From the Department of Chemistry and Biochemistry,
School of Medicine and Revelle College, University of California at
San Diego, La Jolla, California 92093-0601 and the
§ Department of Food Science, Cook College, New Jersey
Agricultural Experimental Station, Rutgers University,
New Brunswick, New Jersey 08901-8520
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ABSTRACT |
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We have found that the novel phospholipid
diacylglycerol pyrophosphate (DGPP), identified in bacteria, yeast, and
plants, but not in mammalian cells, is able to potently activate
macrophages for enhanced secretion of arachidonate metabolites, a key
event in the immunoinflammatory response of leukocytes. Macrophage
responses to DGPP are specific and are not mediated by its conversion
into other putative lipid mediators such as phosphatidic acid,
lysophosphatidic acid, or diacylglycerol. The responses to DGPP are
compatible with a receptor-recognition event because they are blocked
by suramin. Intracellular signaling initiated by DGPP includes
phosphorylation and activation of the Group IV cytosolic phospholipase
A2 and of the extracellular-signal regulated p42
mitogen-activated protein kinase (MAPK) and p44 MAPK, and membrane
translocation of the protein kinase C isoenzymes Leukocytes constitute the primary line of defense against
infection. Recognition of foreign material by specific membrane receptors on the leukocyte surface enables these cells to mount an
immunoinflammatory response that ultimately leads to killing of the
microbe. During the course of these events, leukocyte membrane remodeling generates biologically active lipids that can serve both as
intra- and extracellular mediators (1).
Recently, a novel phospholipid compound, diacylglycerol pyrophosphate
(DGPP),1 has been identified
(2). The biochemical routes leading to biosynthesis and degradation of
DGPP in those cells which produce it have recently been elucidated
(3-8). DGPP is produced by phosphorylation of phosphatidic acid (PA),
a reaction catalyzed by a specific kinase that has been purified from
plants (3). The amount of DGPP in resting cells is barely detectable,
e.g. less than 0.18% of the major phospholipids in
Saccharomyces cerevisiae (9). However, recent studies in
plants have demonstrated that the concentration of DGPP increases
significantly when signaling is activated by the G-protein activating
peptide mastoparan (5). Thus, DGPP has characteristics of a lipid
messenger molecule, suggesting its participation in novel lipid
signaling pathways (9, 10).
DGPP degradation is accounted for by a two-step dephosphorylation
reaction catalyzed by DGPP phosphatase. The enzyme first removes the
DGPP production has not been documented in mammalian cells to date, and
its biological role remains completely unknown (12). The
above-mentioned observations, however, appear to anticipate an
important role for DGPP in cellular signaling. DGPP, like DAG and
lyso-PA, is produced from PA via a single enzymatic step. Because all
these lipids have been shown to potently mediate cellular signaling, we
have now examined the capacity of DGPP to mediate macrophage activation
and the release of arachidonic acid (AA)-derived inflammatory mediators
such as the eicosanoids. The results reported herein demonstrate that
DGPP is a novel, potent macrophage-activating factor and suggest a role
for DGPP in triggering proinflammatory cell responses.
Materials--
The cell line used in this study, termed
P388D1/MAB, is a subclone of the P388D1 cell
line (TIB 63) available from the American Type Culture Collection
(Manassas, VA) that was selected on the basis of high responsivity to
LPS/PAF. A description of the characteristics of this subclone will be
published elsewhere.2
Iscove's modified Dulbecco's medium (endotoxin < 0.05 ng/ml) was from Whittaker Bioproducts (Walkersville, MD). Fetal bovine serum
was from HyClone Labs. (Logan, UT). Nonessential amino acids were from
Irvine Scientific (Santa Ana, CA).
