes (Groups IV and VI). Group IV PLA2, or cPLA2, is Ca2+-dependent and a highly regulated enzyme (4); whereas, Group VI PLA2, or iPLA2, is Ca2+-independent (5). At present it is not known whether Group VI iPLA2 is subjected to post-translational regulation (5). Among these PLA2s, Groups II, V, and IV have been shown to be the responsible enzymes for prostaglandin generation in different systems (6-8). On the other hand, Group VI PLA2 has been implicated in basal fatty acid remodeling reactions (5, 9).
PGs, especially PGE2 and PGF2, are thought
to play a central role in the initiation of spantaneous labor in humans
by mediating physiological effects such as uterine contractions (10) and cervical softening and effacement (11). The human amnion has the
capacity of producing PGE2, and it is known that changes in
this capacity occur in association with parturition (12). Thus,
numerous studies have focused on PG production by amnionic cells,
mostly at the level of PG synthase enzymes (13-15). Surprisingly however, the study of PLA2 in amnion cells has received
much less attention. Myatt and co-workers (16, 17) recently documented the enhancement of cPLA2 protein by interleukin (IL)-1
in amnionic WISH cells after an 8-h treatment, which correlates with
PGE2 production under those conditions. These studies were
conducted at late stages of activation (several hours). Unfortunately,
no information is available on the events that occur immediately after
amnionic cell activation (i.e. up to 1 h). In the
current study, we have investigated the signaling mechanisms that
operate at the early stages of WISH cell activation and lead to
increased PLA2 activity and concomitant AA release.
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MATERIALS AND METHODS |
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Materials--
Human WISH cells (established amnion cell line)
were obtained from the American Type Culture Collection (Rockville,
MD). Iscove's modified Dulbecco's medium (endotoxin <0.05 ng/ml) was
from BioWhittaker (Walkersville, MD). Fetal bovine serum was from
Hyclone Labs. (Logan, UT). Trypsin/EDTA solution was purchased from
Irvine Scientific (Santa Ana, CA).
(5,6,8,9,11,12,1,15-3H)Arachidonic acid (specific activity
100 Ci/mmol), (9,10-3H)palmitic acid (specific activity
43.3 Ci/mmol), and
1-palmitoyl-2-[14C]arachidonyl-sn-glycero-3-phosphocholine
(specific activity 55 mCi/mmol) were obtained from NEN Life Science
Products (Boston, MA).
1-Palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine
(specific activity 59 mCi/mmol). Bromoenol lactone (BEL) was from
Biomol (Plymouth Meeting, PA). Group VI iPLA2 antiserum was
generously provided by Dr. Simon Jones (Genetics Institute, Cambridge,
MA). Group IV cPLA2 antibodies were kindly provided by Dr.
Ruth Kramer (Lilly Research Laboratories, Indianapolis, IN). The
sPLA2 inhibitor LY311727 was kindly provided by Dr. Edward
Mihelich (Lilly Research Laboratories). Rabbit polyclonal anti-ERK-2
that recognizes p42 and p44 MAPKs were a generous gift from Dr. Alan
Saltiel (Parke-Davis, Ann Arbor, MI). Methyl arachidonyl fluorophosphonate (MAFP) was from Cayman (Ann Arbor, MI). Antibodies against PKC, PKC
, and PKC
and the polyconal anti-Raf-1
antibody were from Santa Cruz Biotechnology (Santa Cruz, CA).
Antibodies against PKC
, PKC
, and PKC
were purchased from
Calbiochem.
Cell Culture-- WISH cells (18) were maintained in Iscove's modified Dulbecco's medium suplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere at 90% air and 10% CO2. The cells were subcultured twice weekly by trypsinization and, when used for experiments, were seeded into 24-well (2 × 105 cells/well, NUNC) or 12-well plates (5 × 105 cells/well, Corning Inc.), or 100 × 20-mm dishes (2.5 × 106 cells/dish, Falcon). After a 2-day growth, the cells, at 90% confluency, were rinsed with serum-free medium and incubated for 1-2 h before stimulation.
