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Research Communications |
* Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA;
Department of Chemistry, University of Southern California, Los Angeles, California 90089, USA; and
Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TH, United Kingdom
1Correspondence: Center for Experimental Therapeutics and Reperfusion Injury, Thorn 724, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115, USA. E-mail: CNSerhan{at}zeus.bwh.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: eicosanoids • lipid mediators • inflammation • PMN
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In response to
inflammatory stimuli, PMN phospholipases are activated to remodel cell
membranes and generate bioactive lipids that serve as intra- or
extracellular mediators in the transduction of functional responses
(2)
. Important components of microbicidal and acute inflammatory
responses include reactive oxygen species and granule enzymes that are
targeted to phagocytic vacuoles, but aberrant release of these
potentially toxic agents can lead to amplification of inflammation as
well as tissue injury and are implicated in a wide range of diseases
(3)
. To prevent an over-exuberant inflammatory response and limit
damage to the host, these PMN programs are tightly regulated. The host
mediators serving as endogenous antiinflammatory or protective signals
are only recently being appreciated (4)
.
Bioactive lipids are rapidly generated by activation of cell surface
receptors that carry either specific positive or negative signals to
modulate cellular responses. This is exemplified by the related
eicosanoids leukotriene B4
(LTB4), a potent chemoattractant (5)
, and lipoxin
A4 (LXA4), an endogenous
stop signal for PMN recruitment (4)
. LTB4 and
LXA4 interact with highly specific and distinct
G-protein-coupled membrane receptors (6,
7)
. They each evoke opposing
PMN responses, including LXA4 inhibition of
LTB4-initiated chemotaxis, adhesion, and
transmigration (4)
.
Aspirin is known to affect biosynthesis of lipid mediators and is
widely used clinically for its antiinflammatory properties. Mechanisms
responsible for aspirin's antiinflammatory actions remain of
considerable interest. In particular, new `super-aspirins' are sought
that spare the gastrointestinal tract and do not possess the
deleterious side effects of steroids (8)
. This laboratory has found
that, in addition to inhibiting prostanoid formation, aspirin triggers
the endogenous generation of novel carbon 15 epimers of LX by
transcellular routes (see Fig. 1
A) during inflammation in vivo (e.g., between
tissue resident cells and infiltrating leukocytes) (9)
. These
aspirin-triggered lipoxins (15-epi-LX) are even more potent than the
native LX as inhibitors of PMN responses, in part because they are
active longer (10)
. PMN inhibition by LX and 15-epi-LX is evoked by
specific receptor activation of `inhibitory' signals and not via
direct receptor level antagonism at LTB4
receptors (11)
. Moreover, interest in the regulation of the
LTB4 receptor is heightened by the recent finding
that LTB4 receptors also serve as novel HIV-1
coreceptors (12)
.
|
Despite ~100 years of use, complete knowledge of aspirin's
therapeutic impact is still evolving with many newly discovered
clinical utilities (13)
. Regular ingestion of aspirin decreases the
incidence of myocardial infarction, colorectal carcinoma, and
Alzheimer's disease (reviewed in ref 14
), but side effects from
aspirin, such as gastrointestinal ulceration, can limit its use. The
recent discovery of a second isoform of cyclooxygenase (COX) that is
induced during inflammation has led to a search for super-aspirins that
can selectively inhibit COX-2 without disrupting the protective
constitutive functions of COX-1 (8,
15)
. Of particular interest in this
regard, 15-epi-LX, which inhibit PMN migration, are endogenous products
of aspirin's acetylating ability that may underlie some of the
salutary benefits of aspirin. These findings suggest several novel
strategies of using 15-epi-LX mimetics as new antiinflammatory agents
designed after endogenous mediators. Along these lines, we designed
both LX and 15-epi-LX stable analogs, which, like
15-epi-LXA4, act via the
LXA4 receptor (10,
11)
.
15-epi-16-para-fluoro-phenoxy-lipoxin
A4-methyl ester (15-epi-LXa) is a synthetic
analog of 15-epi-LXA4 (Fig. 1A
, bottom
left) that not only resists rapid inactivation, but acts topically to
inhibit PMN infiltration and vascular permeability in mouse ear skin
inflammation (16)
.