[5,6,8,9,11,12,1,15-3H]Arachidonic acid (specific
activity 100 Ci/mmol) was obtained from NEN Life Science Products. ADP,
pyrophosphate, LPS (Re595), and PAF were from Sigma. Methyl arachidonyl
fluorophosphonate (MAFP) was from Cayman (Ann Arbor, MI). Suramin was
from Biomol (Plymouth Meeting, PA). The MAP kinase kinase inhibitor,
PD098059, was from Calbiochem (San Diego, CA). Group IV
cPLA2 antibodies were kindly provided by Dr. Ruth Kramer
(Lilly Research Laboratories, Indianapolis, IN). Phospho-specific
p42/p44 mitogen-activated protein kinase (MAPK)
(Thr202/Tyr204) antibody was from New England
Biolabs (Beverly, MA). Protein kinase C (PKC) antibodies were from
Santa Cruz Biotechnology (Santa Cruz, CA).
DGPP (dioctanoyl-sn-glycero-3-pyrophosphate,
diC8-DGPP) was chemically synthesized from
dioctanoyl-sn-glycero-3-phosphate (diC8-PA)
(Avanti Polar Lipids, Alabaster, AL) and ortho-phosphoric acid as described by Riedel et al. (8).
DiC8-DGPP was purified by thin-layer chromatography on
potassium oxalate-treated plates using the solvent system
chloroform/acetone/methanol/glacial acetic acid/water (50:15:13:12:4)
(The Rf for DGPP in this system is 0.33, and the Rf for PA is 0.71)
(2). The purified DiC8-DGPP migrated precisely with
enzymatically synthesized DiC8-DGPP using three different
solvent systems (2). In addition, the chemically synthesized
DiC8-DGPP was enzymatically active when employed as a
substrate for pure DGPP phosphatase from Saccharomyces
cerevisiae (6). 32P-labeled ( Cell Culture and Labeling Conditions--
P388D1
cells were maintained at 37 °C in a humidified atmosphere at 90%
air and 10% CO2 in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 2 mM glutamine,
100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were
plated at 106 per well, allowed to adhere overnight, and
used for experiments the following day. All experiments were conducted
in serum-free Iscove's modified Dulbecco's medium.
Stimulation of P388D1 Cells--
Our standard
regimen for activating the P388D1 cells has been described
previously (13-15). Briefly, radiolabeling of the cells with
[3H]AA was achieved by including 0.5 µCi/ml
[3H]AA during the overnight adherence period (20 h). The
cells were placed in serum-free medium for 30-60 min before the
addition of LPS (200 ng/ml) for 1 h. After the LPS incubation, the
cells were exposed to PAF for the time indicated, in the presence of 0.1 mg/ml bovine serum. For DGPP, the LPS priming step was omitted. When suramin was used, it was added 30 min before the stimulants.
After stimulation, supernatants were removed, cleared of detached cells
by centrifugation, and assayed for radioactivity by liquid
scintillation counting. More than 99% of the released radioactive material remains as unmetabolized AA under these experimental conditions.
For the measurement of PGD2 or PGE2 production,
unlabeled cells were used, and the incubations proceeded in the absence
of albumin. Prostaglandins in the supernatants were measured by using radioimmunoassays specific for either PGD2 (Amersham
Pharmacia Biotech) or PGE2 (PerSeptive Diagnostics,
Framingham, MA).
Immunoblotting Studies--
Cells, serum-starved for 1 h,
were stimulated as described above. Afterward, the cells were washed
and lysed in a buffer consisting of 1 mM Hepes, 0.5%
Triton, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and
10 µg/ml leupeptin at 4 °C. Protein was quantified, and a 100-µg
aliquot was analyzed by immunoblot with antibodies against
cPLA2, p42/p44 MAPK, or the protein kinase C isoenzymes
DGPP-induced Arachidonic Acid Metabolism--
When mouse
P388D1 macrophages were exposed to DGPP, an appreciable
formation of prostaglandins such as PGE2 was detected in a
dose- (Fig. 1A) and
time-dependent (Fig. 1B) manner. Positive controls using the standard activation procedure of these cells with
LPS/PAF (13-15) gave a comparable response, indicating that the
DGPP-stimulated prostaglandin response is physiologically relevant.