PLA2 Assays-- Ca2+-dependent PLA2 assay was conducted as described by Kramer et al. (20), with slight modifications. Briefly, aliquots of WISH cell homogenates were incubated for 30 min at 37 °C in 2 mM CaCl2, 50 mM Hepes, pH 7.4, and sonicated liposomes consisting of 2 µM 1-palmitoyl-2-[14C]arachidonyl-sn-glycero-3-phosphocholine and 1 µM 1,2-dioleoyl-sn-glycerol. Products were analyzed by thin-layer chromatography using the system n-hexane/diethyl ether/acetic acid (70:30:1). To measure Ca2+-independent PLA2 activity, aliquots of WISH cell homogenates were incubated for 30 min at 37 °C in 100 mM Hepes, pH 7.5, 5 mM EDTA, 0.8 mM ATP, 400 µM Triton X-100, 100 mM 1-palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine, in a final volume of 500 µl. The substrate was used in the form of mixed micelles of Triton X-100/phospholipid at a molar ratio 4:1, obtained by a combination of heating, vortex mixing, and water bath sonication (21). Products were analyzed by thin-layer chtomatography using the same system described above.
[3H]AA Release-- Radiolabeling of the cells with [3H]AA was achieved by including 0.5 µCi [3H]/106 cells in the culture medium 20 h before stimulation. Cells were stimulated with PMA (25-50 ng/ml) for different periods of time in the presence of 1 mg/ml bovine serum albumin (fatty acid-free). The supernatants were removed and cleared of detached cells by centrifugation, and radioactivity was counted by liquid scintillation. When inhibitors were used, they were added to the cells 30 min before PMA was added to the medium.
PKC Activity--
A Promega kit (PKC assay system, V5910) was
used for this purpose, and the manufacturer instructions were followed.
Briefly, the cells were washed with phosphate-buffered saline,
resuspended in 0.5 ml of extraction buffer (25 mM Tris-HCl,
pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM -mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml
aprotinin, and 0.5 mM PMSF) at 4 °C, and homogenized
using a Dounce homogenizer. Lysates were centrifuged in a
microcentrifuge for 5 min at 4 °C, and supernatants were passed
through a 1-ml column of DEAE cellulose pre-equilibrated with the
extraction buffer. PKC was extracted by using the extraction buffer
plus 200 mM NaCl. PKC activity was then measured with a
biotinylated peptide substrate of PKC that binds to Streptavidin-disks.
PKC was assayed in 25 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.3 mg/ml phosphatidylserine, 30 µg/ml DAG, 25 µM EGTA, 400 µM
CaCl2, and [
-32P]ATP (5000 pmol, 100-200
cpm/pmol). Reactions were run with and without phospholipids and
stopped by adding 7.5 M guanidine-HCl. Aliquots from the
reactions were spotted in streptavidin-disks and washed with 1 M NaCl, and radioactivity was quantified by scintillation
counting.
PKC Translocation Assays--
Experiments were carried out as
described elsewhere (20). Briefly, cells were plated in 100-mm dishes.
Control and PMA-stimulated WISH cells were washed with
phosphate-buffered saline and homogenized with a Dounce homogenizer in
a buffer consisting of 20 mM Tris-HCl, 2 mM
EDTA, 10 mM EGTA, 1 mM PMSF, 20 µM leupeptin, 20 µM aprotinin, and 0.1%
-mercaptoethanol, pH 7.5. Homogenates were centrifuged at 500 × g for 5 min at 4 °C. The resulting supernatant was
centrifuged at 100,000 × g for 1 h at 4 °C to
separate soluble and membrane fractions. Membranes were washed with
buffer, resuspended, and sonicated. After protein quantification, 100 µg were separated by SDS-polyacrylamide gel electrophoresis (10%
gel) and transferred to Immobilon-P membrane (Millipore). Western
blotting analysis was performed by using specific antibodies against
PKC isoforms.
PLD Activation-- The cells were labeled with [3H]palmitic acid (3 µCi/106 cells) for 20 h, and the stimulations were carried out in the presence of 1% ethanol. At the end of the reactions, total lipids were extracted (21, 22) and phosphatidylethanol (PEt), a specific product of PLD activity, was resolved from cellular lipids by thin-layer chromatography on silica-gel G plates (Whatman), using the upper phase of a system consisting of ethyl acetate/isooctane/acetic acid/water (13:2:3:10, v/v/v/v). The lipids were identified by comparison with authentic standards run in the same plate and visualized by iodine vapors. Radioactivity was determined by liquid scintillation counting.
DAG Production-- Cells were labeled overnight with either [3H]palmitic acid (3 µCi/106 cells) or [3H]arachidonic acid (0.5 µCi/106 cells), washed, and were incubated with inhibitors for 30 min prior to stimulation with 25-50 ng/ml PMA. At the indicated times, supernatants were removed, cell monolayers were scraped, and total lipids were extracted (22). For separation of DAG, lipids were separated by thin-layer chromatography with n-hexane/diethyl ether/water (70:30:1, v/v/v). The plates were run twice in this system if monoacylglycerol determination was required as well. Radioactivity in DAG and monoacylglycerol was determined by liquid scintillation counting.
