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Department of Life Science, Pohang University of Science and Technology, Pohang, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor, have
been reported as triggers of superoxide generation in human monocytes
(4, 5, 6), the signaling pathway underlying the activation of
the NADPH oxidase complex, which is followed by a burst of superoxide
production, has not yet been extensively studied. Recently, there have been increasing reports that suggest the involvement of phospholipases in the immune responses of phagocytic cells (7, 8). Phospholipase A2 (PLA2),3 which hydrolyzes the sn-2 fatty acyl bond of phospholipids to generate free fatty acids and lysophospholipids (9), has been implicated in the regulation of several cellular responses, such as the degranulation of phagocytic cells (10, 11). Phagocytic cells are known to contain at least two isoforms of PLA2, a 14-kDa secretory form (sPLA2) and an 85-kDa enzyme referred to as cytosolic PLA2 (cPLA2), which is localized in the cytosol in resting cells and exhibits specificity for arachidonic acid (AA) at the sn-2 position. AA liberated by the action of cPLA2 serves as a physiological cofactor in activation of the NADPH oxidase complex (12). Although substantial evidence supports the idea that PLA2 plays an essential role in the extracellular stimulus-mediated AA release and eicosanoid production during inflammatory responses (13, 14, 15), the signaling mechanism linking cell activation to the activation of PLA2 is only poorly understood.
Phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid (PA), which can subsequently be metabolized to diacylglycerol by phosphatidate phosphohydrolase (16, 17), has also been implicated in the regulation of the immune response by phagocytic cells. Potential roles for the PLD-generated PA and DAG in biological functions of the phagocytic cells were suggested by virtue of the unique enzymatic characteristics of PLD, i.e., the transphosphatidylation reaction by which the phosphatidyl moiety of the phospholipid is transferred to a primary alcohol to produce phosphatidyl alcohol at the expense of PA. The presence of the primary alcohol has been reported to interfere with NADPH oxidase activation, degranulation, and phagocytosis (18, 19, 20, 21). Recently, two isoforms of PLD, PLD1 and PLD2, have been cloned in mammalian cells, and their enzymatic activities have been found to be differentially regulated by polyphosphoinositides, protein kinase C (PKC), and small GTPases, such as ADP-ribosylation factor (ARF) and Rho (22, 23, 24). Nevertheless, the molecular identity of the PLD isoform involved in the activation of phagocytic cells has not yet been revealed. Although many stimuli triggering PLD activation have been reported to also induce AA release in phagocytic cells, the interplay between PLA2 and PLD during immunological responses of phagocytic cells is still unclear.
WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met) was originally identified as a peptide that could stimulate phospholipase C (PLC) activity in several human hemopoietic cells, but not in other cells, such as fibroblasts or neuronal cells (25, 26). The expression of a specific receptor for WKYMVm has been demonstrated on human primary phagocytic cells, including neutrophils and monocytes (27), and recently this peptide has also been reported to stimulate human phagocytes, resulting in superoxide generation and chemotaxis followed by bactericidal activity elicited via an unique signaling mechanism (28). In this report we demonstrate that cPLA2 and PLD1 in human monocytes are both stimulated by the treatment with WKYMVm. Specific inhibition of one of the two phospholipases did not interfere with activation of the other by WKYMVm, while resulting in almost complete abolishment of superoxide production in the human monocytes. Moreover, although the activation of cPLA2 by WKYMVm is partially dependent on both PKC and mitogen-activated protein kinase (MAPK) activity, only the activation of PKC, not that of MAPK, is required for the stimulation of PLD1 by the peptide. The data suggest different, but independent, activation of cPLA2 and PLD1 by WKYMVm. Together with the absolute requirement of their participation in WKYMVm-induced superoxide generation, the results therefore suggest that both phospholipases play crucial roles in the activation of human monocytes in response to treatment with the peptide.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The peptide was synthesized, purified, and prepared in the Peptide Library Support Facility at Pohang University of Science and Technology as described previously (25). PBMC separation medium (Histopaque-1077) was purchased from Sigma (St. Louis, MO). [9,10(N)-3H]Myristic acid (53 Ci/mmol) was purchased from Amersham (Aylesbury, U.K.). Precoated silica gel TLC plates (F-254) were obtained from Merck (Darmstadt, Germany). RPMI 1640 was purchased from Life Technologies (Grand Island, NY). Dialyzed FBS and supplemented bovine calf serum were purchased from HyClone (Logan, UT). Methyl arachidonylfluorophosphonate (MAFP), arachidonyltrifluoromethyl ketone (AACOCF3), and bromoenol lactone (BEL) were from Biomol (Plymouth Meeting, PA). Fura-2/penta-acetoxymethyl ester (fura-2/AM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetoxy- methyl ester (BAPTA/AM) were purchased from Molecular Probes (Eugene, OR). 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide (GF109203X), 3-[1-[3-(amidinothio)propyl]-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide methane sulfonate (Ro-31-8220), and 2'-amino-3'-methoxyflavone (PD98059) were obtained from Calbiochem (San Diego, CA). Anti-cPLA2 Ab was a gift from Dr. D. K. Kim (Chung Ang University, Seoul, Korea). Ab for PLD, C-terminal-specific Ab and PLD1a-specific Ab, were prepared as described in our previous reports (29, 30).