We investigated next the critical components of the DGPP molecular
structure required for stimulation of AA metabolism.
Pyrophosphate-containing compounds such as pyrophosphate itself and ADP
were ineffective (data not shown), suggesting that the DGPP stimulatory
activity arises from its lipidic nature. DGPP was not metabolized to PA or lyso-PA by the macrophages within the time frame of the current experiments (up to 30 min). This was assessed by incubating the cells
with 50 µM [
AA mobilization and subsequent prostaglandin production are events
typically triggered by interaction of agonists with receptors on the
surface of immunoinflammatory cells such as the macrophages. The
characteristics of the DGPP effect depicted in Fig. 1, along with the
fact that its stimulatory potency is similar to that of the receptor
agonists LPS/PAF, prompted us to investigate the possibility of whether
the DGPP effect was receptor-mediated. For these experiments, we used
the receptor antagonist suramin. Because of its effect of blocking
receptor coupling to G-proteins, suramin is regarded as a general
inhibitor of receptor-mediated processes (16). As shown in Fig.
2, prostaglandin production by DGPP was
not observed if the cells were first incubated with suramin. The
suramin inhibition was found to be dose-dependent, with
half-maximal inhibition being observed at 30 µM.
Interestingly, suramin treatment allowed us to distinguish the DGPP
effects from those exerted by exogenous DAG
(dioctanoyl-sn-glycerol), as the effects of the latter on AA
release were largely insensitive to suramin (data not shown). Thus the
effect of DGPP is not mediated via conversion to DAG.
Our investigations into the molecular mechanisms of PAF
receptor-mediated AA mobilization have highlighted the requirement for
a rise in intracellular Ca2+ levels, an event that occurs
within seconds after PAF addition (17). Given the above results
suggesting that DGPP could signal as well in a receptor-mediated
manner, we examined the possibility that DGPP induces intracellular
Ca2+ mobilization in P388D1 cells. To this end,
a protocol identical to that previously used for PAF was used,
i.e. using fura2-loaded cells (17). The results of these
experiments revealed that, unlike PAF, DGPP failed to induce any
detectable change in the intracellular Ca2+ concentration
(not shown). In turn, these data indicate that DGPP does not act as a
Ca2+ ionophore.
DGPP Activates the MAP Kinase Cascade and Cytosolic Phospholipase
A2--
By what mechanism does DGPP stimulate
prostaglandin production? A direct approach to determining the identity
of the PLA2(s) responsible for AA mobilization and
subsequent prostaglandin synthesis is to use selective inhibitors for
the PLA2 forms present in these cells (13). AA mobilization
induced by DGPP was completely blocked by MAFP (13), an inhibitor of
the cytosolic group IV phospholipase A2, or
cPLA2 (Fig. 3A),
suggesting involvement of the latter enzyme in DGPP-induced signaling.
Further support to this notion was given by the finding that DGPP
induced a retardation of the electrophoretic mobility of the small but
measurable amount of unphosphorylated cPLA2 in the resting
cells, indicating phosphorylation of that fraction of enzyme (Fig.
3B). Positive controls using the standard activation
procedure of the P388D1 cells with LPS/PAF (13-15) gave a
similar result.
The kinases responsible for phosphorylation of the cPLA2
have all been identified as members of the MAPK family. In the vast majority of cell types, including P388D1
macrophages,3 the kinases
responsible for phosphorylating the cPLA2 have been identified as the extracellular signal-regulated kinases (ERK) p42 MAPK
and p44 MAPK (18-21). Fig. 4A
shows that DGPP stimulation of the cells led to a robust activation of
both p42 MAPK and p44 MAPK, as judged by increased phosphorylation of
these kinases. The time-course of activation of the ERKs by DGPP is
shown in Fig. 4B. Direct proof that p42/p44 MAPK activation
is involved in prostaglandin synthesis in DGPP-stimulated cells was
established by using the MAP kinase kinase inhibitor PD098059, a
compound that inhibited p42/p44 MAPK phosphorylation (Fig.
