The Mechanism of Docosahexaenoic Acid-induced Phospholipase D
Activation in Human Lymphocytes Involves Exclusion of the Enzyme from
Lipid Rafts*
Olivier
Diaz,
Alexandre
Berquand,
Madeleine
Dubois,
Silvia
Di
Agostino
,
Claudio
Sette,
Sylvain
Bourgoin§,
Michel
Lagarde,
Georges
Némoz, and
Annie-France
Prigent¶
From the Unité INSERM 352, Laboratoire de Biochimie et
Pharmacologie, INSA de Lyon, 69621 Villeurbanne, France,
Dipartimento di Sanità Pubblica e Biologia
Cellulare, Cattedra di Anatomia Umana, Università di Roma Tor
Vergata, 00173 Rome, Italie, and § Centre de Recherche du
CHUL, Laval University, Ste-Foy,
Québec G1V 4G2, Canada
Received for publication, March 12, 2002, and in revised form, July 19, 2002
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ABSTRACT |
Docosahexaenoic acid (DHA), an
n-3 polyunsaturated fatty acid that inhibits T lymphocyte
activation, has been shown to stimulate phospholipase D (PLD) activity
in stimulated human peripheral blood mononuclear cells (PBMC). To
elucidate the mechanisms underlying the DHA-induced PLD activation, we
first characterized the PLD expression pattern of PBMC. We show that
these cells express PLD1 and PLD2 at the protein and mRNA level and
are devoid of oleate-dependent PLD activity. DHA enrichment
of PBMC increased the DHA content of cell phospholipids, which was
directly correlated with the extent of PLD activation. The DHA-induced
PLD activation was independent of conventional protein kinase C but
inhibited by brefeldin A, which suggests ADP-ribosylation factor
(ARF)-dependent mechanism. Furthermore, DHA enrichment
dose-dependently stimulated ARF translocation to cell
membranes. Whereas 50% of the guanosine
5'-3-O-(thio)triphosphate plus ARF-dependent
PLD activity and a substantial part of PLD1 protein were located to the
detergent-insoluble membranes, so-called rafts, of non-enriched PBMC,
DHA treatment strongly displaced them toward detergent-soluble
membranes where ARF is present. Collectively, these results suggest
that the exclusion of PLD1 from lipid rafts, due to their partial
disorganization by DHA, and its relocalization in the vicinity of ARF,
is responsible for its activation. This PLD activation might be
responsible for the immunosuppressive effect of DHA because it is known
to transmit antiproliferative signals in lymphoid cells.
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INTRODUCTION |
Polyunsaturated fatty acids (PUFAs)1 are known to
modulate immune response, particularly by
affecting T cell function (1, 2). Accordingly, dietary supplementation
with n-3 fatty acids decreases the production of T
cell-derived cytokines and lymphoproliferation (3, 4). Although fish
oil-derived fatty acids have found clinical applications for the
treatment of various inflammatory diseases (5) and as adjuvant
immunosuppressive agents (6), the underlying molecular and cellular
mechanisms of PUFA-induced T cell inhibition have not yet been
elucidated in detail.
We have reported previously (7) that in docosahexaenoic acid
(DHA)-enriched peripheral blood mononuclear cells (PBMC), the mitogenic
lectin concanavalin A was able to stimulate phospholipase D (PLD)
activity, whereas no stimulation was observed in control cells. PLD
acts on phosphatidylcholine (PC) to release phosphatidic acid, a
bioactive molecule that has been implicated in the regulation of
numerous cellular functions including cell proliferation and apoptosis
(8). Two mammalian PLD have been cloned from various human and murine
sources. PLD1 has a low basal activity and is activated by
ADP-ribosylation factors (ARF), Rho family GTP-binding proteins, and
protein kinase C (PKC) in the presence of phosphatidylinositol bisphosphate (PIP2). PLD2 has a high basal activity in the
presence of PIP2 but is unresponsive to small G proteins
and PKC (9, 10). Another PLD form that is dependent on unsaturated
fatty acids such as oleate for its activation has also been described, but it has not been cloned yet (10). Interestingly, this
oleate-activated PLD seems to be the major form detected in lysates of
Jurkat T cells where it could be implicated in the apoptotic process
(11). In addition Jurkat T cells also express GTP-dependent
PLD activity (12). PLD protein and mRNA expression has been well
demonstrated in several established lymphocytic cell lines (13), but
only few reports have addressed the PLD of human PBMC. A PLD activity, detected by means of the transphosphatidylation reaction, has been
described in these cells (7, 14). However, the identity of the PLD
isoforms in presence is not precisely known.
