Regulation of Cytosolic Phospholipase A2 Activity in
Macrophages Stimulated with Receptor-recognized Forms of
2-Macroglobulin
ROLE IN MITOGENESIS AND CELL PROLIFERATION*
Uma Kant
Misra and
Salvatore Vincent
Pizzo
From the Department of Pathology, Duke University Medical Center,
Durham, North Carolina 27710
Received for publication, October 9, 2001, and in revised form, November 12, 2001
 |
ABSTRACT |
Macrophages exposed to receptor-recognized forms
of 
2-macroglobulin (2M*)
demonstrate increased DNA synthesis and cell division. In the current
study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to
2M*. Ligation of the 2M* signaling
receptor by 2M*, or its receptor binding fragment,
increased cPLA2 activity 2-3-fold in a concentration and
time-dependent manner. This activation required a
pertussis toxin-insensitive G protein. Cellular binding of
2M* also induced transient translocation of
cPLA2 activity to nuclei and membrane fractions. Inhibition
of protein kinase C activity or chelation of Ca2+ inhibited
2M*-induced increased cPLA2 activity.
Binding of 2M* to macrophages, moreover, increased
phosphorylation of MEK 1/2, ERK 1/2, p38 MAPK, and JNK. Incubation of
macrophages with inhibitors of MEK 1/2 or p38 MAPK before stimulation
with 2M* profoundly decreased phosphorylation of MAPKs,
blocking cPLA2 activation. 2M*-induced
increase in [3H]thymidine uptake and cell proliferation
was completely abolished if activation of cPLA2 was
prevented. The response of macrophages to 2M* requires
transcription factors nuclear factor 
B, and cAMP-responsive
element-binding protein as well as expression of the proto-oncogenes
c-fos and c-myc. These studies indicate that
the activation of cPLA2 plays a crucial role in
2M*-induced mitogenesis and cell proliferation.
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INTRODUCTION |
The plasma proteinase inhibitor 2-macroglobulin
(2M*)1
undergoes a major conformational change when it binds proteinases (1,
2). Each 2M subunit also contains an internal thiol ester, which can be directly attacked by small nucleophiles resulting in a similar conformational change (1). In either event, receptor recognition sites are exposed in each of the subunits (1). These
receptor-recognized forms of 2M, termed
2M*, bind to the low density lipoprotein
receptor-related protein (LRP) present on a variety of cells including
macrophages (1-3). In 1993, we demonstrated that the binding of
2M* to macrophages activated signaling cascades
characterized by an inositol 1,4,5-trisphosphate (IP3)-dependent increase in
[Ca2+]i (4). Subsequent studies
demonstrated that the binding to macrophages activates a pertussis
toxin-insensitive phospholipase C, which hydrolyzes membrane
phosphoinositides generating both IP3 and
diacylglycerol (5, 6). These studies also demonstrated that
2M*-mediated signal transduction was not blocked by
addition of receptor-associated protein (RAP). Because RAP blocks the
binding of all known ligands to LRP, this observation suggested the
presence of a second 2M* receptor on macrophages, which
was termed the 2M* signaling receptor
(2MSR) (1, 5, 6). In support of the identification of a
distinct 2M* receptor were several other observations.
Two classes of binding sites were identified on macrophages, one of
high affinity and low capacity (Kd ~ 50 pM and ~1600 sites/cell) and the other LRP, which
demonstrated lower affinity and higher binding capacity
(Kd ~2-5 nM and ~70,000 sites/cell)
(7-10). Binding of other ligands to LRP, moreover, initiated signaling
cascades that activated a pertussis toxin-sensitive G protein and were
blocked by addition of RAP (4-12). More recent observations, however,
suggest that 2M*-mediated signal transduction requires
the presence of LRP on cells (13). Thus, Backsai et al. (13)
have shown that 2M* binding to LRP on neuronal cells
mediates signaling via N-methyl-D-aspartate receptors. These authors suggest that an adapter protein causes LRP to
associate with this receptor, allowing 2M* to activate signal transduction. Furthermore, Herz and colleagues (14, 15) have
identified a large number of adapter proteins that can associate with
LRP, and Barnes et al. (16) have demonstrated that
Tyr-phosphorylated LRP associates with the adapter protein SHC in
SRC-transformed cells.
Based on our observations with respect to activation of the
p21ras-dependent MAPK and PI
3-kinase signaling cascades and subsequent cell proliferation, we have
proposed that 2M* functions like a growth factor (9,
17-20). Known growth factors activate cytosolic phospholipase
A2 (cPLA2) and the products of
cPLA2 hydrolysis are involved in the growth-promoting
effects of these factors (21-26). We, therefore, have studied the
effect on cPLA2 activation of ligating 2M*
receptors on peritoneal macrophages. Specifically, we studied
activation of PKC, MEK 1/2, ERK 1/2, p38 MAPK, JNK, and
cPLA2; translocation to nuclei and membranes; modulation of cell division by inhibitors of MAPKs and cPLA2; activation
of transcription factors NFB and CREB; and expression of
c-fos and c-myc proto-oncogenes.
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EXPERIMENTAL PROCEDURES |
Materials--
The sources of thioglycollate, cell culture
materials, [3H]thymidine, BAPTA/AM, genistein,
staurosporine, chelerythrine, manumycin A, SB 203580, PD98059, U0126,
AACOCF3, wortmannin, and EGF have previously been described
(6, 10). 1-(6-C17
-3Methoxyestra-1,3,5(10)-trien-17-yl) aminohexyl)-1H-pyrrole-2,5-dione (U73122) was purchased from Biomol
(Plymouth Meeting, PA). Bromoenol lactone (BEL) and pertussis toxin
were procured from Sigma. Endotoxin-free 2M*, binding
site mutants of 2M*, and RAP were prepared as described
previously (9, 10). Antibodies against phosphorylated MEK 1/2, ERK 1/2, p38 MAPK, and JNK were purchased from New England Biolabs (Mississauga, Ontario, Canada). Antibodies against c-Fos, c-Myc, CREB, and NFB proteins were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). [3H]Methylcholine with specific activity of 60-90
Ci/mol was purchased from ARC (St. Louis, MO). Silica gel G plates were
from Analytical Technology (Newark, DE). Lipid standards were purchased
from Avanti Polar Lipids (Alabaster, AL). Xestospongin C was purchased
from Calbiochem (San Diego, CA), GDPS and GTP
S were obtained from Roche Molecular Biochemicals, and AlF3 was from
Sigma. Culture media were from Invitrogen. All other chemicals
and solvents used were of the highest available grade.