Our present interest is the elucidation of signaling pathway(s)
responsible for receptor-operated blockage of PMN responses. Signaling
via phospholipase D (PLD) plays a pivotal role in mounting cellular
responses. Within seconds of exposure to ligands, PLD hydrolyzes
membrane phosphatidylcholine (PC) to generate phosphatidic acid (PA)
(17)
. Formation of PA temporally antecedes functional responses,
including vesicle secretion and assembly of the NADPH oxidase (18,
19)
.
Several isozymes of PLD1 and PLD2 were cloned and characterized (20)
,
with PLD1b identified as a prominent isoform in human granulocytes
(reviewed in ref 21
). Recently, we identified a novel polyisoprenyl
phosphate (PIPP) signaling pathway (Fig. 1A
) and found that,
in PMN, presqualene diphosphate (PSDP) carries biological activity and
serves as a negative intracellular signal that prevents superoxide
anion generation by several stimuli including PA (22)
. Because PLD
activation is linked to superoxide anion generation (23)
, we reasoned
that PIPP signaling might also modulate phospholipase activity critical
to global cellular activation. Here, we report that 1)
LTB4 receptor activation rapidly degrades PSDP, a
key component of PIPP signaling, which is reversed by a
LXA4 receptor agonist, 2) an
aspirin-triggered 15-epi-LXA4 stable analog
potently inhibits LTB4-initiated PLD activation
and superoxide anion generation, and 3) PSDP directly
inhibits both human recombinant and plant PLD. These findings provide
evidence for receptor-initiated PIPP remodeling as a regulatory
signaling pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 22. LTB4
was purchased from Cayman Chemical (Ann Arbor, Mich.), cabbage PLD
(cPLD), farnesyl diphosphate (FDP), squalene, lysis buffer reagents,
and cytochrome c were from Sigma Chemical Co. (St. Louis,
Mo.), and PC and PA were from Avanti Polar Lipids (Alabaster, Ala.).
The integrity and concentration of each bioactive lipid were assessed
just prior to each series of experiments by UV analysis (eicosanoids
and analogs) and phosphorus determinations (polyisoprenyl phosphates),
as described in refs 16
and 22.
Human PMN
Peripheral venous blood (~180 ml) was obtained by venipuncture
from healthy volunteers who denied taking any medication for at least 2
wk and had given written informed consent to a protocol approved by
Brigham and Women's Hospital's Human Research Committee. PMN were
isolated from whole blood and steady-state labeled with
[
-32P]ATP (40
µCiml-1, 90 min, 37°C), as in ref 22
. Labeled
PMN were resuspended (20 x
106ml-1 phosphate-buffered
saline with 1 mM CaCl2, pH 7.40) and exposed to
LTB4 (100 nM), 15-epi-LXa (100 nM), or vehicle
(0.1% EtOH) for 0 to 300 s (37°C). From each incubation,
aliquots were removed at indicated intervals to determine the
radiolabeling of nonsaponifiable lipids (10–12 x
106 PMN) and PLD activity (1–1.25 x
106 PMN). Materials present in each incubation
were saponified, extracted, and separated by thin-layer chromatography
(TLC) with phosphoimaging (model 425E and integration software;
Molecular Dynamics, Sunnyvale, Calif.), which was used for PSDP mass
determination as in ref 22
.
Preparation of recombinant human PLD1b
Spodoptera frugiperda (Sf9) cells were cultured in suspension at
2 x 105 to 2 x
106 cells/ml TC100 medium supplemented with 10%
fetal calf serum (Life Technologies, Inc., Paisley, U.K.). A cDNA
encoding human PLD1b (cloned from placental tissue) was inserted into
the transfer vector pACGHLT (PharMingen, San Diego, Calif.) downstream
of, and in frame with, vector sequences encoding
glutathione-S-transferase (GST), hexahistidine, a protein kinase A
phosphorylation site, and a thrombin cleavage site. The GST-hPLD1b
construct was cotransfected into Sf9 cells with linearized,
polyhedrin-minus (PH-), AcMNPV DNA, Bac-N-Blue according to the
supplier's instructions (Invitrogen, San Diego, Calif.). Homologous
recombination between linearized virus and the transfer vector restored
the function of essential viral gene ORF1629 to yield infectious,
recombinant virus. After two rounds of plaque purification, recombinant
virus was amplified by large-scale infections of Sf9 cells until a
titer of 8 x 107 pfu/ml was obtained. To
generate GST-hPLD1b, 500 ml of Sf9 cells at 2 x
106 cells/ml were infected with virus at a
multiplicity of infection of 10:1. Cells were harvested 72 h
postinfection, lysed, and the expressed GST-hPLD1b was purified on
glutathione agarose beads, according to supplier's instructions
(PharMingen). The purified recombinant protein was identified by
immunoreactivity with goat anti-GST pAb (Amersham Pharmacia Biotech,
Amersham, U.K.); rabbit pAb was raised against the PLD consensus
peptide sequence GSANIN (gift of P. Parker, ICRF, London, U.K.) and by
activity in an in vitro PLD assay (24)
.