MAPK and PLA2 Immunoblotting Studies-- Cells were serum-starved for 24 h, preincubated with 100 µM propranolol for 30 min, and stimulated with 25-50 ng/ml PMA for 1 h. Cells were washed and then lysed in a buffer consisting of 1 mM Hepes, 0.5% Triton X-100, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin at 4 °C. Protein was quantified, and a 100-µg aliquot was analyzed by Western blot under conditions previously described (23), with antibodies against ERK-2 that recognizes both p42 and p44 MAPKs or against PLA2 isoforms.
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RESULTS |
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Phospholipase A2 Activation in Human WISH Cells-- One of the best established systems for the study of lipid mediators in amnionic cells is the human-derived cell line WISH (24). This cell line was established from altered colonies appearing in a subculture of a primary monolayer of amnion cells (18, 19). WISH cells produce large amounts of PGs after prolonged exposure to phorbol esters (18 h) (13, 20, 25). To characterize the steps in the regulation of PG production that occur during the early stages of WISH cell activation, we measured [3H]AA release in these cells after incubation with 50 ng/ml PMA for different time periods (Fig. 1A). After a time lag of approximately 30 min, significant release of [3H]AA was observed at 60 min, reaching a plateau at about 75 min. Typically, a 2-5-fold increase over basal unstimulated release was detected at an optimal PMA concentration of 25 ng/ml (Fig. 1B).
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PAP Is Involved in PMA-induced AA Release in WISH Cells-- The effect of BEL on PMA-induced AA release was examined, and the results are shown in Fig. 4A. BEL was previously identified as a potent iPLA2 inhibitor (5), but more recent results have demonstrated its lack of specificity for iPLA2 in cells, as BEL also potently inhibits another key enzyme in lipid metabolism, i.e. the Mg2+-dependent PA phosphohydrolase (PAP) (27). In fact, the inhibitory effect of BEL on PMA-induced AA release shown in Fig. 4A cannot be attributed to iPLA2 inhibition on the basis of the results presented in Fig. 2A, which show that the iPLA2 activity does not change upon PMA treatment while the cPLA2 activity does. Moreover, BEL inhibited the PMA-induced DAG production in cells labeled with [3H]palmitic acid (Fig. 4B), indicating that BEL is indeed inhibiting the PAP. Thus, the possibility arises that the BEL effect on AA release is due to PAP inhibition. To investigate this possibility, we employed propranolol, a well established PAP inhibitor. Analogous to BEL, propranolol appreciably inhibited the PMA-induced [3H]AA release (Fig. 5A) and [3H]palmitate-labeled DAG production (Fig. 5B).
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Phospholipase D Involvement in PMA-induced AA Mobilization-- One major route for the production of the PA to be used by PAP is the PLD-mediated hydrolysis of phospholipids (28). Fig. 6A shows that, in the presence of ethanol, PMA induced the time-dependent accumulation of PEt in WISH cells, reflecting PLD activation. PEt is a specific product of PLD action in the presence of ethanol. Accumulation of PEt was detected at much earlier time points than AA release (i.e. 15 min), suggesting the possibility that products of PLD may be implicated in cPLA2 activation and attendant AA release. Should this be the case, one would expect that addition of exogenous PLD to the WISH cells would mimic the activating effect of PMA on AA release. Fig. 6, B and C, shows that treatment of the WISH cells with exogenous PLD produced a time- and dose-dependent release of [3H]AA. PLD activation by PMA was unaffected by BEL, confirming that PLD is upstream of the BEL-sensitive step, i.e. the PAP (data not shown).
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Involvement of PKC in PMA-induced AA
Mobilization--
Activation of PKC, particularly the
isoform, has
previously been shown to constitute a major route for PLD activation in a wide variety of cell types (22, 28, 29). WISH cells express PKC
,
,
, and
.3 Of them,
only PKC
was translocated to the membrane fraction after cellular
activation with PMA (Fig. 9A).
The translocation took place very early, being observed at 5 min and
disappearing completely from the cytosolic fraction after 30 min of
stimulation. To assess whether or not PKC
translocation to the
membrane fraction was mediated by PAP-derived DAG, experiments were
conducted in the presence of BEL. BEL affected neither PKC
binding
to the membrane (Fig. 9B) nor PKC activity, as measured
in vitro using a commercial kit (PKC assay system V5910,
Promega) (data not shown). Like BEL, propranolol did not have any
effect on PKC
translocation (data not shown). Involvement of PKC
in PMA-induced AA release was confirmed by the use of the inhibitor
Gö7874, specific for Ca2+-dependent
isoforms, which inhibited [3H]AA release (Fig.