Preparation of human monocytes
Peripheral blood leukocytes were donated by Ulsan Red Cross Blood Center (Ulsan, Korea). PBMCs were separated on a Histopaque-1077 gradient. After two washings with HBSS without Ca2+ and Mg2+, the PBMCs were suspended in RPMI 1640 medium containing 10% FBS and incubated for 60 min at 37°C to let the monocytes attach to the culture dish. Attached monocytes were collected as described previously (31). The purity of the prepared monocytes was >95%, as confirmed by FACS analysis with anti-CD14 Ab-conjugated PE. The isolated cells were then used immediately.
Measurement of [Ca2+]i
The level of [Ca2+]i was determined by Grynkiewicz’s method using fura-2/AM (32). Briefly, prepared cells were incubated with 3 µM fura-2/AM at 37°C for 50 min in serum-free RPMI 1640 medium under continuous stirring. Cells (2 x 106) were aliquoted for each assay in Ca2+-free Locke’s solution (154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 5 mM HEPES (pH 7.3), 10 mM glucose, and 0.2 mM EGTA). Fluorescence changes at the dual excitation wavelengths of 340 and 380 nm were measured, and the calibrated fluorescence ratio was translated into [Ca2+]i.
Measurement of PLA2 activity in cells
Isolated human monocytes (107 cells/ml) were prelabeled with 0.5 µCi/ml of [3H]AA in RPMI 1640 medium containing 10% FBS for 24 h at 37°C in a humidified incubator supplied with 95% air and 5% CO2 as described previously (33). The labeled cells were then washed twice with serum-free RPMI 1640 and incubated in RPMI 1640 medium containing 0.1% fatty acid-free BSA for 15 min at 37°C. After discarding the medium, the cells were stimulated with various concentrations of the peptide for indicated periods of time. The radioactivity in the medium and the collected cells was determined with a liquid scintillation counter. When investigating the effect of inhibitors, the cells were preincubated with the indicated concentrations of each inhibitor or vehicle 15 or 120 min before the stimulation.
Translocation analysis of cPLA2
Monocytes were stimulated with vehicle alone or 100 nM WKYMVm for 2 min, washed with PBS, and resuspended in ice-cold TKM buffer (50 mM Tris-HCl (pH 7.4), 25 mM KCl, 5 mM MgCl2, 1 mM EGTA, 1 mM Na3VO4, 50 mM NaF, 10 µg/ml leupeptin, and 1 mM PMSF) as previously described (34, 35). The cells were permitted to swell by incubation on ice for 10 min and were then disrupted by Dounce homogenization (Kontes, Vineland, NJ). For subcellular fractionation, 0.6 ml of 0.25 M sucrose-TKM was added to the homogenate, and this was centrifuged at 1,000 x g for 5 min. The supernatants were centrifuged at 100,000 x g for 60 min at 4°C, and then the supernatant and pellet were designated the cytosol and nonnucleus membrane, respectively. The low speed pellets were disrupted by sonication and were centrifuged at 100,000 x g for 60 min at 4°C, and then the supernatant and pellet were designated the nucleus supernatant and nucleus membrane, respectively. Twenty-five micrograms of each fractions was separated on 8% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-cPLA2 Abs.