PLD activity and superoxide anion generation
Lysates were generated from cells at rest or after exposure to
agonist using a lysis buffer comprised of 0.1 M HEPES (pH 7.4), 0.7 mM
sodium orthovanadate, 10 µM p-nitrophenylphosphate, 10 mM EGTA, 5.5%
triton X-100, 0.5 M ß-glycerophosphate, 10 mM
phenylmethylsulfonylfluoride, 0.1 mM ammonium molybdate, 12 mM DFP, 5
µgml-1 leupeptin, 2
µgml-1 aprotinin, and 7
µgml-1 pepstatin A (as in ref 25
) and used for
bioassay.
PMN lysates (90–130 µg protein), purified phospholipase D (3–30
units) (EC 3.1.4.4., Sigma Chemical Co.), or recombinant hPLD1b were
warmed (37°C for mammalian enzyme and 30°C for cabbage, 3 min) and
exposed to PSDP, PSMP, or FDP (10–1000 nM, 5 min, 37°C or 30°C),
followed by PC (0.5 to 5 mM) in Tris-HCl (50 mM, pH 7.5) with
CaCl2 (30 mM). Reactions were terminated at
30 s intervals (0–90 s) with Tris-HCl (1 M) plus EDTA (50 mM).
Choline release was quantitated as in ref 26
.
Freshly isolated human PMN (1–3 x 106
PMN/ml HBSS + 1.6 mM CaCl2) were incubated (5
min, 37°C) in the presence of 15-epi-LXa (1–100 nM) or vehicle
(0.1% ethanol), then exposed (10 min) to LTB4
(100 nM) in the presence of cytochrome c (7 mg/ml).
Superoxide anion generation was determined as in ref 22
.
Statistical analysis
Results are expressed as the mean ± SE and
statistical significance was evaluated using the Student's
t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To determine whether LTB4 receptor
activation leads to remodeling of PIPP, and specifically PSDP, cellular
phosphate pools were steady-state labeled with
[-32P]-ATP (see Materials and Methods) and
exposed to either LTB4 (100 nM) or vehicle (0.1%
ethanol) alone. Aliquots were removed at timed intervals from 0 to
300 s (37°C), and nonsaponifiable phosphorylated lipids were
isolated and quantitated by PhosphorImager for
[32P] incorporation. PSDP levels in
unstimulated PMN are ~1.7 nmol/107 PMN (~50
nM) (22)
. PSDP and presqualene monophosphate (PSMP), but not FDP,
incorporated [32P] from ATP (data not shown),
consistent with our recent evidence (22)
. LTB4
initiated a rapid (evident within 30 s) (Fig. 1B
) and
statistically significant decrease in
[32P]-PSDP (28%) within 60 s (Fig. 1C
). Within the ensuing 270 s,
[32P]-PSDP levels returned to baseline amounts
(t=0). Changes in [32P]-PSDP after
LTB4 receptor activation reflected changes in
PSDP mass. These results confirm that PSDP is present in PMN (22)
and
indicate that LTB4 initiates a marked decrement
in PSDP (Fig. 1)
, with a time course of PIPP remodeling concurrent with
LTB4 kinetics of cellular activation (5,
22)
.
15-epimer LX analog switches the LTB4 program to
enhance PSDP
Both LXA4 and
15-epi-LXA4 stable analogs act at the
LXA4 receptor on PMN, inhibiting infiltration
in vivo (11)
. To determine whether LX and 15-epi-LX mediate
inhibitory actions via PIPP signaling, the impact of a
15-epi-LXA4 analog (15-epi-LXa) (100 nM, 5 min,
37°C) on LTB4 (100 nM) -stimulated changes in
PSDP was examined using [32P] labeling of PMN
lipids (vide supra, in parallel incubations). Alone,
15-epi-LXa did not affect the rate of PIPP remodeling (Fig. 1B
). Exposure to LTB4 in the presence
of equimolar 15-epi-LXa not only prevented the
LTB4-initiated decrease in PSDP, but also
stimulated a significant increase (~72%) in
[32P]-PSDP at 60 s (Fig. 1C
).
