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*
Department of Health Chemistry, Showa University School of Pharmaceutical Sciences, Tokyo, Japan; and
Department of Biochemistry, Tokushima University School of Medicine, Tokushima, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Secretory PLA2-IIA is a widely distributed sPLA2 isozyme whose expression level increases dramatically during inflammation (7). In some cells, sPLA2-V, an isozyme closely related to sPLA2-IIA, appears to substitute for sPLA2-IIA (3, 4, 8, 9). The delayed biosynthesis of PG, which is elicited by proinflammatory stimuli such as IL-1, TNF, and LPS, is accompanied by the continuous supply of AA over long periods of culture spanning several hours, during which de novo induction of these two sPLA2s often occurs, depending on cell type (10, 11, 12, 13). Current evidence has suggested that these inducible sPLA2s are crucial for optimal COX-2-dependent PG production (3, 4, 5, 12, 13, 14). Moreover, these sPLA2s often up-regulate COX-2 expression in several cell types (15, 16, 17). The AA-releasing action of these sPLA2s is influenced by the cell activation state, in that only agonist-stimulated cellular membranes become sensitive to them (7, 18). Perturbation of plasma membrane asymmetry by phospholipid scramblase, which carries anionic phospholipids from the inner to the outer leaflet of the plasma membrane, appears to contribute at least in part to sensitization of cells toward the action of sPLA2 (17). More intriguingly, endogenously expressed sPLA2-IIA binds preferentially to glypican, a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan, and accumulates in caveolae and perinuclear sites in cytokine-stimulated cells (19). This particular compartmentalization may allow relatively small amounts of the heparin-binding sPLA2s to be efficiently coupled with perinuclear COX-2.
Several lines of evidence suggest that cPLA2 is required for sPLA2-IIA or -V to act properly (3, 9, 13, 16, 20). Supporting this idea are observations that sPLA2-dependent AA release was blocked by cPLA2 inhibitors and restored by supplementation of exogenous AA in fibroblasts, mast cells, and macrophages (3, 9, 13), and that cotransfection of cPLA2 and sPLA2-IIA augments AA release in a synergistic manner in human embryonic kidney (HEK) 293 cells (3, 20). In addition, we (12) and others (13, 16) have obtained complementary results suggesting that cPLA2 is required for the induction of sPLA2-IIA and sPLA2-V expression in fibroblasts and macrophages, respectively, at the transcriptional level. However, the factor(s) that intermediates between cPLA2 and sPLA2s has remained unclear.
In the present study we show that 12/15-lipoxygenase (LOX), a dual specificity LOX isozyme that produces 12- and 15-hydroperoxyeicosatetraenoic acids (HPETEs) from AA substrate and directly oxidizes membrane lipids (21, 22, 23), lies downstream of cPLA2, contributing to the stimulation of sPLA2-IIA gene transcription. Moreover, 12/15-LOX-mediated lipid oxidation facilitates sPLA2-IIA-mediated membrane hydrolysis, leading to AA release. Our results have revealed unexplored functional cross-talk between the constitutive cPLA2-12/15-LOX and the inducible sPLA2-IIA-COX-2 pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Mouse and human IL-1ßs and human TNF-
were purchased from
Genzyme (Cambridge, MA). Rabbit antiserum to mouse COX-1 was provided
by W. L. Smith (Michigan State University, East Lansing, MI), and
mouse COX-2 cDNA and the COX-2 inhibitor NS-398 were provided by J.