Cell Culture--
Thioglycollate-elicited peritoneal macrophages
were obtained from pathogen-free 6-week-old C57BL/6 mice (Charles River
Laboratories, Raleigh, NC) in Hanks' balanced salt solution containing
10 mM HEPES, pH 7.4, and 3.5 mM
NaHCO3 (HHBSS). The cells were washed with HHBSS and
suspended in RPMI 1640 medium containing 2 mM glutamine, 12.5 units/ml penicillin, 6.25 µg/ml streptomycin, and 5% fetal bovine serum and plated at a cell density of 3.5-4 × 106 cells/4.5 cm2. The monolayers were
incubated for 2 h at 37 °C in a humidified CO2
(5%) incubator. The monolayers were washed with HHBSS three times to
remove nonadherent cells, and monolayers were incubated overnight at
37 °C in RPMI 1640 medium containing the additions listed above
except that 0.2% fatty acid-free BSA replaced the serum.
Measurement of cPLA2 Activity--
Macrophage
monolayers adhered for 2 h in RPMI 1640 medium were radiolabeled
with [3H]methylcholine (2 µCi/ml) for 16-18 h at
37 °C in a humidified CO2 (5%) incubator. The
monolayers were washed three times with cold HHBSS, a volume of the
buffer (300 µl) added, and monolayers (3 × 106
cells/well) incubated for 5 min at 37 °C prior to stimulation with
100 pM 2M, 2M*, the 17-kDa
receptor-binding fragment of 2M* (RBF), or its mutant
K1370A for various periods of time. Studies were also performed with
EGF (10 ng/ml) as a control. The reaction was stopped by aspirating the
medium and adding a volume of chilled methanol. The cells were scraped
into screw cap glass tubes and lipids extracted according to Bligh and
Dyer (27). The organic layer was evaporated to dryness under
N2 at 37 °C, dissolved in a volume of
CHCl3:CH3OH (2:1, v/v), and processed for
lipids fractionation. The choline-labeled lipids were fractionated on
heat activated silica gel G plates in solvent system
CHCl3:CH3OH:acetic acid:H2O
(65:43:1:3, v/v/v/v) (28). Each sample was co-chromatographed with 5 µg of authentic lysoPC. The plates were dried, exposed to
I2 vapors, and gel areas corresponding to the standard
lysoPC scraped into scintillation vials and their radioactivity counted to detect [3H]lysoPC derived from
cPLA2-catalyzed cleavage of
[3H]methylcholine. In experiments, where the effects of
RAP (1 µM/10 min) or BEL, an inhibitor of
Ca2+-independent PLA2 (29), were examined,
[3H]methylcholine-labeled macrophages (3 × 106 cells/well) were incubated with these agents for the
specified time before adding 2M*. Other details were as
described above.
Heterotrimeric G Proteins and cPLA2
Activity--
Macrophages (3 × 106 cells) were
labeled with [3H]methylcholine as described above. The
labeled monolayers were permeabilized with saponin as described
previously and pretreated with GTPS (20 µM) for 5 min
at 37 °C prior to stimulation with 2M* (5). This
concentration was chosen after studies to determine the maximal effect
of GTPS on cPLA2 activation by 2M* (data
not shown). Experimental details for lipid isolation, fractionation,
and counting were as described above. To further examine the
involvement of heterotrimeric G proteins in 2M*-induced
activation of cPLA2 activity, we employed GDPS and
AlF3. GDPS, a nonhydrolyzable analog of GDP,
competitively inhibits G protein activation by GTP and GTP analogs
(30). AlF3, on the other hand, mimics the terminal
phosphate of GTP, such that the structures of the
G-GDP-AlFn complexes resemble that of the
GTP-bound form of the protein (31). [3H]Methylcholine-labeled macrophages were incubated
overnight as above in RPMI 1640 medium, then washed twice with HHBSS. A
volume of permeabilization buffer containing saponin (20 µg/ml) was
added and the cells incubated for 10 min at 25 °C (32). The cells were washed three times with HHBSS and a volume of RPMI 1640 medium added. The cells were preincubated for temperature equilibration, GDPS (1 mM) added, and the cells incubated at 37 °C
for 10 min, followed by the addition of GTPS (20 µM).
The cells were incubated for 10 min and 2M* (100 pM) added, and incubation continued for 20 min as above.
The reaction was terminated by aspirating the medium and adding a
volume of methanol. Other details of lipid extraction, thin layer
chromatography fractionation, and determining the radioactivity in the
[3H]lysoPC fraction were as described above. In
experiments where the effect of AlF3 was studied on
activation of macrophage cPLA2 by 2M*,
AlF3 was added (20 µM) to
[3H]methylcholine-labeled and washed macrophages in RPMI
1640 medium. The cells were incubated for 10 min at 37 °C, followed
by the addition of 2M* (100 pM). The cells
were incubated as above for 20 min. The reaction was terminated by
aspirating the medium and adding a volume of methanol. Other details of
lipid extraction, thin layer chromatography fractionation, and
determination of radioactivity in the [3H]lysoPC fraction
were as described above.
Pertussis Toxin and cPLA2 Activity--
Macrophages
(3 × 106 cells) were obtained and labeled with
[3H]methylcholine as described above. Pertussis toxin (1 µg/ml) was added to the monolayers during the last 12 h of
incubation (5). Pertussis toxin-treated macrophages were washed with
cold HHBSS, a volume of the buffer added, and the cells preincubated
for 5 min at 37 °C prior to stimulation with 2M*.
Other details of lipid isolation, lipid fractionation, and counting
were as described above.