PSDP levels continued to rise for at least 300 s after exposure to
LTB4 (Fig. 1B
). Native
LXA4 and its related LXA4
receptor agonist, 16-phenoxy-LXA4-methyl ester,
gave qualitatively similar responses as 15-epi-LXa with a rank order of
potency of 15-epi-LXa > 16-phenoxy-LXA4 >
LXA4, with 15-epi-LXa 1–2 orders of magnitude
more potent (data not shown). These results indicate that 15-epi-LXa,
which inhibits LTB4 responses in vivo
(11)
, dramatically switches LTB4-initiated PIPP
signaling. Moreover, increases in PSDP levels evoked by coactivation of
the LXA4 and LTB4 receptors
indicate that the time course of PSDP accumulation correlated with
regulation of LTB4's actions by LX and
15-epi-LXa (vide infra).
15-epi-LXa inhibits LTB4-stimulated PLD activity and
O2- generation
LTB4-stimulated PLD activity is associated
with morphological change, degranulation, and
O2- production in PMN (19,
27)
.
To determine whether LT and LX-mediated remodeling of PIPP correlates
with specific cell signaling events, we monitored PLD activity in cell
lysates from the same incubations used in Fig. 1
.
LTB4 yielded increases in PLD activity that were
maximal by 60 s (Fig. 2
A). These values for LTB4 and PLD are
consistent with those of earlier reports (25,
27)
. In the presence of
15-epi-LXa, LTB4-stimulated PLD activity was
inhibited (~81%) at 60 s (Fig. 2A, B
). 15-epi-LXa
also potently inhibited LTB4-stimulated
O2- generation (Fig. 2C
). Together, these findings indicate that ligand–receptor
interactions that signal opposing cellular responses gave an inverse
relationship between [32P]-PSDP levels and PLD
activity, raising the possibility that PSDP might regulate PLD.
|
Direct inhibition of both plant and mammalian PLD
To determine whether polyisoprenyl phosphates act directly on PLD,
PSDP and closely related lipids were incubated with purified plant
enzyme (EC 3.1.4.4; Vm = 0.29 nmol/s, Km =
1.4 mM). As seen in Fig. 3
, PSDP inhibited cPLD in a concentration-dependent fashion (10 to 1000
nM) with a Ki of 20 nM ([PSDP] = 10 nM).
Lineweaver-Burk analyses (Fig. 3)
were consistent with a competitive
inhibition model. Closely related lipids, such as PSMP (minus only one
phosphate), showed a greater than 100-fold loss in inhibitory potency
compared to PSDP (Table 1
).Comparable inhibition was not evident with other polyisoprenoids (i.e.,
FDP and squalene) or a PLD product (PA). We addressed whether PSDP
could also inhibit mammalian PLD by determining recombinant human PLD1b
kinetics in vitro with PSDP. The recombinant enzyme
(Vm = 0.36 nmol/s, Km = 13.8 mM) was also
dramatically inhibited by PSDP with a Ki of 6 nM
(Table 1)
.
|
|
Because PLD activation occurs in vivo in the presence of
many cofactors that modulate its activity, we also determined the
effect of PSDP on PLD activity in PMN lysates. Sixty seconds after
LTB4, PSDP levels decreased (28%, Fig. 1
) and
PLD activity was maximal (Fig. 2)
. Addition of PSDP (100 nM) to PMN
lysates at this time (60 s, LTB4 100 nM) gave
89.5 ± 9.7% inhibition of PLD activity (data not shown).