Measurement of superoxide anion generation
Superoxide anion generation was determined by measuring the reduction of cytochrome c using a spectrophotometer (UV-160A, Shimadzu, Kyoto, Japan) as previously described (36). The human monocytes (2 x 106 cells in RPMI 1640 medium) were preincubated with 50 µM cytochrome c at 37°C for 1 min and then incubated with 100 nM peptide. To study the role of PLA2 or PLD in peptide-induced superoxide generation, aliquots of cells were pretreated with the indicated concentrations of PLA2 inhibitors, butan-1-ol or butan-3-ol, followed by stimulation with the peptide. Superoxide generation was measured as the change in light absorption at 550 nm over 5 min at 1-min intervals.
Measurement of phosphatidylbutanol (PBt) formation in human monocytes
The production of PBt was determined as described in a previous report (28) with a slight modification. Human monocytes were resuspended to 1 x 106 cells/ml in RPMI 1640 medium containing 2.5% FBS and loaded with [3H]myristic acid (5 µCi/ml) for 90 min at 37°C. The loaded monocytes were then washed twice with serum-free RPMI 1640 medium and stimulated with the peptide in the presence of 0.5% butan-1-ol. After 30 min the reactions were quenched by addition of 0.5 ml of ice-cold methanol followed by aspiration of the medium. After adding 1 ml of chloroform and 0.5 ml of 1 M NaCl, total lipids were extracted by vigorous vortex mixing. The lower phase obtained after centrifugation at 550 x g for 10 min was dried under nitrogen gas. The lipids were then solubilized with chloroform/methanol (95/5), spotted onto silica gel 60 TLC plates, and separated using a solvent containing chloroform/methanol/acetic acid (90/10/10) as described previously (37). To determine the amounts of PBt and total lipids, a Fuji BAS-2000 image analyzer (Fuji, Tokyo, Japan) was used.
Immunoprecipitation
Isolated human monocytes were lysed in lysis buffer (20 mM Tris-HCl (pH 7.2), 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1% cholic acid, 1 mM Na3VO4, 50 mM NaF, 10 µg/ml leupeptin, and 1 mM PMSF), and proteins were harvested by centrifugation at 4°C for 5 min at 20,000 x g. For immunoprecipitation, protein A-Sepharose coupled to anti-PLD Ab (5 µg), which is specific to the C-terminal of PLD and recognizes both PLD1 and PLD2 (30, 38), was incubated with the cell lysate (2 mg) for 4 h at 4°C. The immune complexes were then washed five times with lysis buffer, and the immunoprecipitated proteins were released by heating at 95°C for 5 min in Laemmli sample buffer before electrophoresis and immunoblot analysis with anti-PLD Abs (C-terminal-specific Ab and PLD1a-specific Ab).
Statistics
The results are expressed as the mean ± SE from the number of determinations indicated. Student’s t test was used for comparison of individual treatments with their respective control values. In the figure legends, * indicates a significant difference in a probability of p < 0.05 in comparison with values obtained from untreated human monocytes, and # indicates p < 0.05 in comparing the values to those from human monocytes treated with WKYMVm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The treatment with WKYMVm increased
[Ca2+]i in human
monocytes (Fig. 1
) as expected based on
previous reports that had demonstrated expression of a WKYMVm-specific
receptor and a peptide-induced
[Ca2+]i rise in human
cells of hemopoietic origin, U266 B cell lymphoma, HL60 promyelocytic
leukemia, U937 histiocytic lymphoma cells, and neutrophils (26, 27). The dose-response curve for the Ca2+
release triggered by the peptide was very similar to that observed for
other phagocytic cells (26). WKYMVm evoked a rise in the
[Ca2+]i at nanomolar
concentrations, and the maximal response was observed at 100 nM peptide
concentrations.