Modulation of Phosphatidylinositol-dependent
Phospholipase C (PI-PLC) Activity and cPLA2
Activation--
To understand the role of G protein-coupled PI-PLC
activity on cPLA2 activation in
2M*-stimulated macrophages, we employed U73122, which is
an relatively specific inhibitor of G protein-mediated PI-PLC
activation and PI-PLC-linked events (33). To
[3H]methylcholine-labeled and washed macrophages in RPMI
1640 medium, U73122 (2 µM) was added, cells incubated for
10 min as above and then stimulated with 2M* (100 pM/20 min). The reaction was terminated by aspirating the
medium and adding a volume of methanol. Other details for quantifying
radioactivity in the [3H]lysoPC fraction were as
described above. We also examined the role of IP3 generated
upon hydrolysis of phosphatidylinositol 4,5-bisphosphate by G
protein-coupled PI-PLC in cPLA2 activation, by inhibiting
its binding to IP3 receptors (IP3R) and hence
release of endoplasmic reticulum sequestered Ca2+, with
xestospongin C, an antagonist of IP3R (34). To
[3H]methylcholine-labeled macrophages in RPMI 1640 medium
was added xestospongin C (5 µM), and the cells were
incubated for 10 min as above prior to addition of 2M*
(100 pM/20 min). The reaction was terminated by aspirating
the medium and adding a volume of methanol added. Other details of
determining radioactivity in [3H]lysoPC fraction
were as described above.
Measurements of [3H]Thymidine Uptake by
Macrophages--
Murine peritoneal macrophages harvested as above were
allowed to adhere for 2 h in RPMI 1640 medium containing 0.2%
fatty acid-free BSA, penicillin, streptomycin, and glutamine at
37 °C in a humidified CO2 (5%) incubator. The
monolayers were washed twice with HHBSS and a volume of above RPMI
medium added, followed by the addition of [3H]thymidine
(2 µCi/ml) (9, 17). To the respective wells 2M* (100 pM) or PDGF (10 ng/ml) were added. In experiments where the effect of AACOCF3 (20 µM/15 min), PD98059 (50 µM/90 min), or SB 203580 (15 µM/15 min)
were studied, these were added to their respective wells and cells
incubated for the specified time before adding 2M* or
PDGF. The cells were incubated overnight in a humidified CO2 (5%) incubator. The incubations were terminated by
aspirating the medium and washing macrophages twice first with 5%
trichloroacetic acid (15 min/4 °C), and then three times with HHBSS.
The monolayers were lysed with 1 N NaOH and an aliquot used
for liquid scintillation counting and protein estimation (35).
Determination of Macrophage Cell Number--
Because increased
DNA synthesis is generally associated with an increase in total
cellularity, the number of macrophages before and after overnight
exposure to varying concentrations of 2M* was
determined. Peritoneal macrophages were harvested and allowed to adhere
in six-well plates in RPMI 1640 medium containing 5% fetal bovine
serum for 2 h as described above. The adhered cells were carefully
scraped, centrifuged at 1200 rpm for 5 min, and suspended in 15 ml of
RPMI 1640 medium containing 0.2% fatty acid-free BSA, and 0.5-ml
aliquots (2 × 105 cells) were pipetted into 15-ml
siliconized polypropylene tubes. To the respective tubes, a specified
concentration of 2M* was added, the contents mixed
gently, and the tubes incubated overnight as above. After overnight
incubation, 10 µl of trypan blue solution was added to each tube, the
tubes gently shaken during incubation for 2 min, and a 10-µl aliquot
employed for counting the number of cells in a hemocytometer. In
experiments where the modulation in cell numbers of
2M*-exposed macrophages (2 × 105
cells/tube) was studied, SB203580, a specific inhibitor of p38 MAPK (15 µM/30 min) (36); PD98059, a specific inhibitor of MEK 1/2
(50 µM/90 min) (37); U0126, a specific inhibitor of MEK 1/2 (1 µM/10 min) (38); AACOCF3 (20 µM/15 min) (39); and wortmannin, a specific inhibitor of
PI 3-kinase (30 nM/30 min) (40) were added to the
respective tubes, and tubes incubated for the specified time before
adding 2M* (100 pM). The tubes were
incubated and cell numbers counted as described above.
Western Blotting of Phosphorylated MEK 1/2, ERK
1/2, p38 MAPK, and JNK in Macrophages Stimulated with
2M*--
Freshly harvested peritoneal macrophages in
RPMI 1640 medium containing penicillin, streptomycin, glutamine, and
0.2% fatty acid-free BSA were allowed to adhere in six-well plates
(3 × 106 cells/well) for 2 h as above. The
monolayers were washed twice with HHBSS, a volume of above RPMI 1640 medium added, and plates incubated overnight as above. The monolayers
were washed twice, a volume of RPMI medium containing 0.2% fatty
acid-free BSA added, and the cells pretreated with specific
inhibitors/modulators of MAPKs for the specified time period before
exposing to 2M* (100 pM/20 min) or buffer.
The incubations were terminated by aspirating the medium. The lysis of
cells, their electrophoresis, and Western immunoblotting were performed
according to the manufacturer's instruction. In each case, an equal
amount of protein was employed for electrophoresis. The detection
of phosphorylated MAPKs by enhanced chemifluorescence and
quantification of their distribution was performed by phosphorimaging
(Storm®).
Western Blotting of c-Fos, c-Myc, CREB, and NFB Proteins in
Macrophages Exposed to 2M*--
Freshly harvested
peritoneal macrophages in RPMI 1640 medium containing penicillin,
streptomycin, glutamine, and 0.2% BSA were allowed to adhere in
six-well plates (3 × 106 cells/well) for 2 h as
above. The monolayers were washed twice with HHBSSS, a volume of above
RPMI 1640 medium added, and plates incubated overnight as above. The
monolayers were washed, a volume of above RPMI medium added, and the
cells incubated with specific inhibitors/modulators of MAPKs or a
Ca2+ chelator, for the specified time period before
exposing to 2M* (100 pM/20 min) or buffer.
The incubations were terminated by aspirating the medium. The lysis of
cells, their electrophoresis, and Western immunoblotting were performed
according to the manufacturer's instruction. In each case, an equal
amount of protein was employed for electrophoresis. The detection of
immunoblots was performed by enhanced chemifluorescence, and
quantitation of their distribution was done by phosphorimaging
(Storm®).
Translocation of 2M*-induced cPLA2 to
Membrane and Nuclear Fractions of Macrophage--
Two-h adhered cells
(3 × 106 cells/well) in above RPMI 1640 medium were
radiolabeled with [3H]methylcholine (2 µCi/ml) for
16-18 h at 37 °C in a humidified CO2 (5%) incubator.