Trzaskos (Merck-DuPont, Rahway, NJ). Rabbit antiserum to mouse COX-2,
human 12/15-LOX cDNA, AA, 12(S)-HPETE, and the
PGE2 enzyme immunoassay kit were purchased from
Cayman Chemical (Ann Arbor, MI). The LOX inhibitors, including
nordihydroguaiaretic acid (NDGA; general LOX inhibitor), AA-861 (5-LOX
inhibitor), cinnamyl-3,4-dihydroxy--cyanocinnamate (CDC; 12-LOX
inhibitor) and 5,8,11,14-eicosatetraynoic acid (ETYA; 15-LOX
inhibitor), were purchased from Biomol (Plymouth Meeting, PA). The
cDNAs for rat sPLA2-IIA (24) and
porcine leukocyte-type 12-LOX (12/15-LOX) (25) and rabbit
polyclonal Ab against rat sPLA2-IIA
(26) were prepared as described previously. The
cPLA2 inhibitor arachidonoyl trifluoromethyl
ketone (AACOCF3) was purchased from Calbiochem
(La Jolla, CA). Aspirin, n-propyl gallate, butylated
hydroxytoluene (BHT), 1-oleoyl-lysophosphatidylcholine (LysoPC), and
tert-butylhydroperoxide (t-BuOOH) were
purchased from Sigma (St. Louis, MO). Lipofectamine Plus reagent,
Opti-MEM medium, and TRIzol reagent were purchased from Life
Technologies (Gaithersburg, MD). FITC-conjugated goat anti-rabbit
IgG Ab was purchased from Zymed (South San Francisco, CA).
[14C]AA was purchased from NEN Life Science
Products (Boston, MA). Silica gel 60 F254 glass
plates for TLC (20 x 20 cm) were obtained from Merck.
Activation of 3Y1 cells
Rat fibroblastic 3Y1 cells were a gift from Dr. Y. Uehara
(National Institute of Infectious Disease, Tokyo, Japan). The cells
were maintained in culture medium composed of DMEM (Nissui
Pharmaceutical, Tokyo, Japan) supplemented with 10% (v/v) FCS,
penicillin/streptomycin (100 U/ml and 100 µg/ml, respectively; Flow
Laboratories, Rockville, MD), and 2 mM glutamine (Life Technologies) at
37°C in a CO2 incubator flushed with 5%
CO2 in humidified air. The media of 3Y1 cells
that had attained 60–80% confluence in six-well plates (Iwaki Glass,
Tokyo, Japan) were replaced with 2 ml of DMEM supplemented with 2%
FCS. After culture for 24 h, 1 ng/ml mouse IL-1ß and 100 U/ml
human TNF- were added to the cultures to assess the delayed
PGE2 biosynthetic response. The
PGE2 released into the culture medium was
determined by enzyme immunoassay. For the RNA blot analysis, TRIzol was
directly added to the cell monolayer.
Activation of 293 cells
Culture of HEK 293 cells (Human Science Research Resources Bank, Osaka, Japan) and establishment of stable transfectants expressing mouse sPLA2-IIA, rat sPLA2-V, and mouse cPLA2 were detailed in our previous papers (3, 4). The cells (5 x 104 cells in 1 ml of culture medium) were seeded into 24-well plates. To assess AA release, 0.1 µCi/ml of [3H]AA (Amersham, Arlington Heights, IL) was added to the cells on day 3 when they had nearly reached confluence, and culture was continued for another day. After three washes with fresh medium, 250 µl of RPMI 1640 with or without A23187 or human IL-1ß was added to each well, and the amount of free [3H]AA released into the supernatant during culture (30 min with A23187 and 4 h with IL-1ß) was measured. The percent release of AA was calculated using the formula [S/(S + P)] x 100, where S and P are the radioactivities measured in equal portions of the supernatant and cell pellet, respectively.
RNA blotting
All procedures were performed as described previously (14). Briefly, equal amounts (5 µg) of total RNA, purified using TRIzol reagent, were applied to each lane of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore, Bedford, MA). The resulting blots were then sequentially probed with sPLA2-IIA, COX-2, and GAPDH (Clontech, Palo Alto, CA) cDNA probes that had been labeled with [32P]dCTP (NEN Life Science Products) by random priming (Takara Biomedical, Ohtsu, Japan). All hybridizations were conducted at 42°C overnight in a solution comprising 50% (v/v) formamide, 0.75 M NaCl, 75 mM sodium citrate, 0.1% (w/v) SDS, 1 mM EDTA, 10 mM sodium phosphate (pH 6.8), 5x Denhardt’s solution (Nacalai Tesque, Kyoto, Japan), 10% (w/v) dextran sulfate (Sigma), and 100 µg/ml salmon sperm DNA (Sigma).