9C).
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PMA-induced Phosphorylation Cascades in WISH
Cells--
PMA-induced signaling events have been shown to include
activation of p42/p44MAPK downstream of PKC (30). In
keeping with this notion, PMA was able to induce a mobility shift on
SDS-polyacrylamide gel electrophoresis, indicating phosphorylation and
activation of these kinases. cPLA2, which in some instances
lies downstream of p42/p44MAPK (4), also experienced a
mobility shift after PMA treatment (Fig.
10). Interestingly, after BEL or
propranolol treatment, conditions that decrease AA release, the MAPK
and cPLA2 mobility shifts were not prevented (Fig. 10). In
fact, even in the absence of PMA, both inhibitors were able to induce a
cPLA2 mobility shift. Moreover, neither BEL nor propranolol
affected the intrinsic activity of the cPLA2 as measured in
homogenates from PMA-treated cells (26; data not shown). These data
indicate that inhibition of AA mobilization by PAP blockers is not due
to inhibition of the signaling mechanism through which the
cPLA2 increases its intrinsic specific activity, i.e. phosphorylation by MAPKs.
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DISCUSSION |
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Very little is known about how free AA levels are regulated in the amnion, the PLA2s responsible for such a regulation, and the molecular mechanisms involved. In the present study, we have uncovered phosphatidate phosphohydrolase as a novel regulatory element within the signaling cascade that results in cPLA2 activation and AA release during the early stages of activation of the amnionic-like cell line, WISH.
BEL has recently been used as a tool to investigate whether the iPLA2 has a role in AA mobilization in different cell types, as this inhibitor possesses over 1000-fold selectivity for the iPLA2 among other PLA2 forms (31). However, BEL also inhibits the magnesium-dependent PAP (26, 32). In P388D1 cells, the IC50 for inhibition by BEL of the PAP is 8 µM, i.e. almost identical to that for inhibition of iPLA2 in the same cells (21). We have found that BEL appreciably blunts AA release in activated WISH cells; however, it blunts DAG production as well, demonstrating that the drug is affecting the PAP in addition to any effect on the iPLA2. Moreover, the inhibitory effects of BEL on AA release herein reported appear to be a consequence of PAP inhibition, since blockage of this enzyme by two other unrelated strategies, i.e. (i) direct inhibition of the enzyme by propranolol and (ii) PAP substrate depletion by ethanol, gave the same inhibitory effect on AA release. Moreover, unlike the cPLA2, the iPLA2 specific activity does not increase after cell stimulation. Our recent attempts at inhibiting iPLA2 expression by using antisense mRNA technology in WISH cells, similar to those succesfully used in P388D1 macrophages (33), have failed to detect any effect on AA release, which reinforces the notion that the iPLA2 is not an effector in this process.4 It should be noted, however, that unlike P388D1 cells, the WISH cells express very high levels of iPLA2 protein, as judged by immunoblot analysis (results not shown).
Collectively, the aforementioned results constitute, to the best of our knowledge, the first evidence implicating PAP and the metabolite it produces, DAG, in the regulation of AA mobilization. Since PAP is usually coupled to PLD, as is the case in WISH cells as well, and PLD uses phosphatidylcholine as a preferred substrate, it seems logical to assume that the DAG involved in cPLA2 activation in WISH cells arises mainly from phosphatidylcholine.
Besides the identification of PAP as an important regulator of the AA release response in WISH cells, another striking feature of the current work is the finding that none of the PAP-inhibition strategies used resulted in alteration of the intrinsic activity of the cPLA2, as measured by both phosphorylation and in vitro activity. This finding raises interesting questions as to the role of DAG in cPLA2 activation in WISH cells. The currently accepted paradigm of cPLA2 activation by stimuli considers the involvement of two different signaling branches that converge at the cPLA2 itself (4). The first one is a phosphorylation cascade that culminates in the phosphorylation of the cPLA2 and serves to increase the intrinsic activity of the enzyme. Both the nature of the kinase involved as well as the site of phosphorylation remain controversial (4). The second branch for cPLA2 signaling involves the translocation of the enzyme from the cytosol to the membrane, where its substrate is localized. This translocation, which does not modulate the cPLA2 activity itself, is currently believed to be mediated by increased Ca2+ availability although other factors may also be involved, especially in the case of stimuli like PMA which do not promote Ca2+ increases (34, 35). The two pathways for cPLA2 activation are independent of each other, but both appear to be required for proper cPLA2 activation and subsequent AA release (4).