The radiolabeled monolayers were washed three times with cold HHBSS, a
volume of the buffer added, and monolayers incubated for 5 min at
37 °C prior to stimulation with 2M* (100 pM) for various periods of time. The reaction was stopped
by aspirating the medium, and a volume of "buffer A" containing 20 mM Tris-HCl, pH 7.4, 10 mM KCl, 2 mM MgCl2, 1 mM phenylmethylsulfonyl
fluoride, 20 µg/ml leupeptin, 0.3 mM CaCl2, 1 mM NaF, and 1 mM sodium orthovanadate was
added. Cells were allowed to swell for 10 min on ice, followed by the
addition of three volumes of "buffer B" containing 50 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl2, 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 0.2 mM EGTA, 0.2 mM CaCl2, 1 mM NaF, 1 mM sodium orthovanadate, and 100 mM benzamidine. The cells were scraped into glass tubes
containing sodium orthovanadate and 100 mM benzamidine and
homogenized in a Potter-Elvehjem homogenizer by 15 up-and-down strokes
at 4 °C. The homogenates were centrifuged for 10 min at 800 × g, 40 °C. Supernatant was carefully removed, nuclear
pellets washed with cold buffer B twice by centrifuging, and the
pellets suspended in a volume of buffer B. The supernatant was layered
on a 30-70% sucrose gradient and centrifuged at 200,000 × g for 60 min at 40 °C. The membrane-enriched fraction at
the sucrose interface was collected and pelleted at 200,000 × g, and the pellet, containing both endoplasmic reticulum and
plasma membranes, was suspended in a volume of buffer B. The
supernatant remaining after membrane isolation is considered as
"cytosol." Nuclei, membrane fractions, and cytosol were prepared
under identical conditions in all experiments. The purity of cell
fractions was evaluated by electron microscopy as described previously
(41-43). Equal amounts of cytosol, membrane, or nuclear protein was
processed in each experiment for lipid isolation. The details of lipid
isolation, lysoPC fractionation by thin layer chromatography, and
counting of radioactivity in the lysoPC fraction were as described
above. In experiments where the effects of inhibition of MEK 1/2, p38
MAPK, or cPLA2 were examined on 2M*-induced
translocation of cPLA2 to cellular membranes, the
[3H]methylcholine-labeled macrophages were incubated with
inhibitors of these enzymes for the specified time period before adding
2M* as described above. The reactions were terminated by
aspirating the media. Other details of preparing subcellular fractions,
lipid isolation, lysoPC fractionation, and counting of radioactivity were as described above.
| |
RESULTS |
Binding of 2M* to Macrophages Induces
cPLA2 Activity--
cPLA2 catalyzes the
hydrolysis of phosphatidylcholine generating arachidonic acid and
lysoPC as its products (21-26). Thus, the quantification of either
product has been employed to study cPLA2 activation
(21-26). In our studies, macrophage cPLA2 activity was
quantified by measuring the levels of [3H]lysoPC
generated upon hydrolysis of [3H]methylcholine-labeled
phosphatidylcholine. Stimulation of
[3H]methylcholine-labeled macrophages with 100 pM 2M*, or RBF, increased cPLA2
activity by ~2-fold compared with buffer or native nonactivated
2M-treated macrophages (Fig.
1A). Under our experimental conditions, the maximal increase in [3H]lysoPC in
2M*-stimulated macrophages occurred at about 10 min after stimulation and it plateaued thereafter (Fig. 1A). RBF
binding site mutant K1370A induces signal transduction when it binds to macrophage 2M* receptors, which belong to the class
defined as 2MSR (7, 8). Addition of this mutant RBF also
increased [3H]lysoPC generation comparably to
2M* or EGF (Fig. 1B). 2M* binds to growth factors and cytokines, but RBF and K1370A lack the
growth factor binding domain (44). Thus, these studies demonstrate that
the observed effects are related directly to 2M*
cellular binding. cPLA2 activity increased nearly linearly
up to a concentration of 50-100 pM 2M* and
it plateaued at higher concentrations (Fig. 1C). The
kinetics of 2M*-induced increase in cPLA2
activity parallels those observed in 2M*-induced
increase in [Ca2+]i,
IP3 (17), phospholipase D activation, PI 3-kinase activation (10), and p21ras activation reported
previously (20).
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Fig. 1.
2M*-induced increase in
cPLA2 activity as measured by quantification of
[3H]lysoPC in macrophages. Panel A, effect of
time of incubation on cPLA2 activity of 2M*
(100 pM) ( ), native 2 M (100 pM) ( ), or buffer ( ). Panel B,
column 1, buffer; column 2,
2M* (100 pM/20 min); column
3, RBF (100 pM/20 min); column
4, K1370A (100 pM/20 min); column
5, EGF (10 ng/ml/20 min). Panel C, effect of
concentration of 2M* on cellular cPLA2
activity. Details are described under "Experimental Procedures."
Values in each panel are mean ± S.E. from three to
five independent experiments and are expressed as percentage of change
over basal value, which is taken as 100%.
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|
2M*-induced Activation of cPLA2 Is RAP-
and BEL-insensitive--
RAP, the 39-kDa receptor-associated protein
does not block 2M*-induced signal transduction, but does
block signaling by all other LRP ligands so far studied (5, 17). The
results presented in Fig. 2A
show that the ligation of macrophage receptors with either
2M* or the RBF binding site mutant K1370A, activates
cPLA2 activity in the presence of RAP, as expected from
this fact. Chelation of [Ca2+]i
with BAPTA/AM, completely abolished 2M*-stimulated cPLA2 activity (Fig. 2B). We further evaluated
the Ca2+ sensitivity of 2M*-induced increase
in cPLA2 activity by using BEL, a very potent inhibitor of
Ca2+ independent PLA2 (Fig. 2C).
Preincubation of labeled cells with BEL had no effect on
cPLA2 activity stimulated by 2M* or K1370A (Fig. 2C), which demonstrates that 2M*
induces the activity of a Ca2+-dependent
cytosolic PLA2. To rule out the contribution of endotoxin in 2M* or K1370A-induced activation of cPLA2
activity, the labeled cells were treated with boiled 2M*
and cPLA2 activation measured (Fig. 2C). In
addition all preparations were endotoxin-free as determined by assay
(data not shown). The results indicate that endotoxins do not
contribute to agonist-induced stimulation of cPLA2
activity.