Collectively, these results indicate that PSDP is a potent inhibitor of
both plant and mammalian PLDs and establish a critical role for both
the terminal phosphate and the isoprenoid chain length in PSDP's
action with PLD activity.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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that coincided temporally with increased PLD activity (Fig. 2)
. As PSDP
remodeling returned toward baseline values, PLD activity decreased,
revealing an inverse relationship and suggesting a role for PSDP in the
regulation of this pivotal lipid-modifying enzyme. Cells exposed to
LTB4 and an LXA4 receptor
agonist (15-epi-LXa) showed a dramatic switch in PSDP remodeling to
give increased [32P]-PSDP and marked inhibition
of both PLD activity and superoxide anion generation (Figs. 1,
2)
. In
addition, synthetic PSDP was a selective and potent inhibitor of
isolated PLD (Fig. 3
, Table 1
), a property not shared by other closely
related lipids. The reciprocal relationship between PSDP levels and PLD
activity as well as direct inhibition of recombinant human PLD1b,
purified cPLD, and PLD activity in PMN lysates support a role for PSDP
as an endogenous lipid regulator of PMN PLD activity. The different
temporal profiles of PIPP remodeling initiated on receptor activation
by PMN ligands with opposing actions (i.e., stimulation and inhibition)
suggest that PIPP remodeling and PSDP itself may serve as important
components in intracellular signaling, particularly as stop signals.
Cholesterol is not a biosynthetic product in PMN, as they lack a mixed
function oxidase and cyclase necessary for its endogenous formation
from acetate (28)
. In view of the present findings, the resultant
biosynthetic termination at squalene in PMN suggests that products such
as squalene's direct precursor, PSDP, carries functions distinct from
cholesterol biosynthesis. Hence, it is likely that the PIPP signaling
pathway uncovered here in human PMN may extend to other cell types. In
addition to dietary influences known to affect mevalonate and
polyisoprenyl phosphate biosynthesis, PSDP formation is also actively
regulated by soluble immune stimuli and growth factors (Fig. 1B, C
; ref 22
). Granulocyte/macrophage-colony stimulating factor, for
example, increases PSDP remodeling in PMN, whereas the chemotactic
peptide fMLP triggers (within seconds) rapid decrements in PSDP and
reciprocal increments in PSMP, which return to baseline within 5–10
min (22)
. This time course of PIPP remodeling is similar in magnitude
and extent to LTB4-initiated decrements in PSDP
(Fig. 1B, C
) and correlates well with the time course of
activating neutrophil responses such as
O2- generation, which is
inhibited by PSDP (22)
. The presence of PSDP in peripheral blood PMN
despite their inability to generate cholesterol from endogenous
sources, its rapid remodeling in response to receptor-mediated
inflammatory stimuli of diverse classes of receptor agonist, and its
ability to inhibit PLD activity and NADPH oxidase at nanomolar levels
support a role for PSDP as a novel negative intracellular signal. Thus,
this newly uncovered PIPP signaling might function to decrease negative
signal levels, in contrast to the well-appreciated phosphatidylinositol
signaling pathways (reviewed in ref 24
) that, when activated, rapidly
generate positive intracellular stimuli (e.g., inositol triphosphate,
diacylglycerol, and Ca2+).
Aspirin, the leading nonsteroidal antiinflammatory drug, also effects
cholesterol biosynthesis by mechanisms that remain to be completely
elucidated (29)
. Beyond its well-appreciated inhibition of COX, aspirin
can pirate this system to set in place an antiinflammatory circuit
generating 15-epi-LX, carbon 15-R-epimers of the natural
15-S-containing-LX, during cell–cell interactions by
aspirin-acetylated COX-2 and 5-lipoxygenase (Fig. 1A
and ref 9
). These aspirin-triggered LX carry antiinflammatory and
antiproliferative properties (30,
31)
, and may mediate a component of
aspirin's beneficial therapeutic actions. As observed in these
experiments, LXA4 receptor activation by a
15-epi-LX mimetic reversed PSDP remodeling initiated by
LTB4 receptors, leading to increases in PSDP
levels (Fig. 1)
. Since the 15-epi-LXa inhibited both PLD activity and
superoxide anion generation (Fig. 2)
, these results implicate PIPP
remodeling as a component of the cellular basis for aspirin's
inhibition of excessive inflammatory responses. In addition to
regulating LTB4's stimulatory actions, this
novel mechanism of inhibition of LTB4 receptor
signaling may also play broader roles in host defense, as this receptor
was recently identified as a coreceptor for HIV-1 (12)
.