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WKYMVm has been shown to stimulate superoxide generation and kill
infected micro-organisms in human monocytes by interacting with a
unique cell surface receptor (28). Unsaturated fatty
acids, especially AA generated by the action of
PLA2, have been known to play an essential role
in superoxide production catalyzed by the NADPH oxidase complex
(12). In this context, we investigated the possible role
of PLA2 as a downstream effector of the
peptide-specific receptor. When stimulated with various concentrations
of the peptide, the human monocytes responded with a
concentration-dependent increase in AA release, with a maximal effect
at a peptide concentration of 
100 nM, at which level the maximal
effect on Ca2+ mobilization was also observed
(Fig. 2A). An inactive
analogue of the peptide,
D-Trp-D-Lys-D-Tyr-D-Met-D-Val-D-Met (wkymvm), did
not induce any significant AA release at concentrations of up to 1
µM. At 100 nM, WKYMVm caused a rapid release of AA from human
monocytes, which peaked after 5 min (Fig. 2B). To address
the question of which isoform of PLA2 is
responsible for the WKYMVm-induced increase in AA release, we
introduced several isoform-specific inhibitors of
PLA2. Pretreatment of the cells with the
cPLA2-specific inhibitors, MAFP and
AACOCF3, blocked the peptide-induced liberation
of AA in a concentration-dependent manner (Fig. 3A). At a concentration of 10
µM MAFP or AACOCF3, the peptide-induced AA
release was almost completely prevented, while another
PLA2 inhibitor, BEL, that is known to be specific
for iPLA2, did not interfere with the
peptide-induced AA release. The peptide-stimulated AA release was also
inhibited by chelation of intracellular Ca2+ with
BAPTA/AM, which supports the idea of cPLA2
activation (data not shown). Mobilization of Ca2+
has been shown to be necessary for the activation of
cPLA2 and its translocation to the nuclear
membrane (39, 40, 41). Moreover, stimulation of human
monocytes with 100 nM peptide led to cPLA2
translocation to the nuclear membrane fraction (Fig. 3B).
These results, therefore, indicate that WKYMVm evokes AA release by
stimulating cPLA2 , but not
iPLA2, in human monocytes.
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As expected from our previous results that demonstrated
PLC/PKC-dependent activation of PLD in human monocytes by WKYMVm
(28), preincubation of the cells with various
concentrations of two PKC inhibitors (Ro-31-8220 and GF109203X) before
stimulation with WKYMVm blocked PLD activation in terms of PBt
formation (Fig. 4A). Hereby,
the identity of PLD isoform, which might be involved in
WKYMVm-dependent PBt formation in human monocytes, was scrutinized
using PLD Abs (C-terminal-specific Ab and PLD1a-specific Ab) as
immunological probes. By immunoprecipitation using protein A-Sepharose
coupled to C-terminal-specific Ab, which can react with both
PLD1 and PLD2
(38), followed by immunoblot analysis with the
anti-PLD1a-specific Ab or the C-terminal PLD Ab, we identified
PLD1, but not PLD2, in
monocytes (Fig. 4B). Recalling the fact that PKC has been
shown to be involved in the regulation of PLD1 in
a number of cells (23, 42), we concluded that WKYMVm
induced the generation of PBt in human monocytes via the PKC-dependent
activation of PLD1.
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A PLC inhibitor (U-73122), a PKC inhibitor (GF109203X), and the
intracellular calcium chelator BAPTA/AM have been shown to blunt the
cellular responses triggered by WKYMVm in human monocytes, the
activation of PLD, as well as superoxide generation (28).
Moreover, a number of previous publications have reported that
stimulation of phagocytic cells with AA, the product of
PLA2 activity, leads to the activation of NADPH
oxidase, which then results in superoxide generation (31, 43). The involvement of the two phospholipases,
cPLA2 and PLD1, in
superoxide generation after treatment of human monocytes with WKYMVm
was, therefore, further investigated. The inhibitors (MAFP and
AACOCF3 for cPLA2, and
butan-1-ol and GF109203X for PLD1), which
diminished the WKYMVm-induced activation of the phospholipases,
displayed similar potencies of inhibition in terms of superoxide
generation (Fig. 5, A and
B). Since the inhibition of one of the two phospholipases
(cPLA2 or PLD1) almost
completely interfered with WKYMVm-induced superoxide generation, it is
reasonable to conclude that the activation of both phospholipases plays
a pivotal role in peptide-induced superoxide production by human
monocytes.
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PA, the product of PLD activity, has been known to participate in
the activation of cPLA2 in phagocytic cells such
as neutrophils (44). Treatment of neutrophils with
ethanol, an inhibitor of PA formation by PLD, suppressed
FMLP-stimulated AA release, suggesting that PLD acts upstream of
PLA2 in neutrophils (45). However,
the interrelationship between PLA2 and PLD in the
context of superoxide generation has not been studied extensively in
human monocytes. Stimulation of the cells with 100 nM WKYMVm in the
presence of 0.5% butan-1-ol did not affect the peptide-induced AA
release in monocytes (Fig. 6A). Ethanol also did not
inhibit the AA release induced by the peptide even at concentrations up
to 1.5% (data not shown). When the monocytes were preincubated with
various concentrations of cPLA2 inhibitor
(AACOCF3 or MAFP) up to a concentration of 20
µM before stimulation with 100 nM WKYMVm, the peptide-induced PLD
activation was not affected (Fig. 6B). Although activation
of both phospholipases was observed after treatment of human monocytes
with WKYMVm, and it was demonstrated that this was required to activate
the cells to generate superoxides (Fig. 5
), no hierarchy was evident
between PLA2 and PLD in the peptide-induced
signaling pathway.