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Fig. 2.
Modulation of cPLA2 activity in
macrophages. Panel A, effect of RAP on
2M*-induced increase in cellular cPLA2
activity. Column 1, buffer; column
2, RBF (100 pM/20 min); column
3, RAP (1 µM/10 min) then RBF (100 pM/20 min); column 4, K1370A (100 pM/20 min); column 5, RAP (1 µM/10 min) then K1370A (100 pM/20 min).
Panel B, effect of
[Ca2+]i on
2M*-induced increase in cPLA2 activity.
Column 1, buffer; column 2,
2M* (100 pM/20 min); column
3, BAPTA/AM (10 µM/30 min) then
2M (100 pM/20 min). Panel C,
effect of boiling of ligands and BEL on 2M*-induced
increase in cPLA2 activity. Column 1,
unboiled buffer; column 2, boiled buffer;
column 3, BEL (0.5 µM/15 min) then
buffer; column 4, 2M* (100 pM/20 min); column 5,
2M (100 pM; boiled for 3 min, centrifuged
and supernatant added); column 6, BEL (0.5 µM/15 min) then 2M*. Columns
7-9 are identical to columns 4-6 except that
K1370A (100 pM) replaced 2M*. Experimental
details are given under "Experimental Procedures." The values
represented are mean ± S.E. from at least four individual
experiments in each case and are expressed as percentage of change in
[3H]lysoPC formation compared with buffer-treated control
(100%).
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|
2M*-induced cPLA2 Activity
Is Coupled to a Pertussis Toxin-insensitive G Protein--
Addition of
2M* and GTPS (20 µM) to
saponin-permeabilized cells caused a nearly 2-fold increase in
cPLA2 activity compared with that observed with
2M* alone (Fig.
3A). Preincubation of cells
with GDPS prior to addition of GTPS and 2M* nearly
abolished 2M*-stimulated cPLA2 activity
(Fig. 3B). Further, treatment of cells with
AlF3, as expected, potentiated the
2M*-induced increase in cPLA2 activity (Fig.
3C). These results strongly support the involvement of a
heterotrimeric G protein in the activation of cPLA2 by
2M*. We propose that cPLA2 activation in
2M*-stimulated cells requires a heterotrimeric G protein
coupled to PI-PLC. If this hypothesis is correct, inhibition of PI-PLC
activation and blocking the binding of second messenger generated
consequent to PI-PLC activation to the effector molecules should
inhibit agonist-induced cPLA2 activation. The results shown
in Fig. 3C demonstrate that, indeed, either the inhibition
of PI-PLC with U73122 or the inhibition of binding of newly generated
IP3 to IP3R by xestospongin C inhibited
c-PLA2 activation in 2M*-stimulated cells.
We next treated pertussis toxin preincubated cells with 2M* and quantified the release of
[3H]lysoPC (Fig. 3D). The results demonstrate
that the G protein involved in 2M*-mediated stimulation
of cPLA2 activity is pertussis toxin-insensitive.
Activation of cPLA2 by receptor-coupled G proteins may be
mediated by direct interaction of these G proteins with cPLA2 (21-26). In addition, G
protein-dependent activation of PLC increases DAG and
IP3, the latter of which raises
[Ca2+]i. Activation of PKC,
activation of MAPKs, and subsequent phosphorylation and activation of
cPLA2 then occur (21, 26).
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Fig. 3.
Effect of heterotrimeric G proteins on
2M*-induced increase in
cPLA2 activity. Panel A, Effect of time of
incubation of macrophages with 2M* (100 pM)
in absence () or presence () of GTPS (20 µM).
Panel B, effect of incubating macrophages with GDPS
before adding GTPS and 2M* on cPLA2
activity. Column 1, buffer; column
2, 2M* (100 pM/20 min);
column 3, GTPS (20 µM/10 min);
column 4, GTPS (20 µM/10 min)
then 2M* (100 pm/20 min); column
5, GDPS (1 mM/10 min); column
6, GDPS (1 mM/10 min) then GTPS (20 µM/10 min) then 2M* (100 pm/20 min).
Panel C, effect of AlF3 and PI-PLC
modulation on cPLA2 activity. Column
1, buffer; column 2,
2M* (100 pM/20 min); column
3, AlF3 (20 µM/15 min);
column 4, AlF3, (20 µM/15 min) then 2M* (100 pM/20
min); column 5, xestospongin C (5 µM/10 min); column 6, xestospongin
C (5 µM/10 min) then 2M* (100 pM/20 min); column 7, U73122 (2 µM/10 min); column 8, U73122 (2 µM/10 min) then 2M (100 pM/20
min). Panel D, effect of time of incubation of macrophages
with 2M* (100 pM) in absence () and
presence () of pertussis toxin (1 µg/ml/16 h). Details are given
under "Experimental Procedures." Values are mean ± S.E. from
two individual experiments performed in duplicate and are expressed as
percentage of change over zero time (100%).
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Phosphorylation of MEK 1/2, ERK 1/2, PKC, p38 MAPK, and JNK in
2M* Induces Activation of cPLA2--
We
next examined the involvement of MEK 1/2, ERK 1/2, p38 MAPK, and JNK in
cPLA2 activation in two ways, namely by quantifying the
levels of phosphorylated (activated) forms of these MAPKs by Western
blotting and by inhibiting MEK 1/2 or p38 MAPK with their specific
inhibitors before stimulating macrophages with 2M* and
quantifying [3H]lysoPC. Inhibition of p38 MAPK with
SB203580, and of MEK 1/2 with PD98059 or U0126, nearly abolished
2M*-induced increase in cPLA2 activity and
release of [3H]lysoPC, an effect comparable with treating
cells with AACOCF3, a cPLA2 inhibitor (Fig.
4A). Treatment of labeled
macrophages with genistein, a tyrosine kinase inhibitor, had no effect
on 2M*-induced increase in cPLA2 activity
(Fig. 4B). The binding of 2M* to
2MSR activates the hydrolysis of membrane
phosphatidylinositol bisphosphate via both PLC and PLC (4, 12).