In the past few years, several groups have reported the presence of PLD
enzymes in caveolae, which are specialized domains of the plasma
membrane related to sphingolipid- and cholesterol-rich microdomains or
rafts. Both PLD1 and PLD2 have been found in caveolar membranes
depending on the type of cell considered. Thus, PLD1 has been detected
in the caveolin-rich membrane fractions of 3Y1 rat fibroblasts (15) and
C2C12 mouse skeletal myotubes (16). In human keratinocytes, PLD2 was
located in the caveolin-rich low density fractions, whereas PLD1 was
clearly excluded from the caveolar fractions (17). In contrast, both
PLD1 and PLD2 were found in caveolae of NIH 3T3 cells transfected by
activated oncogenic forms of src, ras, and
raf (18). Although lymphocytes are devoid of caveolae and
caveolin expression (19), their plasma membrane contains typical
sphingolipid- and cholesterol-rich microdomains, which have a key role
in immune cell signaling (20, 21). It has been shown that upon
cross-linking, the T cell antigen receptor associates with lipid rafts
and promotes the recruitment and aggregation of raft-associated
proteins such as Src family kinases while excluding others such as
tyrosine phosphatase, which initiates phosphorylation signaling
cascades. Interestingly, in PUFA-enriched T cells the Src kinases Lck
and Fyn appeared to be excluded from rafts suggesting that PUFA had
partially disrupted these structures (22), which might explain their
immunosuppressive effect.
The goal of the present study was to characterize further the PLD
activity of human PBMC to investigate the possible association of PLD
enzymes with lipid rafts and to determine the mechanisms involved in
the increase of PLD activity induced by DHA in mitogen-stimulated cells. In particular, we considered the possibility that the location of PLD isoforms inside or outside lipid rafts might be altered under
the influence of DHA with, as a consequence, changes in the impact of T
cell stimulation on PLD activity.
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EXPERIMENTAL PROCEDURES |
Preparation of Human PBMC--
Peripheral blood was obtained
from healthy subjects who had not taken any medication for 2 weeks
prior to blood donation (Etablissement Français du Sang, Lyon,
France). Venous blood was drawn into citrate-phosphate-dextran
anticoagulant. PBMC were separated by dextran sedimentation and density
gradient centrifugation through Histopaque 1077 (Sigma) and then washed
three times with RPMI 1640 by low speed centrifugation in order to more
thoroughly eliminate the contaminating platelets. PBMC were then
adjusted to a concentration of 2 × 107 cells/ml in
RPMI 1640 (with Hepes and bicarbonate) medium. All steps were carried
out at room temperature. Under such conditions, cell viability
established by the trypan blue exclusion test was always greater than
95%. Flow cytometry analyses of cell preparations after staining with
specific monoclonal antibodies showed that about 65-70% of the
isolated cells were CD3+ T cells (T3 Coulter clone), 4-6% were CD19+
B cells (B4 Coulter clone), 16-24% were CD11b+ monocytes (MO1 Coulter
clone), and 4-6% were CD41a+ platelets (GP IIb IIIa, Immunotech,
Marseille, France).
DHA Enrichment of the Cells--
For the preparation of fatty
acid-albumin complexes, docosahexaenoic acid (Sigma) was stored at
20 °C in ethanol solution under nitrogen. Different amounts of the
ethanolic solution were evaporated to dryness under reduced pressure. 5 µM human delipidated serum albumin (HSA) in RPMI 1640 medium was added to give final fatty acid concentrations ranging from 5 to 15 µM and an albumin to fatty acid ratio varying from
1 to 3. The mixtures were incubated under nitrogen for 4 h at
37 °C.
DHA enrichment of PBMC was achieved by incubating the cells
(2 × 107cells/ml) for 2 h at 37 °C in RPMI
1640 medium containing increasing concentrations of DHA bound to 5 µM delipidated HSA.
The fatty acid composition of cell phospholipids was analyzed by gas
chromatography. Cell lipid extracts or lipids extracted from the
gradient fractions were separated on Silica Gel G60 plates (Merck) with
the solvent system hexane/diethyl ether/acetic acid (60:40:1, by
volume). The silica gel areas corresponding to phospholipids were
scraped off and transmethylated. Briefly, 1 volume of 5% H2SO4 in methanol was added to the scraped
silica gel, and transmethylation was carried out at 100 °C for 90 min in screw-capped tubes. The reaction was terminated by the addition
of 1.5 volume of ice-cold 5% (w/v) K2CO3, and
the fatty acid methyl esters were extracted with isooctane and analyzed
using a PerkinElmer Life Sciences chromatograph model 5830, equipped
with a capillary column (30 m × 0.32 mm, Supelco) and a flame
ionization detection. The column was two-step programmed from 135 to
160 °C at 2 °C/min and from 160 to 205 °C at 1.5 °C/min;
the detection temperature was maintained at 250 °C. The vector gas
was helium at a pressure of 0.8 pounds/square inch (5520 pascals).
Peaks were identified using standard fatty acid methyl esters.