RT-PCR
Synthesis of cDNA was performed using 1 µg of total RNA and AMV reverse transcriptase, according to the manufacturer’s instructions supplied with the RNA PCR kit (AMV, version 2.1, Takara Biomedical). Subsequent amplifications of the partial cDNA encoding LOX isozymes were performed using 1 µl of the reverse transcribed mixture as a template with specific oligonucleotide primers (Greiner Japan, Tokyo, Japan) as follows: rat 5-LOX: sense, 5'-CTT CCT ACA CTG TCA CCG TAG-3'; and antisense, 5'-GTC CAC TCC CTT TTC ACT ATC-3'; and rat 12/15-LOX: sense, 5'-CCA GTA GAA CCA ATC CAG CTG-3'; and antisense, 5'-TTG ATG GCT GAG CTC TTT GCT G-3'. The expected sizes of the PCR products for rat 5-LOX and rat 12/15-LOX were 523 and 563 bp, respectively. The PCR mixtures were subjected to 30 cycles of amplification by denaturation (30 s at 94°C), annealing (30 s at 60°C), and elongation (1 min at 72°C). The PCR products were analyzed by 1% agarose gel electrophoresis with ethidium bromide. The gels were further subjected to Southern blot hybridization using specific LOX probes as required for the experiments.
Transfection of 12/15-LOX cDNA
Porcine 12/15-LOX cDNA was inserted into a mammalian expression vector pcDNA3.1 at the XbaI site and transfected into rat fibroblastic 3Y1 cells and HEK 293 cells using Lipofectamine Plus reagent according to the manufacturer’s instructions. Briefly, 1 µg of 12/15-LOX cDNA was mixed with 6 µl of Lipofectamine Plus reagent in 200 µl of Opti-MEM medium, incubated for 30 min at room temperature, and then added to cells that had attained 70% confluence in six-well dishes (Iwaki Glass) containing 1 ml of Opti-MEM. After incubation for 3 h, the medium was replaced with 2 ml of fresh culture medium. After overnight culture, the medium was replaced again with 2 ml of fresh medium. To obtain stable transfectants, the cells were harvested 3 days after transfection and were cloned by limiting dilution in 96-well plates in culture medium supplemented with 800 µg/ml Geneticin (Life Technologies). After culture for 2–4 wk, wells containing a single colony were selected, and the expression was checked by RNA blotting and LOX activity. The established clones were expanded and used for the subsequent experiments.
To establish HEK 293 transfectants that expressed both sPLA2 and 12/15-LOX, sPLA2-IIA-expressing 293 cells (3, 4) were secondarily transfected with 12/15-LOX cDNA, which had been subcloned into pcDNA3.1/hyg+ (Invitrogen, San Diego, CA) at the XbaI site. Three days after transfection, the cells were seeded into 96-well plates in the presence of 50 µg/ml hygromycin (Invitrogen) to establish stable transformants expressing both sPLA2-IIA and 12/15-LOX.
Immunocytostaining
The cells grown on collagen-coated cover glasses (Iwaki Glass) were fixed for 30 min in 10% formalin in PBS. After three washes with PBS, the fixed cells were sequentially treated with 3% BSA (for blocking) in PBS for 1 h, with an anti-sPLA2-IIA Ab (1/500 dilution) for 1 h, and then with FITC-goat anti-rabbit IgG (1/50 dilution) for 1 h. After six washes with PBS, the cells were mounted on glass slips using Perma Fluor (Japan Tanner, Suita, Japan), and the sPLA2-IIA signal was visualized using a laser scanning confocal microscope (IX70, Olympus, New Hyde Park, NY).
Measurement of 12/15-LOX activity
The lysate was incubated with 0.1 µCi of [14C]AA for 10 min at 24°C in 100 µl of PBS in the presence or the absence of inhibitors. The reaction was terminated by addition of 0.3 ml of diethyl ether/methanol/1 M citric acid (30/4/1, v/v). The extracts were separated by TLC with a solvent system of diethyl ether/petroleum ether/acetic acid (85/15/0.1, v/v) at -20°C (25). Distribution of radioactivities on the TLC plates was detected by BAS2000 imaging analyzer (Fujix, Tokyo, Japan). The positions of [14C]AA, 12-HETE, and 15-HETE were determined by comparing their Rf values with those of authentic standards (Cayman Chemical).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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When 3Y1 cells were treated with IL-1ß and TNF-, delayed
PGE2 generation occurred over 48 h, which
was regulated by functional coupling of two inducible enzymes,
sPLA2-IIA and COX-2 (12).