We have shown that the ligation of 2MSR with
2M* stimulates PLC activity, which is sensitive to
genistein (12). Because genistein treatment did not affect
2M*-induced cPLA2 activity, the studies
suggest that PLC is involved in the activation of cPLA2.
Inhibition of PI 3-kinase with wortmannin, however, did not affect
cPLA2 activation. These results are consistent with
published studies demonstrating that both ERK 1/2 and p38 MAPK are
involved in the phosphorylation of cPLA2 at Ser-505, which
is essential for its activation (21-26).
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Fig. 4.
Regulation of
2M*-induced cPLA2 activity.
Panel A, modulation of cPLA2 activity by MAPKs.
Column 1, buffer; column 2,
2M* (100 pM); column
3, SB203580 (15 µM/30 min) then
2M; column 4, PD98059 (50 µM/90 min) then 2M*; column
5, U0126 (1 µM/10 min) then
2M*; column 6, AACOCF3
(20 µM/15 min) then 2M*; column
7, wortmannin (30 nM/30 min) then
2M. In each case, after addition of 2M*
cells were incubated for 20 min. Panel B,
modulation of cPLA2 activity by PKC. Column
1, buffer; column 2,
2M* (100 pM/20 min); column
3, chelerythrine (200 nM/15 min) then
2M*; column 4, staurosporine (20 nM/16 h) then 2M*; column
5, genistein (20 µM/16 h) then
2M*; column 6, PMA (50 nM/16 h) then 2M*; column
7, PMA (1 µM/30 min) then 2M*.
Details are described under "Experimental Procedures." Values are
mean ± S.E. from three individual experiments and are expressed
as percentage of change in [3H]lysoPC formation compared
with controls (100%). The values for [3H]lysoPC
formation in cells pretreated with various inhibitors and then
stimulated with buffer were ±10% of basal values (data not
shown).
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Stimulation of murine peritoneal macrophages with 2M*
caused ~2-fold increase in the expression of phosphorylated MEK 1/2 protein (Fig. 5A),
phosphorylated ERK 1/2 protein (Fig. 5B), phosphorylated p38
MAPK protein (Fig. 6A), and
phosphorylated JNK protein (Fig. 6B).
2M*-induced increased phosphorylation of MEK 1/2 was
inhibited by chelation of Ca2+ with BAPTA/AM or inhibition
of PKC with chelerythrine (Fig. 5A). 2M*-induced increase in ERK 1/2 phosphorylation was
drastically inhibited by PD98059, U0126, chelation of
[Ca2+]i with BAPTA/AM, or
inhibition of PKC with chelerythrine (Fig. 5B). Inhibition
of PKC as well as chelation of
[Ca2+]i abrogated
2M*-induced increase in phosphorylated p38 MAPK protein
(Fig. 6A). The increase in JNK protein phosphorylation in
2M*-stimulated macrophages was reduced by BAPTA/AM or
chelerythrine (Fig. 6B).
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Fig. 5.
Modulation of MEK 1/2 and ERK 1/2
phosphorylation in macrophages stimulated with
2M*. See
"Experimental Procedures" for details. Panel A, MEK 1/2
phosphorylation. Column/lane 1,
buffer; column/lane 2, 2M* (100 pM); column/lane 3, BAPTA/AM (10 µM/30 min) then 2M*;
column/lane 4, chelerythrine (200 nM/15 min) then 2M*. The results shown are
representative of at least four individual experiments. Quantification
of immunoblots was performed by PhosphorImager (Storm®),
and the results are expressed as changes in phosphorylated MEK 1/2 in
arbitrary units. Panel B, ERK 1/2 phosphorylation.
Column/lane 1, buffer; column/lane 2,
2M* (100 pM); column/lane 3,
U0126 (1 µM/15 min) then 2M*;
column/lane 4, PD98059 (50 µM/90 min) then
2M*; column/lane 5, BAPTA/AM (10 µM/30 min) then 2M*; column/lane
6, chelerythrine (200 nM/15 min) then
2M. Results shown are representative of three or four
individual experiments and are expressed as change in phosphorylated
ERK 1/2 levels in arbitrary units.
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Fig. 6.
Regulation of phosphorylated p38 MAPK and JNK
in macrophages stimulated with
2M*. See
"Experimental Procedures" for details. Panel
A, p38 MAPK phosphorylation. Column/lane
1, buffer; column/lane 2, 2M* (100 pM); column/lane 3, BAPTA/AM (10 µM/30 min) then 2M*; column/lane
4, chelerythrine (200 nM/15 min) then
2M*. Results shown are representative of three to four
individual experiments and are expressed as changes in phosphorylated
p38 MAPK levels in arbitrary units. Panel B, JNK
phosphorylation. Column/lane 1, buffer;
column/lane 2, 2M* (100 pM);
column/lane 3, BAPTA/AM (10 µM/30 min) then
2M*. Results shown are representative of three or four
individual experiments and are expressed as changes in phosphorylated
JNK levels in arbitrary units.
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In many cells, PKC regulates MAPK pathways either alone or
in combination with other mechanisms (21-26, 45-47). To further
examine the involvement of PKC, we quantified the activity of
cPLA2 in macrophages in which PKC activity was
down-regulated with PMA, or inhibited by chelerythrine or
staurosporine, respectively (Fig. 4B). Inhibition of PKC
with its inhibitors or its down-regulation nearly abolished
2M*-induced increase in cPLA2 activity, as
determined by quantifying of [3H]lysoPC (Fig.
4B). In contrast, up-regulation of PKC significantly increased cPLA2 activity in 2M*-treated
cells (Fig. 4B).
2M*-induced Translocation of cPLA2 to
Nuclei and Membranes--
cPLA2 activity as determined was
present both in nuclei and the membrane fractions (Fig.
7). Immediately after 2M*
stimulation, cPLA2 activity increased in the nuclear
fraction reaching a maximal level at 15 min and plateauing at longer
periods of incubation (Fig. 7A). In contrast, the peak
cPLA2 activity in the membrane fraction occurred between 2 and 5 min after 2M* stimulation and then declining to a
steady state level. Very little cPLA2 activity was present
in the cytosol after 2M* stimulation. The results demonstrate that, in 2M*-stimulated macrophages, the
nuclear membranes are the main target for cPLA2
translocation, although some activity is associated with other
membranes. Treatment of macrophages with PD98059 or SB203580 before
stimulating them with 2M* inhibited cPLA2
activity in nuclei (Fig. 7B). No inhibition in
2M*-induced increase in
[Ca2+]i was observed in these
studies; hence, the reduced nuclear cPLA2 activity most
likely reflects phosphorylation by MAPKs.