PLD Assay in Intact Cells--
PLD was determined on the basis
of its transphosphatidylation activity. Freshly isolated cells were
incubated for 1 h at 37 °C in the presence of
[3H]arachidonic acid (37 kBq/ml, specific activity 7400 GBq/mmol, Amersham Biosciences) in 0.1% ethanol and then washed three
times in RPMI 1640 medium. Labeled cells were further enriched with DHA
as described above. Control cells were incubated without fatty acid in
the presence of 5 µM HSA alone. DHA-treated and control cells were then incubated for 30 min at 37 °C in the presence of 1%
butanol or 1% ethanol, and in the absence (unstimulated) or presence
of 5 µg/106 cells concanavalin A (+ConA), a specific
activator of T cells. In some experiments, control and DHA-enriched
cells were stimulated with 0.1 µg/ml OKT3 (Cilag Laboratories,
Levallois Perret, France) for 30 min in the presence of 1% butanol. In
experiments designed to investigate the influence of inhibitors, the
following conditions were used: bisindolylmaleimide (BIM, 1 µM) and calphostin C (126 nM) were added 30 min before ConA activation, and brefeldin A (BFA, 1-25 µg/ml) was
added 10 min before ConA activation. Incubations were terminated by
addition of ethanol and acidification of the medium to pH 3-4 with 2 N HCl. Lipids were extracted with chloroform/ethanol (6:3,
by volume) according to Boukhchache and Lagarde (23) in the presence of
50 µM butylhydroxylated toluene. Phosphatidylalcohols were separated on bidimensional TLC (Silica Gel G60 plates, Merck) using chloroform, methanol, 28% ammonia (65:35:5.5, by volume) for the
first migration, and ethyl acetate/isooctane/acetic acid (9:5:2, by
volume) for migration in the second dimension. Spots stained by
Coomassie Brilliant Blue R were scraped off and mixed with Picofluor
(Packard Instrument Co.), and the radioactivity was determined by
liquid scintillation counting. The radioactivity associated with
phosphatidylalcohols was expressed as percentage of the radioactivity
incorporated in total phospholipids.
Cell-free PLD Assay--
Oleate-dependent PLD
activity was measured as described by Chalifa et al. (24).
Briefly, the standard reaction mixture (120 µl) contained 50 mM Na-Hepes buffer, pH 7.2, 2.7 mM
(107 cpm/assay)
[3H]dipalmitoylphosphatidylcholine
([3H]DPPC, specific activity 3700 GBq/mmol, PerkinElmer
Life Sciences), 4 mM sodium oleate, 1 mM EGTA,
1 mM MgCl2, 2% ethanol, and 60-100 µg of
proteins from total cell lysates. After 30 min of incubation the
reaction was stopped by the addition of 2 ml of ice-cold
chloroform/methanol/HCl (1:1:0.002) and mixing. Phase separation was
achieved by further addition of 0.1 N HCl, 1 mM
EGTA, mixing, and centrifugation (2,000 rpm, 10 min). The organic phase
was collected, dried under nitrogen, and redissolved in
chloroform/methanol containing standard phosphatidylethanol as a
carrier. Phosphatidylethanol was separated on TLC using ethyl acetate/isooctane/acetic acid (9:5:2). Plates were stained by Coomassie
Brilliant Blue R, and spots corresponding to phosphatidylethanol were
scraped off and mixed with Picofluor, and the radioactivity was
determined by liquid scintillation counting. The radioactivity associated with phosphatidylalcohols was expressed as percentage of the
radioactivity of the substrate [3H]DPPC.
PIP2-dependent PLD activity was determined
according to Brown et al. (25). Briefly, 10 µl of mixed
lipid vesicles (phosphatidylethanolamine/phosphatidylinositol bisphosphate/phosphatidylcholine, molar ratio 16:1.5:1) with
[choline-methyl-3H]dipalmitoylphosphatidylcholine
(specific activity 2200 GBq/mmol, PerkinElmer Life Sciences) to give
106 cpm per assay was added to 40 µl of cell lysate in a
total of 100 µl containing 50 mM Hepes, pH 7.5, 80 mM KCl, 2.5 mM MgCl2, 2 mM CaCl2, 1 mM dithiothreitol.
Assays were done with or without 50 µM GTP
S and 1 µM rARF. The final PC concentration was 8 µM. Assays were incubated for 30 min at 37 °C and
stopped as described above. After centrifugation, the radioactivity of
an aliquot of the aqueous phase was determined by liquid scintillation
counting. Recombinant ARF was kindly provided by Dr. Blandine Geny,
INSERM U332, Paris, France.
RNA Extraction and Reverse Transcription (RT)-PCR--
Total RNA
was isolated from human PBMC using Trizol Reagent (Invitrogen) and
reverse-transcribed with Moloney murine leukemia virus-reverse
transcriptase (Promega, Lyon, France) using random hexamer mixed
oligonucleotides. Specific primers for the amplification of PLD1
transcripts were designed on the basis of published human sequences
(26) to discriminate between PLD1a and PLD1b. The sense and antisense
primers were 5'-GGGATCCGTGTGAAGCGGGTCACTTCAGGACCG-3' and
5'-GGGAATTCTCTGGTTTCCCCATGCAGCTCTCCCAC-3', respectively. Primers to specifically amplify PLD2 transcripts were designed on the basis of
published human sequences (27) and chosen in a domain corresponding to
the N-terminal part of the protein. The sense and antisense primers
were 5'-GGGAATTCGACGGCGACCCCTGAGAGCCTCTTC-3' and
5'-GGGAATTCACGGTATTTCTTCTTGGTTGTCCAGG-3', respectively.
Amplification conditions for PLD1 were 94 °C for 45 s, 60 °C
for 45 s, and 72 °C for 45 s, for 35 cycles. Amplification
conditions for PLD2 were 94 °C for 45 s, 57 °C for 45 s, and 72 °C for 45 s, for 35 cycles. PCDNA3 plasmids
carrying the hPLD1b and hPLD2 cDNAs originating from Dr. M. Frohman's laboratory and kindly provided to us by Dr. Michel Record
(Toulouse, France) were used for positive controls.