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The upstream signaling pathway leading to the peptide-induced
stimulation of cPLA2 activity was compared with
that resulting in PLD1 activation. The
Ca2+-chelating agent BAPTA/AM has been shown to
almost completely block PBt formation in cells stimulated with WKYMVm
(28). Mobilization of Ca2+, which is
indispensable for superoxide generation, must be required for the
activation of PLD1 as well as
cPLA2, because WKYMVm was not able to stimulate
the generation of PBt or AA in human monocytes in the presence of
BAPTA/AM (Fig. 7). Although the
peptide-induced PBt formation dropped to near basal level after
preincubation of the cells with 5 µM GF109203X, a specific PKC
inhibitor, AA release was only partially inhibited, as shown in Fig. 7.
MAPK activation by extracellular stimulation generally leads to
cPLA2 activation in immune cells, including
monocytes, and WKYMVm also stimulates the activation of MAPK in human
monocytes (data not shown). We therefore examined the effect of MAPK
kinase inhibitor PD98059 on WKYMVm-stimulated AA release and PBt
formation in monocytes. Upon the introduction of PD98059, the
inhibitory effect was different for the two phospholipases: no effect
on PLD1 and about 50% inhibition of
cPLA2 (Fig. 7). These results suggest that the
stimulation of cPLA2 activity by WKYMVm is
partially dependent on the activation of PKC and MAPK, although only
PKC activation, and not MAPK activation, is required for
PLD1 stimulation by the peptide. Calcium
mobilization is required for the activation of both phospholipases.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although AA has been widely used to stimulate the phagocytic NADPH oxidase activity in a cell-free system, its physiological role in superoxide generation in intact cells has not yet been established. The roles of the PLA2 enzyme(s), although critical to cell activation, have not been thoroughly characterized as of yet. The existence of several isoforms of PLA2 makes it difficult to define the specific PLA2 molecule responsible for the release of AA following phagocyte stimulation (46, 47, 48). In this report, the peptide triggered AA release in a concentration- and time-dependent manner, and the peptide-induced AA release was attenuated by pretreatment with cPLA2-specific inhibitors, MAFP and AACOCF3, but not with BEL, which is specific for iPLA2. This suggests an essential role for cPLA2. The implication of cPLA2 in WKYMVm-induced AA release is further supported by the translocation of cPLA2 to the membrane fraction, which is a widely accepted indicator of the activation of cPLA2. According to our results, cPLA2, rather than iPLA2, appears to be involved in the WKYMVm-induced AA release and superoxide generation in human monocytes. Recently, Dana et al. demonstrated that cPLA2-deficient PBL-985 cells failed to be stimulated into releasing AA and activating the NADPH oxidase enzyme in response to a variety of soluble and particulate stimuli. Their data, therefore, support the idea of a major physiological role for cPLA2-generated AA in the activation of phagocytic cells (12). On the contrary, Tithof et al. have shown that iPLA2, but not cPLA2, regulates the release of AA for superoxide generation in neutrophils by using a mixture of polychlorinated biphenyls (Aroclor 1242), which let them rule out a cPLA2 effect on NADPH oxidase activation (11).
PLD-derived PA and its PA phosphatase-mediated hydrolysis product DAG
have been implicated in the regulation of NADPH oxidase activity in
cell-free systems and in intact phagocytic cells (49, 50, 51, 52, 53, 54).