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Fig. 7.
Intracellular translocation of
cPLA2 stimulated with
2M*. Experimental details are
given under "Experimental Procedures." Panel
A, translocation of cPLA2 activity as measured
in quantifying the levels of [3H]lysoPC at various times
after 2M* stimulation in nuclei (), membranes ( ),
and cytosol (). Values are mean ± S.E. from three independent
experiments and are expressed as changes in [3H]lysoPC levels
compared with zero time (100%). Panel B, effect
of inhibition of cPLA2 phosphorylation by MAPKs on its
nuclear translocation in macrophages stimulated with
2M*. Column 1,
[3H]lysoPC levels in nuclei at zero time;
column 2, [3H]lysoPC levels in
nuclei at 20 min after 2M* stimulation;
column 3, effect of treatment of macrophages with
PD98059 (50 µM/90 min); column 4,
SB203580 (15 µM/30 min) before 2M*
stimulation on nuclear [3H]lysoPC levels. Values are
mean ± S.E. from at least two experiments done in duplicate and
are expressed as percentage of change compared with zero time
(100%).
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2M*-induced Increase in [2H]Thymidine
Uptake and Peritoneal Macrophage Proliferation Are Attenuated by
Inhibitors of cPLA2, MEK 1/2, and p38 MAPK--
In view of
the role of cPLA2 in cell proliferation (21-26), we have
examined the involvement of cPLA2 in
2M*-induced [3H]thymidine uptake and cell
proliferation (Fig. 8). Like PDGF, 2M* and K1370A increased [3H]thymidine
uptake by macrophages by ~2-fold (Fig. 8A). The
agonist-induced increase in [3H]thymidine uptake was
abolished by AACOCF3, a specific inhibitor of
cPLA2, by PD98059, or by SB 203580 (Fig. 8B).
These results demonstrate that cPLA2 activity and MAPK
activation is intimately involved in 2M*-induced
increases in [3H]thymidine uptake.
[3H]Thymidine uptake may indicate enhanced DNA synthesis,
but there are other potential mechanisms of enhanced uptake independent of new nucleic acid synthesis. Because new DNA synthesis is normally associated with an increase in total cellularity, we determined the
macrophage cell number before and after overnight incubation with
varying concentrations of 2M* (Fig. 8B). The
maximal increase in macrophage numbers occurred at 50-100
pM 2M*, and the cell numbers plateaued at
higher concentrations of 2M* (Fig. 8B). The
kinetics of 2M*-induced increase in cell number is
similar to that observed for 2M*-induced increase in
protein and DNA synthesis (17). Inhibition of cPLA2 by
AACOCF3 or inhibition of MAPK-dependent
phosphorylation of cPLA2 abolished
2M*-induced increase in macrophage cell number (Fig.
8C).
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Fig. 8.
Involvement of
2M*-induced increase in
cPLA2 activity in macrophages on
[3H]thymidine incorporation and cell proliferation.
See "Experimental Procedures" for details. Panel A,
[3H]thymidine incorporation into macrophages stimulated
with 2M*. Column 1, buffer;
column 2, 2M (100 pM);
column 3, PD98059 then 2M*;
column 4, SB203580 then 2M*;
column 5, AACOCF3 then
2M*; column 6, K1370A (100 pM); column 7, PD98059 then K1370A;
column 8, SB20358 then K1370A; column
9, AACOCF3 then K1370A; column
10, PDGF (10 µg/ml); column 11,
PD98059 then PDGF; column 12, SB203580 then PDGF;
column 13, AACOCF3 then PDGF. Values
are mean ± S.E. from two individual experiments performed in
quadruplicate and are expressed as femtomoles of
[3H]thymidine uptake. Addition of PD98059, SB203580, or
AACOCF3 to the buffer control exerted very little effect
(data not shown). Panel B, increase in macrophage
cell number after overnight treatment with various concentrations of
2M*. See "Experimental Procedures" for details.
Panel C, effect of inhibition of MAPKs and
cPLA2 on 2M*-induced increase in cell
number. Column 1, buffer; column
2, 2M* (100 pM/16 h);
column 3, SB203580 (15 µM/30 min)
then 2M*; column 4, PD98059 (50 µM/90 min) then 2M*; column
5, U0126 (1 µM/15 min) then
2M*; column 6, AACOCF3
(20 µm/15 min) then 2M*. Values are the mean ± S.E. from three experiments and are expressed as cell number/ml after
overnight incubation with 2M* in the absence or presence
of inhibitors.
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2M* Exposure Elevates the Levels of NFB and CREB
Transcription Factors in Peritoneal Macrophages--
Stimulation of
macrophages with 2M* (100 pM/20 min) caused
a 2-fold increase in the expression of NFB (Fig.
9A). Ligation of
2MSR on macrophages elevates cyclic AMP (cAMP) levels by
~50% (4). cAMP, an important intracellular second messenger,
mediates the transcriptional induction of many genes through
PKA-dependent phosphorylation of transcription factor CREB
(48-50). This reaction modulates its nuclear transport, and DNA
binding affinity, thus enhancing its transactivation potential (49,
50). Stimulation of macrophages with 2M* (100 pM/20 min) increased the expression of CREB by ~2-fold
(Fig. 9B), suggesting thereby the involvement of CREB in
2M*-induced mitogenic responses in macrophages.
Inhibition of MEK 1/2 or p38 MAPK in 2M*-stimulated
macrophages reduced 2M-induced increased expression of
NFB protein by ~25-30% (Fig. 9A) and profoundly
reduced 2M*-induced increase in CREB protein expression
(Fig. 9B). These results suggest that both translational and
transcriptional regulation of 2M*-induced cellular
responses are mediated by cPLA2 activation.
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Fig. 9.
Effect of
2M* treatment on transcription
factor NFB and CREB proteins and
their modulation by MAPKs. The immunoblot of NFB protein and
its quantification by PhosphorImager are shown in panel A.