Preparation of Cytosolic and Particulate Cell Fractions for
Western Blotting Experiments--
Pelleted cells were washed three
times with Phillips' buffer and disrupted by glycerol lysis according
to Caldwell et al. (28) as described previously (29). After
glycerol treatment, the cells were pelleted at 900 × g
for 10 min, resuspended in lysis buffer (20 mM Hepes, 25 mM sucrose, 0.1 mM EGTA, 0.05 mM phenylmethylsulfonyl fluoride, 10 units/ml aprotinin, 2 µg/ml pepstatin A, pH 7.4), and stored frozen at 80 °C. After thawing, the cells were homogenized in a glass/Teflon homogenizer (40 strokes at
maximal speed), and homogenates were centrifuged at 42,000 × g for 20 min. Particulate and cytosolic fractions (3 volumes) were mixed with 4× concentrated Laemmli
buffer (1 volume), and proteins were determined by the method of
Schaffner and Weissmann (30) using bovine serum albumin as a standard.
Proteins were separated on a 10 (PKC
) or 15% (ARF) acrylamide gel
and electrotransfered onto an Immobilon P membrane (Millipore, St.
Quentin Yvelines, France). For PLD Western blot analyses, proteins
were denatured by 4 M urea in Laemmli buffer and separated
on 8% acrylamide gel in 4 M urea. Aspecific
antibody-binding sites were saturated by incubating membranes in TBS-T
(20 mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween
20) containing 5% bovine serum albumin, overnight at 4 °C. All
following steps were performed in TBS-T at room temperature. After
washing, the blots were incubated for 90 min either with an anti-PKC
monoclonal antibody (Santa Cruz Biotechnology, diluted 1:2000) or with
an anti-ARF 1:3 polyclonal antibody (sheep IgG, Upstate Biotechnology
Inc., diluted 1:1000). The PLD isoforms were detected with PLD1 and
PLD2 antisera (dilution 1:2000) prepared by Dr. S. Bourgoin (Laval
University, Canada). PLD1 antiserum was raised against the following
short human PLD1 peptides: residues 1-16, 144-162, 967-981, and
1027-1040. PLD2 antiserum was raised against the N-terminal sequence
of human PLD2 (residues 13-33). After washing, membranes were
incubated for 1 h either with a horseradish peroxidase-conjugated
goat anti-mouse antibody (1:15,000) or a donkey anti-sheep antibody
(Sigma, 1:10,000) or a goat anti-rabbit antibody (Bio-Rad, 1:20,000).
Immunoreactive proteins were visualized using the ECL detection system
(Amersham Biosciences) and x-ray film autoradiography. Bands were then
quantified with a videodensitometric analyzer (Bioprofil,
Vilbert-Lourmat, Germany).
Isolation and Characterization of Lipid Rafts from Human
PBMC--
Lipid rafts were isolated according to the procedure of
Montixi et al. (31) based on the insolubility of these
structures in cold non-ionic detergent, with slight modifications.
Briefly, control or DHA-enriched PBMC, either stimulated or not (5 × 108 cells), were homogenized in 1 ml of ice-cold lysis
buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA) supplemented with a mixture of protease inhibitors
(protease inhibitor mixture Sigma) and 1 mM orthovanadate.
After centrifugation at 800 × g at 4 °C for 10 min,
the post-nuclear supernatant was incubated with Triton X-100 at a final
concentration of 1% for 1 h at 4 °C. The lysate was then
adjusted to 1.3 M sucrose by the addition of an equal volume of 2.6 M sucrose and placed at the bottom of an
ultracentrifuge tube, and a step sucrose gradient (0.2-0.9
M with 0.1 M steps, 1 ml each) was placed on
top. It was centrifuged at 200,000 × g for 16 h
in an SW41 rotor (Kontron) at 4 °C. One-ml fractions were recovered
from the bottom to the top of the gradient. The sucrose concentration
of each fraction was determined with a Brix refractometer (Merck).
Aliquots were immediately used for PLD and 5'-nucleotidase assays, and
the remaining was stored at 80 °C until lipid analyses.
5'-Nucleotidase activity of the glycosylphosphatidylinositol-anchored
CD73, used as a marker of lipid rafts, was assayed as described by
Gentry and Olsson (32) with slight modifications. Aliquots of the
gradient fractions were incubated with 1 µM
[3H]5'AMP (specific activity 722 GBq/mmol, Amersham
Biosciences) in 1 ml of reaction mixture containing 60 mM
Tris, pH 7.4, 1 µM erythro-9-(2-hydroxy-3-nonyl)adenine
to inhibit endogenous adenosine deaminase, 20,000 cpm
[14C]adenosine as an internal standard, with or
without 25 µM adenosine 5'-(,
-methylene)diphosphate
(AMPCP) to inhibit 5'-nucleotidase activity, for 30 min at 37 °C.
The incubation was terminated by addition of 200 µl of 5%
ZnSO4 and 200 µl of 0.3 M
Ba(OH)2. After centrifugation, the radioactivity of
supernatant containing [3H]adenosine was measured by
liquid scintillation counting. 5'-Nucleotidase activity obtained by
difference between values with and without AMPCP was expressed
as nmol of 5'-AMP hydrolyzed per min per mg of proteins.