In a previous report we demonstrated that WKYMVm induced PLD activation
in human monocytes and that pretreatment of the cells with PI-specific
PLC inhibitor (U-73122) or PKC inhibitor (GF109293X) before peptide
stimulation prevented PLD activation, superoxide generation, and
bacteria-killing activity (28). This suggests that there
is an essential requirement for PLD activation in the WKYMVm-induced
immune response. To delineate the identity of the PLD isoform(s)
involved in peptide-stimulated superoxide generation, the expression of
PLD in human monocytes was analyzed by immunological methods. Monocytes
express PLD1 and do not express
PLD2. We also confirmed PLD activity in the
presence of ADP-ribosylation factor (ARF) and guanosine
5'-[-thio]triphosphase (GTP[S]) with
PLD1 immunoprecipitates (data not shown). The
peptide-induced PLD activation and superoxide generation are PKC
dependent (Fig. 4A). From the results and keeping in mind
that the PKC dependency is a characteristic property of
PLD1, it must be PLD1 that
is activated by the novel chemoattractant peptide and whose activation
is required for superoxide generation in human monocytes.
Although more and more reports have demonstrated that
PLA2 and PLD are important molecules in
regulation of the activity of phagocytic cells, the interrelationship
between PLA2 and PLD is only poorly understood.
In several recent studies carried out to define the interrelationship
between PLD and PLA2 in the process of phagocytic
cell activation, different lines of evidence have been reported.
Bauldry et al. (44) observed that the release of AA and
the generation of platelet-activating factor correlated with the
enhanced PA and DAG production that occurred upon stimulation with FMLP
in intact PMN cells primed with TNF-
. The introduction of a
combination of PA and DAG reconstituted full
cPLA2 activity in permeabilized human PMN cells,
while addition of either PA or DAG alone evoked only partial activation
of cPLA2, suggesting that PLD acts as an upstream
regulator in the induction of PLA2 activity.
Fujita et al. showed that treatment of neutrophils with ethanol (PA
acceptor) or propranolol (PA phosphohydrolase inhibitor) suppressed the
FMLP-stimulated AA release, and they suggested that
cPLA2 may be downstream of the PLD-dependent
signaling pathway in FMLP-stimulated neutrophils (45). In
this study we demonstrated that the inhibition of PA production via a
transphosphatidylation reaction in the presence of a primary alcohol
does not affect the WKYMVm-induced cPLA2
activation (Fig. 6A). Although PA-dependent Raf-1-MEK
(MAPKK/mitogen-activated protein kinase kinase)-MAPK-mediated
cPLA2 activation pathway is present in monocytes, the
involvement of uncharacterized component(s) in the signaling pathways
induced by the peptide cannot be excluded. In nonphagocytic cells, AA
or melittin, a PLA2 stimulator, enhanced PLD
transphosphatidylation activity in rat heart sarcolemma
(55). Meanwhile, differential regulation of PLD and
PLA2 by PKC activated with PMA has been
demonstrated in P388D1 macrophages; PLA2
activation took place independently of PKC, which contrasted with PLD,
which was PKC dependent for priming and enhancement of its activity
(56). In our study MAFP and AACOCF3
did not interfere with the peptide-induced PLD activation, suggesting
cPLA2-independent PLD activation (Fig. 6B). The relationship between PLA2 and
PLD may depend on the stimulus and the cell type. In human monocytes
challenged with WKYMVm, independent activation of
cPLA2 and PLD1 has been
observed.
The signal transduction pathway between cell activation by extracellular stimulus and activation of cPLA2 remains unknown, although significant progress has been made in understanding the biochemical mechanisms involved in the stimulation of cPLA2 activity in phagocytic and other cells. Activation of PKC and MAP kinase cascades has been implicated in the phosphorylation of cPLA2, which increases the enzyme’s activity (57, 58, 59). However, there is a substantial debate over the involvement of PKC and MAPK in the activation of cPLA2. The direct activation of PKC by PMA or cell-permeating DAG evoked little AA release (55, 60). We investigated the upstream signal transduction pathway activated by WKYMVm that results in the stimulation of cPLA2 and compared it with the one leading to PLD activation. Internal Ca2+ mobilization is absolutely required for the activation of PLD1 as well as cPLA2. However, the activities of the two phospholipases are differentially modulated by PKC and MAPK. PKC activation, but not MAPK activation, is absolutely required for the peptide stimulation of PLD1, while stimulation of cPLA2 activity by WKYMVm is only partially dependent on the activation of PKC and MAPK. We also checked the effect of PKC or MAPK kinase inhibitor on the peptide-induced cPLA2 translocation. GF109203X and PD98059 partially inhibited cPLA2 translocation to nuclear membrane by WKYMVm (data not shown), supporting the idea that the peptide stimulates cPLA2 via PKC and MAPK activation.