AACOCF3, a cPLA2 inhibitor,
attenuated the expression of sPLA2-IIA without
affecting that of COX-2 (Fig. 1
A), accompanied by reduction
of PGE2 generation (12). Addition of
AA and LysoPC modestly, but significantly, restored this
AACOCF3 suppression of
sPLA2-IIA induction (Fig. 1A),
suggesting that cPLA2-derived AA, LysoPC, or
their metabolites play some role in the induction of
sPLA2-IIA. Cytokine-induced
sPLA2-IIA expression was also markedly reduced by
NDGA (Fig. 1B), which suppresses several LOX isozymes
(27), but not by COX inhibitors such as aspirin and NS-398
(12). As NDGA is an antioxidant, the effects of other
general antioxidants (n-propyl gallate and BHT) on
sPLA2-IIA expression were next examined. As shown
in Fig. 1C, n-propyl gallate, but not BHT,
markedly suppressed cytokine induction of
sPLA2-IIA (as described below, this suppressive
effect was correlated with potency to inhibit 12/15-LOX). Consistent
with these observations, PGE2 generation 24
h after stimulation with IL-1ß/TNF- was reduced by >80% by
either NDGA or n-propyl gallate.
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A, top), which was
assessed by the conversion of [14C]AA to
[14C]12-HETE using cell lysates, and its mRNA
expression, which was assessed by RNA blotting (Fig. 3A,
bottom). The expression level of 12/15-LOX in the
transfectants was estimated to be >20 times higher than that in
control cells. NDGA and n-propyl gallate each markedly
decreased 12/15-LOX activity, whereas BHT showed little effect (Fig. 3B). Thus, the antioxidants that decreased
sPLA2-IIA expression (Fig. 1
B) had the
capacity to inhibit 12/15-LOX activity (Fig. 3B).
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alone or in combination, the induction of
sPLA2-IIA mRNA was markedly increased relative to
that of replicate mock-transfected cells, and these two cytokines acted
in synergy (Fig. 4A). On the
basis of quantification of sPLA2-IIA expression,
it increased 
16- and 120-fold in IL-1ß/TNF--stimulated control
and 12/15-LOX-transfected cells, respectively, over that in
unstimulated control cells. Low, but significant,
sPLA2-IIA expression was already detectable in
12/15-LOX-overexpressing, but not in control, cells even without
cytokine stimulation. In contrast, cytokine-induced expression of COX-2
mRNA was increased only minimally (Fig. 4A), and
constitutive expression of cPLA2 and COX-1 was
unchanged (data not shown), following overexpression of 12/15-LOX.
IL-1ß/TNF--stimulated PGE2 generation
reached 1.8 ± 0.3 (mean ± SE; n = 3;
p < 0.05 vs unstimulated control cells) and 17.5
± 2.8 (n = 3; p < 0.05 vs
unstimulated 12/15-LOX-transfected cells and IL-1ß/TNF--stimulated
control cells) ng/ml PGE2 in mock- and
12/15-LOX-transfected cells, respectively. Thus, increased
sPLA2-IIA expression in
IL-1ß/TNF--stimulated 12/15-LOX-transfected cells appeared to be
roughly correlated with increased PGE2
biosynthesis (Fig. 4B). Furthermore, 12/15-LOX-enhanced
sPLA2-IIA expression (Fig. 4C) and
PGE2 generation (Fig. 4D) were
suppressed dramatically by AACOCF3 or NDGA almost
in parallel, whereas the expression of COX-2 was unaffected by these
inhibitors (Fig. 4C). The addition of 10 µM
AACOCF3 also reduced the production of 12-HETE in
12/15-LOX-transfected cells by 80% (data not shown), suggesting a role
of endogenous cPLA2 in supplying AA to
12/15-LOX.
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, A and B).