GM1 was quantitated on dot blots according to Ilangumaran et
al. (33). Briefly, 20 µl of each gradient fraction were dotted onto Immobilon using a Hybri-Dot Manifold apparatus (Bethesda). Membranes were rinsed with distilled water and blocked with 5% bovine
serum albumin in TBS-T for 1 h. After three 10-min washes with
TBS-T, membranes were incubated with horseradish peroxidase-conjugated cholera toxin in TBS-T containing 1% bovine serum albumin for 90 min
and then rinsed 7 times with TBS-T and developed with the ECL reagent.
The luminograms were quantitated using cooled digital CCD camera system
(ImageMaster VDS-CL, Amersham Biosciences) and ImageQuant software.
PIP2 was quantitated on dot blots performed as described
above using a mouse PIP2 antiserum (Assay Designs,
Euromedex, Souffelweyersheim, France, dilution 1:500) for
immunodetection and horseradish peroxidase-conjugated goat anti-mouse
antibody diluted 1:10,000 as the secondary antibody.
Total phospholipids were assayed with the ammonium ferrothiocyanate
method according to Stewart (34).
Cholesterol was determined enzymatically using a commercial assay kit
(Sigma) according to the manufacturer's recommendations.
PLD Activities and Western Blotting--
PLD activities were
determined on 40-µl aliquots of each gradient fraction as described
above. For PLD1 Western blotting, proteins from 400 µl of each
fraction were precipitated with ice-cold acetone, and pellets were
dissolved in 40 µl of Laemmli buffer containing 4 M urea.
PLD2 proteins were immunoprecipitated from 400 µl of gradient
fractions using the PLD2 antiserum described above according to Marcil
et al. (35).
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RESULTS |
Dose Dependence of the DHA-induced PLD Activation in Stimulated
PBMC--
To define more precisely the PLD-stimulating effect of DHA
that we have observed previously (7) in human PBMC, cells were preincubated for 2 h with increasing concentrations of DHA
complexed to human delipidated albumin (DHA to albumin ratio from 0 to
3) before T cell stimulation with ConA in the presence of 1% butanol. A marked and dose-dependent increase of PLD activity was
observed in DHA-treated cells compared with control cells incubated in the presence of delipidated albumin (Fig.
1A). Gas chromatography analysis of the fatty acid composition of cell phospholipids showed that the relative amount of esterified DHA was concomitantly increased (Fig. 1B). Furthermore, PLD activity was significantly
correlated to the percentage of DHA present in cell phospholipids (Fig.
1C, r = 0.68, p = 0.015, n = 12). These results suggest that the extent of PLD
activation was directly related to DHA enrichment of membrane phospholipids. A PLD-stimulating effect of DHA was also observed when
PBMC were stimulated with the anti-CD3 monoclonal antibody OKT3,
although the extent of PLD activation was lower than that induced by
ConA (Fig. 1D).
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Fig. 1.
Effect of DHA enrichment on PLD activation
and on the DHA content of total phospholipids from human PBMC.
A, [3H]arachidonate-labeled PBMC were
incubated for 2 h with 5 µM HSA or with increasing
concentrations of DHA bound to HSA. Cells were then stimulated with
ConA (5 µg/106 cells) in the presence of 1% butanol for
30 min. At the end of the incubation period, lipids were extracted from
cells plus medium, and the lipid extracts were separated by TLC as
described under "Experimental Procedures." The radioactivity
associated to phosphatidylbutanol was measured by liquid scintillation
counting. Results are expressed relative to the radioactivity
incorporated in total phospholipids and are means ± S.E. of four
separate experiments. Data were analyzed by ANOVA, and means were
compared by a protected t test. *, significantly different
from control cells incubated without DHA. p < 0.05. B, lipids were extracted and total phospholipids
separated on TLC. Phospholipid spots were scraped off, and the fatty
acids were transmethylated and analyzed as described under
"Experimental Procedures." Results are expressed as mol % and are
means ± S.E. of four separate experiments. Data were analyzed by
ANOVA and means were compared by a protected t test. *,
significantly different from control cells incubated without DHA.
p < 0.05. C, the radioactivity
associated to phosphatidylbutanol expressed as percent of total
phospholipid radioactivity was plotted as a function of the DHA content
of cell phospholipids. Data are from A and B. D, [3H]arachidonate-labeled PBMC were
incubated for 2 h with 5 µM HSA (control cells) or
with 15 µM DHA bound to HSA. Cells were then stimulated
or not with the anti-CD3 monoclonal antibody OKT3 (0.1 µg/ml), in the
presence of 1% butanol for 30 min. Lipids were extracted and analyzed
as described above. Results are means ± S.E. of three separate
experiments. *, significantly different from unstimulated control
cells. p < 0.05.
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|
Characterization of PLD in Human PBMC--
To investigate the PLD
isoform expression pattern of human PBMC, we performed RT-PCR using
primers for PLD1, allowing us to discriminate between the two splice
variants PLD1a and PLD1b, and primers specific for PLD2. As shown in
Fig. 2A, human PBMC clearly
express the three PLD transcripts. It is noteworthy that the intensity
of the PLD1a band appeared to be roughly 2-fold higher than that of
PLD1b, suggesting that human PBMC preferentially express the former
variant. Western blot analyses of total cell lysates showed that both
PLD1 and PLD2 are also expressed at the protein level (Fig.