Hydrolysis of PC to PA by PLD appears crucial in transmembrane
signaling by a wide range of receptor classes during PMN activation
(19)
. Both G-protein-linked receptors and receptor tyrosine kinases
activate PLD. In leukocytes, several factors, including PKC
(in a
kinase-independent manner) and increased intracellular calcium, can
activate PLD1 (32)
. FMLP-stimulated PLD activity in PMN is increased by
membrane association of the ADP-ribosylation factor and small GTPase
RhoA (33)
. PSDP directly inhibited recombinant hPLD1b in the absence of
regulatory proteins (see Table 1
). These results suggest that PSDP may
inhibit PLD at its catalytic center and is likely to act at other PLD
isoforms, such as PLD1a and PLD2 isoforms, where the catalytic centers
are conserved. PSDP's ability to serve as an endogenous inhibitor of
PLD likely results from its unique 3-dimensional and physical chemical
properties, which might now serve as a template for the preparation of
more potent PLD inhibitors designed to fulfill the structure activity
relationship uncovered here.
Regulation of PMN activation in complex host responses is controlled in
part by soluble mediators and, in particular, by autocoids with
opposing actions (2)
such as LT and LX, which here gave markedly
different profiles for PIPP remodeling (Fig. 1)
. In most cell types,
PSDP is appreciated as a biosynthetic intermediate in cholesterol
production by microsomal squalene synthase, which catalyzes
head-to-head condensation of two FDP (34)
. Ligand-operated rapid
remodeling of PSDP in PMN is likely to occur in membranes in proximity
to LTB4 and LXA4 receptors,
and suggests a nonmicrosomal pool of PSDP that may result from
1) novel biosynthetic and/or metabolic pathways or
2) intracellular trafficking of PIPP with proteins from
endoplasmic reticulum to membrane domains. Incorporation of
[32P] from ATP into PSDP, but not FDP (see
Results), is further evidence in support of a novel route for PSDP
formation in PMN. Our results suggest that PIPP remodeling is linked to
cell surface receptor activation and is involved in the intracellular
transmission of extracellular ligands with opposing biological actions.
In our working model, a `negative lipid signal' (i.e., PSDP) is held
at a set point, like a ratchet, in `resting' cells. Incoming positive
signals (LTB4, fMLP, etc.) initiate the
degradation and inactivation of this inhibitory lipid (e.g., remodeling
PSDP to the inactive monophosphate species, PSMP) (Fig. 1A
and ref 22
). Thus, PIPP remodeling enables mounting of intracellular
positive signals that threshold for activation of select cellular
processes. This type of signaling may explain the selectivity and tight
coupling required by agonists such as LTB4 that
stimulate highly specialized functional responses of PMN such as
chemotaxis, granule mobilization, and superoxide anion generation. The
extent to which this model of cell signaling, namely,
receptor-initiated degradation of negative lipid signals, occurs with
other receptors and cell types remains for future studies.
In summary, ligand-operated rapid remodeling of PIPPs in human PMN and
direct inhibition of PLD activity at nanomolar levels support a role
for PSDP as an intracellular signal (22)
and provide novel
intracellular targets by which PSDP can regulate cellular responses.
Given the wide occurrence of PIPP and critical role of PLD in the plant
and animal kingdoms (21,
35)
, PIPP remodeling and direct inhibition of
PLD established here in human PMN may have wider implications in cell
signaling in other cell types and species. The present results are the
first to show direct inhibition of a phospholipase involved in signal
transduction by an endogenous intracellular lipid; they set forth a new
paradigm for lipid–protein interactions in the control of cellular
responses, namely, receptor-initiated degradation of a repressor lipid,
which is also subject to regulation by aspirin ingestion via the
actions of aspirin-triggered 15-epimer LX. Together, these results
suggest that PIPP signaling pathways might also be of interest in
pharmacologic interventions and, specifically, that the conformation of
PSDP can serve as a template for design of novel inhibitors.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication December 9, 1998.
Revision received January 12, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
100 nM), 15-epi-LXa (
, 100 nM), vehicle (
, 0.1% ethanol), or
15-epi-LXa (100 nM, 5 min), followed by LTB4 (
, 100 nM).
Nonsaponifiable lipids were extracted and separated by TLC, and
[32P] incorporation was quantitated by phosphoimaging
(see Materials and Methods). Values are densitometric measurements.
0.0% for peripheral blood PMN in the absence of agonist is ~1.7
nmol/107 PMN (see text), therefore a 30% change in PSDP
represents ~ 0.51 nmol/107 PMN. Panel
B reports a representative time course
(n=5) and panel C shows the change
(mean ± SE) at 60 s. *P<0.05 by
Student's t test.
4 for reactions in the absence of PSDP (