A previous study has shown by colocalization that these compartments
are caveolae vesicles (19). This particular
sPLA2-IIA signal was markedly elevated in the
cells overexpressing 12/15-LOX, in which the
sPLA2-IIA signal was already detectable under
unstimulated conditions (Fig. 5C) and was increased markedly
after cytokine stimulation (Fig. 5D). Colocalization of
sPLA2-IIA, an AA donor, and COX-2, an AA
acceptor, in the same perinuclear area may facilitate their efficient
coupling during the delayed PGE2-biosynthetic
response.
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stimulation of 3Y1 cells (12) and the
present observation that cPLA2 and 12/15-LOX are
functionally linked (also see Fig. 7D) suggest that the
cPLA2-12/15-LOX pathway precedes the
sPLA2-IIA/COX-2 pathway, contributing to
up-regulation of cytokine-induced sPLA2-IIA
expression and accompanying PGE2 biosynthesis.
This regulatory mechanism is not peculiar in 3Y1 cells, because IL-1ß
induction of sPLA2-IIA gene expression in rat
aortic smooth muscle cells is also NDGA sensitive (28). As
the ligands for the nuclear receptor peroxisome proliferator-activated
receptor (PPAR
) are fatty acids and their oxidative derivatives,
including 12/15-LOX metabolites (29), and the
sPLA2-IIA gene promoter region contains a motif
showing homology with the PPAR-binding sites (positions -160 to -133)
(28), we conducted EMSA to address the involvement of this
putative PPAR binding site in 12/15-LOX-mediated hyperinduction of
sPLA2-IIA. However, activation of PPAR in
cytokine-stimulated 3Y1 cells was unchanged regardless of 12/15-LOX
overexpression (data not shown). Moreover, activation of NF-
B, which
is crucial for sPLA2-IIA induction by cytokines
(30), was also unaffected by 12/15-LOX overexpression
(data not shown). Thus, activation of these transcription factors alone
appears to be insufficient to fully explain the 12/15-LOX-mediated
induction of sPLA2-IIA. Whether some other
regulatory elements are responsible for 12/15-LOX induction of
sPLA2-IIA is now under investigation.
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In view of the fact that AA release by sPLA2s from activated cells often occurs even without accompanying de novo induction of its expression, it has been suggested that membrane rearrangement during cell activation also represents a crucial event leading to efficient sPLA2-mediated membrane phospholipid hydrolysis (7, 18). Among the models proposed to date (3, 9, 13, 16, 17, 18, 19, 20, 31, 32, 33, 34), several studies have shown that treatment of cells with agents provoking the oxidative response makes the membranes susceptible to sPLA2-IIA (34, 35, 36).
To verify that membrane oxidation indeed induces such an alteration, we
exploited HEK293 transfectants that stably overexpressed each
PLA2 isozyme (3, 4). As shown in
Fig. 6A,
sPLA2-IIA-expressing 293 cells released
approximately four times more AA (reaching >10% release) than
parental cells after 4 h of treatment with t-BuOOH, a
potent membrane oxidant (37), whereas replicate cells
transfected with cPLA2 exhibited only a modest
increase in AA release. Coexpression of cPLA2 and
sPLA2-IIA increased AA release in synergy
(reaching >20% release), in agreement with previous observations that
these two enzymes cooperate functionally (3, 9, 13, 16, 20). The increased AA release observed in
sPLA2-IIA-expressing, but not control, cells
after t-BuOOH treatment was markedly suppressed by the
antioxidant n-propyl gallate (Fig. 6B). A similar
increase in t-BuOOH-induced AA release and its suppression
by n-propyl gallate were seen in cells expressing
sPLA2-V (Fig. 6B). Thus, the cells
became more sensitive to the signaling sPLA2s
after membrane lipid oxidation.