2B). To characterize further the PLD present in human PBMC,
we then measured the enzyme activity of cell lysates using specific
assay conditions. Although the above results clearly denote the
presence of PLD1 and PLD2 in human PBMC, they do not rule out the
possible presence of an oleate-dependent enzyme, which has
not yet been characterized at the molecular level. Because the PLD
activity of human PBMC was up-regulated by DHA enrichment prior to
mitogenic activation, it was important to look for an oleate-dependent activity in PBMC lysates. As shown in Fig.
2C, PBMC lysates did not show any oleate-stimulated PLD
activity. In contrast, a marked synthesis of phosphatidylethanol was
observed when PIP2 was included in the substrate
phospholipid vesicles confirming the presence of a
PIP2-dependent PLD activity in these cells.
This PIP2- dependent synthesis was further
increased by more than 3.5-fold by GTPS. This latter result also
confirms that a G protein-dependent PLD1 activity is present in
PBMC lysates and suggests that the amount of G protein present in
lysates is sufficient to allow marked PLD1 activation. The membrane
fraction of PBMC exhibited a PIP2-dependent PLD
activity that was only modestly increased by GTPS addition due to
the loss of cytosolic G proteins (Fig. 2D). Full activation
was restored by the addition of recombinant ARF. Taken together these
results indicate that PIP2-dependent PLD1a,
PLD1b, and PLD2 are present in human PBMC and that these cells are
clearly devoid of oleate-dependent PLD activity.
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Fig. 2.
Characterization of PLD in human PBMC.
A, RT-PCRs were carried out as described under
"Experimental Procedures" using specific primers for PLD1, which
allowed us to discriminate the splice variants PLD1a and -1b
(lanes 2-4) and primers for PLD2 (lanes 6-8).
Positive controls were obtained by using pCDNA3 plasmids carrying
hPLD1b (lane 2) and hPLD2 (lane 6) cDNAs as
templates. Negative controls were obtained by using PBMC RNA untreated
by reverse transcriptase (lanes 4 and 8).
Lane 3, the larger product of 446 bp and the smaller
one of 332 bp derived from PLD1a and PLD1b, respectively; lane
6, the band at 329 bp has the expected size for amplification of a
PLD2 transcript. B, total cell lysates from 6 × 106 PBMC were subjected to SDS-PAGE and analyzed by Western
blotting using PLD1 and PLD2 antisera. Lysates from COS-1 cells
transfected with pCDNA3-hPLD1b and pCDNA3-hPLD2 were used as
positive controls. C, aliquots of cell lysates (8 × 106 cells) were added to a reaction mixture containing
2.7 mM [3H]DPPC in the absence () or
presence (+) of 2 mM sodium oleate or to reaction mixtures
containing 8 µM [3H]DPPC in the absence
() or presence (+) of 12 µM PIP2, with or
without 50 µM GTPS.
[3H]Phosphatidylethanol was separated by TLC.
D, aliquots of the 100,000 × g pellet
fraction (from 8 × 106 cells) were added to reaction
mixtures containing substrate phospholipid vesicles
(PE/PIP2/PC, 16:1.5:1) and incubated at 37 °C for 30 min
in the absence () or presence (+) of 50 µM GTPS with
or without recombinant ARF. [3H]Choline released from
dipalmitoylphosphatidyl[methyl-3H]choline was
measured as described under "Experimental Procedures." Data are
means ± S.E. of three separate experiments.
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The DHA-induced PLD Activation in ConA-stimulated PBMC Is
Independent of PKC--
PLD1 activity is known to be regulated through
activation by PKC and, in particular, by the PKC isoform (9). Thus,
we sought to determine whether the DHA-induced PLD activation observed in ConA-stimulated PBMC was dependent on PKC. Several studies have
shown that polyunsaturated fatty acids can either activate (36) or
inhibit (37) protein kinase C activity depending on the cell type
considered. It has also been shown that DHA rapidly induces the
translocation of several PKC isoenzymes to the particulate fraction of
human monocytes (38). We thus examined whether DHA treatment of PBMC
affects PKC expression. Results of Western blot experiments show
that DHA enrichment of the cells dose-dependently decreased
the PKC amount in the cytosolic fractions (Fig.
3A, left) while the
amount in the particulate ones was maintained and even increased at the
highest DHA concentration used (Fig. 3B, left).