Because PAP is not involved in regulating the cPLA2 phosphorylation cascade, it appears logical to suggest that PAP is involved in regulating binding of the cPLA2 to the membrane. DAG is long known to cause perturbations in membrane bilayers, rendering them susceptible to PLA2 attack (36). DAG accumulation in membranes has the effect of spreading apart the phospholipid headgroups, thereby making the glycerol backbone more accessible to the PLA2. Indeed, one of the most commonly utilized methods for detecting cPLA2 activity is based on this principle (20). Thus, at the same time the specific activity of the cPLA2 is increased as a result of its enhanced phosphorylation, local accumulations of DAG in the membrane allow for an appropriate substrate presentation for the enzyme. When the cPLA2 translocates to the membrane as a a result of increased Ca2+ availability and/or other factors, the enzyme will find optimal conditions to initiate AA release. The key role for DAG in this process is strengthened by the observation that, in the presence of BEL or propranolol, both p42/p44MAPK and cPLA2 become phosphorylated normally in WISH cells but no AA release is induced.
DAG is regarded as a universal activator of PKCs. However it is
highly unlikely that the PAP-derived DAG is playing such a role in
PMA-induced AA release in WISH cells because, in this system, the only
PKC that becomes activated is the isoform, which is the one that
PMA directly activates. PMA-induced PKC
is most likely the upstream
event that triggers DAG production by activating the PLD, which in turn
generates the PA substrate for the PAP. Therefore, despite the presence
in WISH cells of the DAG-activable isoforms
and
, none of them
become activated after DAG levels increase. This is consistent with our
suggestion that the PAP-derived DAG serves a structural, not messenger,
role for AA release in PMA-activated WISH cells. In agreement with this
view is the recent work by Pettitt et al. (37) in activated endothelial cells. These investigators provided compelling evidence that only the DAG which derives from inositol lipids, i.e.
the one that PMA mimics in our WISH cell system, is able to activate PKC; whereas, the DAG arising from the PLD/PAP pathway does not serve a
messenger role but rather a structural/metabolic role (37).
An interesting but yet unresolved question regarding AA metabolism by amnionic WISH cells relates to the fact that apreciable free AA mobilization is already detectable after a 30-min cell challenge, whereas PGE2 is released only after 4-6 h of stimulation with the phorbol ester (13). It has been suggested that COX-2 is the enzyme responsible for PG production and that its synthesis by PMA-activated WISH cells requires at least 4 h (13). Thus the question arises as to why free AA is produced much before it can be metabolized to PGE2. It seems likely that at short times, the free AA may act as a signaler rather than an intermediary metabolite. As a matter of fact, it has been suggested that exogenous AA up-regulates the expression of COX-2 in uterine stromal cells (38). The induction is not due to conversion of AA to prostaglandin by COX-1 because it occurs even in the presence of aspirin, a well known inhibitor of COX activity. Studies are currently underway in our laboratory to investigate this intriguing possibility.
In conclusion, this study has shown that PAP plays an important role in
the regulation of cPLA2, possibly by facilitating interaction of the enzyme with its substrate and not by increasing the
specific enzyme activity. Our data suggest the mechanism for AA
mobilization in PMA-activated WISH cells depicted in Fig.
11. According to this model, the
phorbol ester activates PKC which, in turn, activates PLD. The PLD
gives rise to PA that will be converted to DAG by PAP. The DAG produced
by this pathway will act to allow a good substrate presentation for the
cPLA2. Parallel to but independent of this sequence of
events, PKC
and perhaps PA as well (39) act to activate the MAP
kinase pathway, which leads to the phosphorylation of
cPLA2. When these two signaling routes are turned on, the
cPLA2 will start hydrolyzing phospholipids, resulting in
the early generation of free AA.
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FOOTNOTES |
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* This work was supported by Grants HD26171 and GM20501 from the National Institutes of Health.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.: 619-534-3055;
Fax: 619-534-7390; E-mail: edennis{at}ucsd.edu.
1 The abbreviations used are: PLA2, phospholipase A2; AA, arachidonic acid; BEL, bromoenol lactone; cPLA2, cytosolic PLA2; DAG, diacylglycerol; iPLA2, Ca2+-independent PLA2; MAFP, methyl arachidonyl fluorophosphonate; MAPK, mitogen-activated protein kinase; PA, phosphatidic acid; PAP, phosphatidate phosphohydrolase; PKC, protein kinase C; PLD, phospholipase D; PG, prostaglandin; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; PEt, phosphatidylethanol.
2 B. Johansen and E. A. Dennis, unpublished data.
3 M. A. Balboa and E. A. Dennis, unpublished data.
4 M. A. Balboa, J. Balsinde, and E. A. Dennis, unpublished data.
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REFERENCES |
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