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A, the
IL-1ß-stimulated increase in AA release by
sPLA2-IIA-transfected 293 cells was suppressed
dose dependently by several agents that inhibit 12/15-LOX (NDGA, CDC,
and ETYA), but not by 5-LOX (AA-861). AA release by
cPLA2/sPLA2-IIA
cotransfectants was also suppressed by these 12/15-LOX inhibitors to a
similar extent (data not shown). Our previous study has shown that
these 12/15-LOX inhibitors suppress sPLA2-IIA
induction in 3Y1 cells (12). Thus, these results suggest
that endogenous 12/15-LOX may participate in IL-1ß-induced activation
of sPLA2-IIA-mediated membrane hydrolysis. In
support of this idea, supplementation of the
sPLA2-IIA-expressing, but not parental, cells
with 12-HPETE, a primary product of 12/15-LOX, resulted in a
significant increase in AA release even without stimulation (Fig. 7B). Moreover, the cells cotransfected with
sPLA2-IIA and 12/15-LOX released more AA than the
cells expressing sPLA2-IIA alone following A23187
stimulation, even though the sPLA2-IIA expression
levels in both clones were comparable, and AA release by the clone
expressing 12/15-LOX alone did not increase significantly (Fig. 7C). More [14C]12/15-HETE was
produced in A23187-stimulated cells coexpressing
sPLA2-IIA and 12/15-LOX than in replicate cells
expressing 12/15-LOX alone (Fig. 7D). These results suggest
that 12/15-HPETE or some other oxidative lipid metabolite produced by
12/15-LOX during cell activation may cause membrane perturbation,
leading to accelerated AA release by sPLA2s.
The finding that AA released by either cPLA2 or
sPLA2-IIA is efficiently converted by 12/15-LOX
to 12/15-HETE (Fig. 7D) implies the presence of an autocrine
amplification loop in the sPLA2-dependent
response. Thus, cPLA2 activation immediately
after cell activation leads to the production of 12/15-LOX metabolites,
which, in turn, trigger sPLA2-IIA-mediated AA
release. The AA thus released is further oxidized by 12/15-LOX, thereby
amplifying the lipid oxidation-directed membrane rearrangement process
and eventually leading to sustained activation of
sPLA2-IIA. As the cellular actions of
sPLA2-V are very similar (3, 4, 12, 13, 15, 16, 17), if not identical (38), to those of
sPLA2-IIA, we speculate that cellular functions
of the group II subfamily of sPLA2s may be
generally influenced by 12/15-LOX.
Conclusion: on the mechanisms by which signaling sPLA2s are activated
Although several studies have suggested that cPLA2, a well-recognized initiator of AA metabolism, precedes the prolonged PG biosynthetic response mediated by the two related heparin-binding signaling sPLA2s, sPLA2-IIA and sPLA2-V (3, 9, 13, 16, 20), the mechanisms by which cPLA2 modulates the functions of sPLA2s have remained unclear. In search of a regulatory molecule that links cPLA2 and sPLA2, we have found that 12/15-LOX, a LOX isozyme that oxygenates free AA as well as esterified polyunsaturated fatty acids in the cellular membranes (21, 22, 23), may play a pivotal role in the regulation of signaling sPLA2s. Although overexpression experiments indicate only the possibility that a 12/15-LOX-sPLA2 pathway could operate in parental cells, they are the best recourse open to us in view of the nature of LOX inhibitors. 12/15-LOX regulation of sPLA2 occurs in two ways: 1) it up-regulates the induction of sPLA2-IIA expression; and 2) it accelerates sPLA2-IIA-mediated membrane phospholipid hydrolysis, probably through oxidizing and thereby sensitizing the cellular membranes. This scenario may also occur in immunologically relevant cells such as macrophages, in which 12/15-LOX is expressed (21, 22, 23), and sPLA2 expression and function require prior activation of cPLA2 (8, 13). Thus, our present results have revealed a functional array of enzymes in separate arms of the AA cascade, the LOX and COX pathways, and substantiated a long-held hypothesis that membrane perturbation following cell activation is a prerequisite for sPLA2s to exert their proper actions on cells. Moreover, our results provide new insight into the biological importance of LOX-directed lipid oxidation signaling in regulating the expression and function of particular lipid-metabolizing enzymes and imply that the intracellular redox state affects the AA metabolic activity of cells. Thus, 12/15-LOX inhibitors would be a potential target for the development of therapeutic and prophylactic drugs for diseases or tissue disorders in which sPLA2s are significantly involved.