This pattern suggests that DHA favors PKC translocation in the
absence of cell activation. In marked contrast, the PKC distribution
pattern was totally different when PBMC were further stimulated with
ConA after DHA enrichment (Fig. 3, right). The large PKC
translocation induced by ConA in non-enriched cells was substantially
impaired in DHA-enriched cells. At the highest DHA concentration used
for enrichment, PKC remained mainly cytosolic. Thus, under the
conditions of maximal PLD activation (DHA plus ConA), the translocation
of PKC to the particulate fraction was strongly counteracted. This
result strongly suggests that the mechanism of DHA-induced PLD
activation in stimulated PBMC is PKC independent. To support this
hypothesis further, experiments were performed in the presence of PKC
inhibitors and in cells treated with the phorbol ester TPA for a
prolonged period to down-regulate conventional PKC isoforms. Control or
DHA-enriched cells were pretreated with the PKC inhibitor BIM that
competes with ATP at the catalytic site, prior to ConA activation. As
shown in Fig. 4A, BIM did not
induce any significant PLD inhibition whatever the dose of DHA used for
cell enrichment. Similar results were obtained with another PKC
inhibitor, calphostin C, that inhibits the binding of phorbol esters to
the PKC regulatory domain (not shown). In addition, the
DHA-induced PLD activation was not affected by PKC
down-regulation. Fig. 4B shows that PLD activation was similar in cells pretreated for 14 h with 100 nM TPA
prior to ConA activation and in cells incubated for the same period in RPMI alone. In contrast, the strong PLD activation induced by 10 nM TPA in control non-enriched cells was totally lost after PKC down-regulation (Fig. 4C). Collectively, these results
indicate that PLD activation by DHA in the presence of mitogens neither involves PKC activation nor requires the presence of an intact PKC.
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Fig. 3.
Effect of DHA on the translocation of
PKC in human PBMC. PBMC were incubated
for 2 h with 5 µM HSA or with 5 or 15 µM of DHA bound to HSA. Cells were then incubated for 30 min in the absence or presence of ConA (5 µg/106 cells).
At the end of incubation, cells were lysed, and the cytosolic and
particulate fractions were separated by ultracentrifugation. Cytosolic
(A) and particulate (B) proteins (8 µg per
lane) were analyzed by Western blotting, using an anti-PKC
monoclonal antibody for immunodetection. Spots were analyzed by
videodensitometry. Bar diagrams show results expressed as
arbitrary units normalized to values obtained in the cytosol of control
unstimulated cells and are means ± S.D. of five separate
experiments. *, significantly different from non-enriched unstimulated
cells (DHA 0, ConA) p < 0.05;
 , significantly different from non-enriched stimulated cells
(DHA 0, +ConA) p < 0.05. Blots shown in the upper part of the two panels
are from one typical experiment.
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Fig. 4.
The DHA-induced PLD activation in
ConA-stimulated PBMC is independent of PKC. A,
[3H]arachidonate-labeled PBMC were incubated for 2 h
with 5 µM HSA or with 5 or 15 µM of DHA
bound to HSA. Cells were then incubated in the absence or presence of 1 µM BIM for 30 min at 37 °C and further stimulated with
ConA (5 µg/106 cells), in the presence of 1% butanol for
30 min. Results are expressed relative to the radioactivity
incorporated in total phospholipids and are means ± S.E. of four
separate experiments. Data were analyzed by ANOVA, and means were
compared by a protected t test. *, significantly different
from control non-enriched cells. p < 0.05. B, [3H]arachidonate-labeled control or
DHA-enriched (5 µM) PBMC were incubated in the absence or
presence of 100 nM TPA for 14 h and then stimulated
with ConA (5 µg/106 cells) in the presence of 1% butanol
for 30 min. Results are expressed relative to the radioactivity
incorporated in total phospholipids and are means ± S.E. of five
separate experiments. Data were analyzed by ANOVA, and means were
compared by a protected t test. *, significantly different
from control non-enriched cells without TPA pretreatment.
p < 0.05. C,
[3H]arachidonate-labeled control PBMC were
incubated in the absence or presence of 100 nM TPA for
14 h and then stimulated with 10 nM TPA in the
presence of 1% butanol for 30 min. Results are expressed relative to
the radioactivity incorporated in total phospholipids and are
means ± range of two separate experiments.
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The DHA-induced PLD Activation in ConA-stimulated PBMC Is Dependent
on Small G Proteins--
To look for the possible involvement of small
G proteins in the DHA-induced activation of PLD in stimulated PBMC,
DHA-enriched cells were permeabilized with digitonin in the presence or
absence of GTPS before ConA activation. Permeabilization in the
absence of GTPS allows macromolecules such as ARF or Rho to leak out of the cells, whereas such leakage can be prevented when digitonin and
GTPS are added simultaneously. As shown in Fig.
5A, cells permeabilized in the
absence of GTPS lost their ability to respond to ConA, whereas PLD
activation was maintained when the permeabilization was done in the
presence of GTPS. These observations show that the combined effects
of DHA and ConA require soluble factors that are removed during
permeabilization. To examine whether ARF proteins could be involved in
the DHA-induced PLD activation of stimulated PBMC, DHA-enriched cells
were preincubated for 10 min with BFA prior to ConA activation. As
shown in Fig. 5B, BFA, which acts primarily as an inhibitor
of ARF activation, was able to block PLD activation. This inhibitory
effect was already observed for a BFA concentration of 5 µg/ml, and
the inhibition was total at 10 and 25 µg/ml BFA. These results
suggest that ARF is a mediator of PLD activation by ConA in
DHA-enriched cells.
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Fig. 5.
The DHA-induced PLD activation in
ConA-stimulated PBMC is dependent on small G proteins.
A, GTPS is required for ConA activation of PLD in
DHA-enriched cells permeabilized with digitonin.
[3H]Arachidonate-labeled control or DHA-enriched (5 µM) PBMC were either incubated in RPMI (unpermeabilized)
or with 8 µM digitonin in the absence or presence of 100 µM GTPS and then stimulated with ConA (5 µg/106 cells) in the presence of 1% butanol for 30 min.