5A), and PGE2 release (Fig. 5C). PD098059 also slightly inhibited the
phosphorylation of the small fraction of cPLA2 that was not
phosphorylated under basal conditions (Fig. 5B). It is
important to note that phosphorylation of the cPLA2 at
Ser505 (i.e. the one causing the gel shift) has
been demonstrated not to be required for AA mobilization and
prostaglandin release under certain conditions (21, 22). Hence,
cPLA2 phosphorylation does not necessarily have to
correlate with enhanced AA release, as other factors may also be
involved (21-24). As a matter of fact, no correlation between
DGPP-induced cPLA2 gel shift (Fig. 5B) and
PGE2 release (Fig. 5C) is evident from our data.
Therefore DGPP-induced cPLA2 phosphorylation leading to a
gel shift is just interpreted as a consequence of DGPP-induced MAP
kinase activation, without necessarily having a role in AA release.
DGPP Activates Protein Kinase C--
Having thus established that
DGPP behaves as an activator of the "classical" MAPK cascade module
and its downstream effector cPLA2, we sought to investigate
next the effect of DGPP on signaling elements that are known to be
placed upstream of the MAPK cascade. PKC has often been documented as
an upstream activator of the classical MAPK kinase pathway and the
cPLA2 (23, 24). When activated, protein kinase C
translocates to membranes as this is its cellular site of action.
Therefore, activation of PKC by DGPP could be easily followed by
monitoring its increased appearance in membranes. Translocation of the
PKC isoenzymes Phosphatidic acid and the compounds that derive from it in a
single enzymatic step, i.e. lyso-PA and DAG, are recognized
to play key roles in cell physiology, not only as intermediate lipid metabolites, but as potent signaling mediators (26, 27). In this paper,
we have identified a signaling pathway, initiated by another direct
metabolite of PA, DGPP, through which leukocytes may be activated to
generate inmunoinflammatory lipid mediators. Such a signaling pathway
includes activation of several PKC isoenzymes, p42/p44 MAPK, and
finally the cPLA2. Thus DGPP adds to the number of
biological structures capable of triggering a proinflammatory response
by phagocytic cells.
Although identified in 1993 (2), the cellular roles of DGPP and the
enzymes involved in its metabolism, namely PA kinase and DGPP
phosphatase, have remained obscure. It has been speculated that DGPP
might simply represent a product of signal attenuation of PA-mediated
signaling (2, 3). Thus plasma membrane increases in PA as a consequence
of activation of the phosphoinositide turnover could be attenuated by
its conversion to DGPP via PA kinase, which has been demonstrated to
reside in the plasma membrane (2, 3). It has been speculated as well
that DGPP might represent a precursor species for the PA to be utilized
either for specific signaling functions or phospholipid synthesis (6).
Studies in plants have shown DGPP to be a minor polar lipid that
dramatically increases and then decreases in concentration when cells
are activated (5, 10). This behavior, typical of lipid messenger
formation and attenuation, suggests that DGPP is itself a signaling
molecule and not just a by-product of PA metabolism.
PA and lyso-PA have been long known to possess stimulatory activity on
cells when applied as an extracellular stimulus. lyso-PA, acting via a
specific surface receptor, appears to mediate a myriad of biological
effects under physiological settings (26). On the other hand, it seems
logical to rationalize that the extracellular signaling ability of PA
is more relevant under pathophysiological settings, where relatively
large amounts of PA arising from damaged tissue membranes can be made
accessible for stimulation of cells in the vicinity. Given the obvious
structural similarities among DGPP, PA, and lyso-PA, we wondered
whether DGPP would have extracellular activating capacity as well. Our
results have provided strong evidence to document that DGPP is capable
on its own of activating immunoinflammatory signaling in macrophages.