Finally, our current studies have led to the identification of several factors that affect the cellular actions of sPLA2-IIA. Glypican delivers endogenous heparin-binding sPLA2s into caveola signalsomes of activated cells, a process crucial for their efficient functional coupling with COX in the PG biosynthetic response (19). Alteration of plasma membrane asymmetry by phospholipid scramblase enhances AA release by signaling sPLA2s (17). The M-type sPLA2 receptor appears to mediate some biological actions of sPLA2-IIA, at least in some animal species (39). Here we have shown that the cPLA2-12/15-LOX pathway is a key regulatory step for sPLA2 functions. Given that sensitivity to sPLA2s differs according to cell type, it is likely that cells showing limited expression of either of these modifying factors may be refractory to the actions of sPLA2s even in the presence of the appropriate stimuli.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ichiro Kudo, Department of Health Chemistry, Showa University School of Pharmaceutical Sciences, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
3 Abbreviations used in this paper: PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; AA, arachidonic acid; COX, cyclo-oxygenase; CDC, cinnamyl-3,4-dihydroxy--cyanocinnamate; ETYA, 5,8,11,14-eicosatetraynoic acid; LOX, lipoxygenase; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid, NDGA, nordihydroguaiaretic acid; HEK, human embryonic kidney; LysoPC, lysophosphatidylcholine; AACOCF3, arachidonoyl trifluoromethyl ketone; t-BuOOH, tert-butylhydroperoxide; BHT, butylated hydroxytoluene; PPAR, peroxisome proliferator-activated receptor.
Received for publication March 3, 2000. Accepted for publication July 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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B and peroxisome proliferator-activated receptor-mediated process. J. Biol. Chem. 274:23085. ligands in macrophages by 12/15-lipoxygenase. Nature 400:378.[Medline]
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W. K. Han, A. Sapirstein, C. C. Hung, A. Alessandrini, and J. V. Bonventre Cross-talk between Cytosolic Phospholipase A2{alpha} (cPLA2{alpha}) and Secretory Phospholipase A2 (sPLA2) in Hydrogen Peroxide-induced Arachidonic Acid Release in Murine Mesangial Cells: sPLA2 REGULATES cPLA2{alpha} ACTIVITY THAT IS RESPONSIBLE FOR ARACHIDONIC ACID RELEASE J. Biol. Chem., June 20, 2003; 278(26): 24153 - 24163. [Abstract] [Full Text] [PDF]
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M. Murakami, S. Masuda, S. Shimbara, S. Bezzine, M. Lazdunski, G. Lambeau, M. H. Gelb, S. Matsukura, F. Kokubu, M. Adachi, et al. Cellular Arachidonate-releasing Function of Novel Classes of Secretory Phospholipase A2s (Groups III and XII) J. Biol. Chem., March 14, 2003; 278(12): 10657 - 10667. [Abstract] [Full Text] [PDF]
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 C. C. Silliman, E. E. Moore, G. Zallen, R. Gonzalez, J. L. Johnson, D. J. Elzi, X. Meng, K. Hanasaki, J. Ishizaki, H. Arita, et al. Presence of the M-type sPLA2 receptor on neutrophils and its role in elastase release and adhesion Am J Physiol Cell Physiol, October 1, 2002; 283(4): C1102 - C1113. [Abstract] [Full Text] [PDF]
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M. Murakami, K. Yoshihara, S. Shimbara, G. Lambeau, M. H. Gelb, A. G. Singer, M. Sawada, N. Inagaki, H. Nagai, M. Ishihara, et al. Cellular Arachidonate-releasing Function and Inflammation-associated Expression of Group IIF Secretory Phospholipase A2 J. Biol. Chem., May 17, 2002; 277(21): 19145 - 19155. [Abstract] [Full Text] [PDF]
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M. Murakami, R. S. Koduri, A. Enomoto, S. Shimbara, M. Seki, K. Yoshihara, A. Singer, E. Valentin, F. Ghomashchi, G. Lambeau, et al. Distinct Arachidonate-releasing Functions of Mammalian Secreted Phospholipase A2s in Human Embryonic Kidney 293 and Rat Mastocytoma RBL-2H3 Cells through Heparan Sulfate Shuttling and External Plasma Membrane Mechanisms J. Biol. Chem., March 23, 2001; 276(13): 10083 - 10096. [Abstract] [Full Text] [PDF]
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