The immunoblot and quantification of CREB protein and its
quantification by PhosphorImager are shown in panel B. Lane/column 1, buffer; lane/column
2, 2M* (100 pM);
lane/column 3, U0126 then 2M*;
lane/column 4, PD98059 then 2M;
lane/column 5, SB203580 then 2M*.
Values are the mean ± S.E. from three or four individual
experiments in each case and are expressed as changes in NFB and
CREB protein, respectively, in arbitrary units.
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c-Fos and c-Myc Protein Levels in Macrophages--
To understand
the involvement of two early genes, namely c-fos and
c-myc, in 2M*-induced mitogenesis and cell
proliferation, we next quantified the c-Fos and c-Myc proteins in
2M*-stimulated macrophages by Western blotting and
phosphorimaging (Fig. 10, A and B). Incubation of macrophages with inhibitors of MEK 1/2
or p38 MAPK, or chelation of
[Ca2+]i, before stimulating with
2M* caused inhibition in the expression of c-Fos and
c-Myc proteins. The results presented show that the early genes
c-fos and c-myc participate in
2M*-induced new protein and DNA synthesis and MAPKs
regulate these activities both posttranslationally and
transcriptionally.
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Fig. 10.
Effect of
2M* treatment on expression of
c-Fos and c-Myc
proteins and their modulation by calcium and
MAPKs. The immunoblot and the quantification of c-Fos protein are
shown in panel A. Immunoblot of c-Myc protein and the
quantification are shown in panel B. Lane/column
1, buffer; lane/column 2,
2M* (100 pM); lane/column
3, U0126 then 2M*; lane/column
4, PD98059 then 2M*; lane/column
5, SB203580 then 2M*; lane/column
6, BAPTA/AM then 2M*. Values are the
mean ± S.E. from three or four individual experiments in each
case and are expressed as changes in c-Fos and c-Myc protein levels,
respectively, in arbitrary units.
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DISCUSSION |
In this study we show that ligation of macrophage
receptors with 2M*, RBF, or its mutant K1370A
up-regulates the Ca2+-dependent 85-kDa
cPLA2 activity in a concentration- and
time-dependent manner. cPLA2 activity is RAP-
and BEL-insensitive and Ca2+-dependent. A
pertussis toxin-insensitive G protein is involved in
2M*-induced activation of cPLA2 activity.
Binding of 2M* to its receptors on peritoneal
macrophages activates MEK 1/2, ERK 1/2, p38 MAPK, and JNK by inducing
their phosphorylation. Incubation of macrophages with inhibitors of MEK
1/2 or p38 MAPK before stimulation with 2M* profoundly
decreased the expression of the respective phosphorylated MAPKs and
nearly abolished 2M*-induced increase in
cPLA2 activity. Similar effects were observed by inhibition of PKC or chelation of [Ca2+]i.
Binding of 2M* to macrophages induced the transient translocation of cPLA2 activity primarily to nuclei and
also to other membrane fractions. Concomitant with these results,
inhibition of cPLA2, PKC, or the MAPKs blocked
2M*-induced increase in [3H]thymidine
uptake and cell proliferation. Finally, ligation of 2M*
receptors increased the levels of transcription factors NFB and
CREB, as well as c-Fos and c-Myc proteins. 2M* induced
increased expression of transcription factors, and these proto-oncogene proteins were also affected to varying degrees by inhibitors of MAPKs
and chelation of [Ca2+]i.
Multifactorial regulation of cPLA2 activity has been
reported, which is cell- and agonist-specific. These factors include transcriptional regulation involving heterotrimeric G proteins, increase in [Ca2+]i, activation of
PKC, cPLA2 translocation and membrane localization, and
phosphorylation by MAPKs (21-26). Involvement of G proteins in
cPLA2 activation may occur directly through interaction of
heterotrimeric G proteins with the enzymes or indirectly by activation
of signaling cascades subsequent to receptor ligation (21-26).
Phosphorylation at Ser-505 of cPLA2 is essential for
agonist-induced arachidonic acid release in many, but not all, types of
cells (21-26). MEK 1/2, ERK 1/2, p38 MAPK, and JNK are involved in
this phosphorylation reaction (45-47). Certain agonists such A23187 or
okadaic acid can stimulate cPLA2 activity in a
Ca2+- and PKC-independent manner (51-55). In some types of
cells, these agents may also promote translocation of cPLA2
to nuclei (56).
We show here that, after ligation of 2M* receptors, a
pertussis toxin-insensitive G protein indirectly affects
cPLA2 activity in 2M*-stimulated
macrophages. Ligation of the receptor with 2M* activates
both PLC and genistein-sensitive PLC (5, 6, 12). Because
genistein did not affect 2M*-induced cPLA2
activity, it appears that PLC activity is involved in the activation
of cPLA2 in macrophages stimulated with 2M*.
PLC-catalyzed hydrolysis of phosphatidylinositol bisphosphate
generates IP3 and DAG (5, 6, 12). The former elevates
[Ca2+]i and the latter activates
PKC. Soluble cPLA2 binds Ca2+ at CaLB domains,
and translocates to nuclei and membrane fractions (21-26).
Phosphorylation-dependent activation of
2M*-induced cPLA2 activity involves the
participation of PKC because the inhibition of PKC severely reduced the
release of lysoPC as well as activation of MEK 1/2, ERK 1/2, p38 MAPK,
and JNK.
Agonists may regulate cPLA2 activity by two possible
mechanisms, namely agonist-induced de novo protein synthesis
of the enzyme or activation of the enzyme. The data collected in this
study suggest that triggering of signaling cascades by
2M* promotes activation of cPLA2. Various
agonists, however, also increase steady state cPLA2
mRNA levels and promote stability of this mRNA (57-59). Thus,
IFN- initiates cPLA2 gene transcription and induces cPLA2 synthesis (60). Accumulation of cPLA2 was
completely abrogated by actinomycin D or cycloheximide, and this
correlated with loss of cPLA2 activity (61). Our recent
observations suggest that the binding of 2M* to murine
peritoneal macrophages also causes a 2-3-fold increase in de
novo synthesis of cPLA2 compared with unstimulated
cells (62). In the present study, we also demonstrate inhibition of
2M*-induced cPLA2 activity by inhibition of
MEK 1/2, p38 MAPK, PKC, or chelation of
[Ca2+]i
TOP
ABSTRACT
INTRODUCTION