This is a very remarkable discovery because DGPP has not been
identified in mammalian cells (10). Thus it is possible that
immunocompetent cells may have evolved to recognize a distinctive lipid
on the membranes of foreign organisms, thus adding to the specificity
of the immunoinflammatory reaction. Two other pieces of evidence herein
reported are consistent with the aforementioned possibility. First, the
DGPP effect arises from its lipidic nature and is specific
(i.e. is not mediated by its conversion to either PA or
lyso-PA); second, the DGPP effects are strongly inhibited by
suramin, which suggests the involvement of a proteinaceous
component of the plasma membrane in DGPP recognition. We cannot rule
out, however, that suramin may have acted in our experiments in a
receptor-independent manner.
The current results raise an intriguing question as to the identity of
the membrane receptor to which DGPP binds to activate macrophage AA
metabolism. Although it is possible that DGPP interacts with a specific
surface receptor, the structural similarities between DGPP and lyso-PA
raise, as well, the possibility that DGPP is acting as a surrogate for
lyso-PA and hence interacting with a lyso-PA receptor. A receptor for
lyso-PA, vzg-1/lpA1/edg-2, has
recently been identified (28, 29). Northern blot analysis of mRNA
from P388D1 macrophages failed to detect expression of the
vzg-1/lpA1/edg-2 lyso-PA receptor (27,
28) on these cells.4
Moreover, this receptor does not bind DGPP when assayed by a specific
receptor binding assay (28, 29).4 Therefore, the DGPP
effects reported herein are not mediated by this lyso-PA receptor.
Recently, two other lyso-PA-like receptors have been identified (30,
31). It will be interesting to investigate their relationship, if any,
to DGPP signaling. It is interesting to note in this regard that the
effects of DGPP herein reported do not involve intracellular
Ca2+ movements, whereas signaling through the lyso-PA
receptor is usually accompanied by this response (26).
In summary, the current results demonstrate that DGPP is a potent and
specific extracellular mediator and provide keys to understanding the
biological significance of this novel phospholipid compound. The
studies reported here provide the foundation for future molecular
studies directed toward understanding the intriguing biological role of DGPP.
,
,
. These
results establish DGPP as a novel macrophage-activating factor and
suggest a potential role for this compound in triggering homeostatic
cellular responses.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-phosphate from DGPP to form PA and then removes the phosphate from
PA to form diacylglycerol (DAG) (6). The reactions catalyzed by DGPP
phosphatase are Mg2+-independent and
N-ethylmaleimide-insensitive (6, 7). The properties of the
DGPP phosphatase reactions in yeast, bacteria, and plants are
strikingly similar to the Mg2+-independent PA phosphatase
purified from mammalian cells (9). In fact, the
Mg2+-independent PA phosphatase from rat liver also
displays DGPP phosphatase activity (11). The
Mg2+-independent phosphatase is postulated to play
important roles in cellular signaling by modulating the cellular levels
of several bioactive lipids including diacylglycerol, PA, lyso-PA, and
sphingosine-1-phosphate (9).
EXPERIMENTAL PROCEDURES
-32P) DGPP
was synthesized enzymatically using purified Catharanthus roseus PA kinase as described by Wu et al. (6).
,
, and
.
RESULTS
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Fig. 1.
DGPP induces PGE2
production in P388D1 macrophages. A, effect
of increasing DGPP concentrations on PGE2 production
(30-min stimulation). B, time-course of PGE2
production (50 µM DGPP).
-32P]DGPP for different
periods of time (5, 10, 15, and 30 min). Analysis of the distribution
of 32P radioactivity after the incubations by thin-layer
chromatography (6) revealed that all the label was recovered as DGPP.