In our study we demonstrated that the activation of PLA2 and PLD by the chemoattractant hexapeptide WKYMVm is necessary for the activation of NADPH oxidase in human monocytes and that the two phospholipases act independently in this process. The biochemical mechanisms that link the peptide-induced enhancement of each phospholipase activity to increased superoxide generation, i.e., the stimulation of the NADPH oxidase complex, remains to be elucidated.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sung Ho Ryu, Department of Life Science, Pohang University of Science and Technology, San 31, Hyojadong, Pohang 790-784, Korea.
3 Abbreviations used in this paper: PLA2, phospholipase A2; WKYMVm, Trp-Lys-Tyr-Met-Val-D-Met-NH2; AA, arachidonic acid; PLD, phospholipase D; PBt, phosphatidylbutanol; PA, phosphatidic acid; MAFP, methyl arachidonyl fluorophosphonate; AACOCF3, arachidonyltrifluoromethyl ketone; BEL, bromoenol lactone; PLA2, Ca2+-independent PLA2; PKC, protein kinase C; DAG, diacylglycerol; [Ca2+]i, intracellular calcium concentration; MAPK, mitogen-activated protein kinase; fura-2/AM, fura-2/pentaacetoxymethyl ester; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetoxymethyl ester; GF109203X, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide; Ro-31-8220, 3-[1-[3-(amidinothio)propyl]-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide methanesulfonate; PD98059, 2'-amino-3'-methoxyflavone.
Received for publication September 8, 1999. Accepted for publication February 9, 2000.
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 H. K. Kang, H.-Y. Lee, M.-K. Kim, K. S. Park, Y. M. Park, J.-Y. Kwak, and Y.-S. Bae The Synthetic Peptide Trp-Lys-Tyr-Met-Val-D-Met Inhibits Human Monocyte-Derived Dendritic Cell Maturation via Formyl Peptide Receptor and Formyl Peptide Receptor-Like 2 J. Immunol., July 15, 2005; 175(2): 685 - 692. [Abstract] [Full Text] [PDF]
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 Y.-S. Bae, J. C. Park, R. He, R. D. Ye, J.-Y. Kwak, P.-G. Suh, and S. Ho Ryu Differential Signaling of Formyl Peptide Receptor-Like 1 by Trp-Lys-Tyr-Met-Val-Met-CONH2 or Lipoxin A4 in Human Neutrophils Mol. Pharmacol., September 1, 2003; 64(3): 721 - 730. [Abstract] [Full Text] [PDF]
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 Z. Shmelzer, N. Haddad, E. Admon, I. Pessach, T. L. Leto, Z. Eitan-Hazan, M. Hershfinkel, and R. Levy Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes J. Cell Biol., August 18, 2003; 162(4): 683 - 692. [Abstract] [Full Text] [PDF]
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Y.-S. Bae, T. G. Lee, J. C. Park, J. H. Hur, Y. Kim, K. Heo, J.-Y. Kwak, P.-G. Suh, and S. H. Ryu Identification of a Compound That Directly Stimulates Phospholipase C Activity Mol. Pharmacol., May 1, 2003; 63(5): 1043 - 1050. [Abstract] [Full Text] [PDF]
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Y.-S. Bae, Y. Kim, J. C. Park, P.-G. Suh, and S. H. Ryu The synthetic chemoattractant peptide, Trp-Lys-Tyr-Met-Val-D-Met, enhances monocyte survival via PKC-dependent Akt activation J. Leukoc. Biol., February 1, 2002; 71(2): 329 - 338. [Abstract] [Full Text] [PDF]
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, basal
PBt formation in monocytes. The data shown are the mean ± SE
(n = 6; A). Isolated monocytes were
lysed in lysis buffer (20 mM Tris-HCl (pH 7.2), 10% glycerol, 150 mM
NaCl, 1% Triton X-100, 1% cholic acid, 1 mM
Na3VO4, 50 mM NaF, 10 µg/ml leupeptin, and 1
mM PMSF), and 2 mg aliquots of cell lysate were subjected to
immunoprecipitation with protein A-Sepharose coupled to
C-terminal-specific Ab followed by immunoblot analysis with
anti-PLD Abs (anti-C-terminal-specific and
anti-PLD1a-specific Abs; B).