Neither 32P-labeled PA nor 32P-labeled lyso-PA
could be detected. In addition, no uptake of DGPP by the cells could be
detected, as quantitated by measuring the amount of cell-associated
32P radioactivity after the incubations. Thus the effect of
DGPP on AA mobilization appears to be specific and not mediated via its
putative conversion to other bioactive lipids such as PA or lyso-PA.
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Fig. 2.
Effect of suramin on DGPP-induced
PGD2 production (50 µM, 30-min stimulation)
(closed circles) compared with unstimulated control cells
(open circles).
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Fig. 3.
cPLA2 involvement on DGPP
signaling. A, effect of MAFP on DGPP-induced AA
release. The cells were incubated for 20 min without ( ) or with (+)
25 µM MAFP as indicated. Afterward, the cells were either
untreated (
or treated with (+) 50 µM DGPP as
indicated, and AA release was determined after 30 min. B,
phosphorylation of cPLA2 by different stimuli in
P388D1 macrophages. Cells were incubated with DGPP (50 µM, 30 min), LPS/PAF (1 h with 200 ng/ml LPS followed by
10 min with 100 nM PAF), or neither (control).
Afterward, cells were lysed, and cPLA2 mobility shift was
analyzed by immunoblot.
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Fig. 4.
Involvement of the MAPK cascade in DGPP
signaling. A, phosphorylation of p42/p44 MAPK by
different stimuli in P388D1 macrophages. Cells were
incubated with DGPP (50 µM for 30 min), LPS/PAF (1 h with
200 ng/ml LPS followed by 10 min with 100 nM PAF), or
neither. Afterward, cells were lysed, and p42/p44 MAPK phosphorylation
was analyzed by immunoblot. B, time-course of the effect of
DGPP (50 µM) on p42/p44 MAPK phosphorylation.
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Fig. 5.
Effects of PD098059, a MAP kinase kinase
inhibitor. A, dose-dependent inhibition by
PD098059 of DGPP-induced phosphorylation of p42/p44 MAPK. B,
effect of PD098059 on cPLA2 phosphorylation induced by
DGPP. C, effect of PD098059 on DGPP-induced PGE2
production (close circles) and controls lacking DGPP
(open circles). PD098059 concentrations are expressed as
micromolar in all panels.
,
, and
was readily observed within 1 min of
cell treatment with DGPP, i.e. earlier than the activation
of p42/p44 MAPK (Fig. 6). The one other
PKC isoenzyme present in these cells, PKC
, (25) did not translocate
to membranes in response to DGPP (not shown).
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Fig. 6.
Translocation of three PKC isoenzymes to the
membrane fraction of cells stimulated with DGPP (50 µM)
for the times indicated.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We are indebted to Prof. Jerold Chun
(Pharmacology Dept., UCSD) for conducting the lyso-PA receptor studies.
We are indebted as well to Dr. Juan Llopis (Pharmacology Dept., UCSD)
for help with the intracellular Ca2+ measurements. We thank
June Oshiro (Rutgers University) for making the
[-32P]DGPP.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HD 26171 and GM 20501 (to E. A. D.) and GM 28140 (to G. M. C.).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.
2 H. Shinohara, M. A. Balboa, C. A. Johnson, J. Balsinde, and E. A. Dennis, manuscript in preparation.
3 J. Balsinde, M. A. Balboa, and E. A. Dennis, unpublished observation.
4 J. Chun, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: DGPP, diacylglycerol pyrophosphate; AA, arachidonic acid; PLA2, phospholipase A2; cPLA2, cytosolic phospholipase A2; MAFP, methyl arachidonyl fluorophosphonate; PGE2, prostaglandin E2; PGD2, prostaglandin D2; PA, phosphatidic acid; lyso-PA, lysophosphatidic acid; PD098059, (2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one); DAG, diacylglycerol; LPS, lipopolysaccharide; PAF, platelet-activating factor; PKC, protein kinase C; ERK, extracellular signal-regulated kinase..
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