Results expressed relative to the radioactivity incorporated in total
phospholipids are from one experiment representative of two.
B, effect of brefeldin A on the ConA-induced PLD
activation in DHA-enriched PBMC. [3H]Arachidonate-labeled
PBMC were incubated for 2 h with 5 µM DHA bound to 5 µM HSA. DHA-enriched cells were then incubated for 10 min
before the addition of ConA (5 µg/106 cells) and 1%
butanol. Results are expressed relative to the radioactivity
incorporated in total phospholipids and are means ± S.E. of five
separate experiments. Data were analyzed by ANOVA, and means were
compared by a protected t test. *, significantly different
from control cells incubated without inhibitor, p < 0.05; , significantly different from DHA-enriched cells incubated
without inhibitor, p < 0.05.
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DHA Stimulates ARF Translocation--
Because ARF
activation was correlated with translocation of the protein from
cytosol to membranes, cytosolic and particulate fractions were prepared
from control and DHA-enriched PBMC either stimulated by ConA or not,
and the presence of ARF proteins was investigated by Western blotting.
Fig. 6A shows that the
relative amount of ARF in the particulate fraction was about 3-fold
higher in DHA-enriched cells stimulated with ConA than in control
unstimulated cells. In control PBMC, ConA activation tended to increase
the proportion of ARF in the particulate fraction, but this effect did
not reach significance. These results suggest that DHA and ConA
synergize to translocate ARF to the particulate fractions. In addition,
the effect of DHA on ARF translocation was clearly dose-dependent (Fig. 6B). Together with BFA
inhibition, these results suggest that DHA may activate PLD by favoring
ARF translocation to membranes.
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Fig. 6.
Effect of DHA on ARF translocation.
A, PBMC enriched with 15 µM DHA or
control PBMC were incubated in the absence or presence of ConA (5 µg/106 cells). At the end of the incubation, cells were
lysed and the cytosolic and particulate fractions separated by
ultracentrifugation. Particulate proteins (10 µg per lane) were
analyzed by Western blotting using an anti-ARF polyclonal antibody for
immunodetection. Spots were analyzed by videodensitometry. Bar
diagrams show results expressed as arbitrary units normalized to
values obtained for unstimulated control cells and are means ± S.D. of three separate experiments. Data were analyzed by ANOVA, and
means were compared by a protected t test. *, significantly
different from control unstimulated cells, p < 0.05. B, PBMC enriched with DHA concentrations increasing
from 5 to 15 µM, or control cells were incubated in the
presence of ConA (5 µg/106 cells) and processed as
described above for Western blotting experiments with an anti-ARF
antibody. A typical blot representative of three separate experiments
is shown.
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PLD1 Activity Is Located to the Detergent-insoluble Membrane (DIM)
Domains of PBMC--
Detergent-insoluble membrane domains from PBMC
were obtained by fractionation of Triton X-100-treated cell lysates on
a discontinuous sucrose density gradient. As shown in Fig.
7A greater than 80% of the
total proteins were recovered in the first three high density fractions
containing from 36 to 31% sucrose (Fig. 7D). In contrast, the main part of the 5'-nucleotidase activity of CD73, a
glycosylphosphatidylinositol-anchored enzyme (39), was distributed in
the low density fractions containing 21 to 15% sucrose (Fig.
7A). Moreover, the ganglioside GM1 was also distributed in
the same low density fractions of the gradient as 5'-nucleotidase
activity (Fig. 7A, inset). As observed for protein distribution, greater than 70% of the total phospholipids were
recovered in the high density sucrose fractions, whereas cholesterol
was present both in the high density fractions, which contained Triton
X-100-soluble material, and in the low density fractions (Fig.
7B). Thus, the phospholipid to cholesterol molar ratio which
was higher than 10 in the high density fractions was decreased to 1-2
in the low density fractions, as it has been observed in
detergent-resistant microdomains isolated from Madin-Darby canine
kidney cells (40). In contrast, PIP2 was mainly
immunodetected in the low density fractions of the gradient (Fig.
7B, inset). Taken together these results indicate
that the low density fractions fulfill the criteria of
detergent-insoluble microdomains. Samples of each gradient fraction
were then tested for PLD activity using [choline-methyl-3H]phosphatidylcholine, in
mixed phospholipid vesicles including PIP2, as a substrate,
in the presence or absence of rARF protein plus GTPS. Basal PLD
activity measured in the absence of ARF plus GTPS was mainly present
(76%) in the high density sucrose fractions, whereas the activity
recovered in the low density fractions (6-8) consisted of less than
8% of total (Fig. 7D). PLD2 protein was then
immunoprecipitated from each gradient fraction and analyzed by Western
blotting using a PLD2-specific antiserum. A 
90-kDa, PLD2-immunoreactive band, in the high density fractions of the gradient, coincided with basal PLD activity (Fig. 7D,
inset). No PLD2 band could be detected in the low density
detergent-insoluble microdomains. In contrast, GTPS plus
ARF-dependent PLD activity, estimated as the difference
between assays with and without ARF plus GTPS, was mainly associated
with the GM1, PIP2, and cholesterol-rich low density
fractions which also contained most of the 5'-nucleotidase activity.
Thus, about 50% of total GTPS plus ARF-dependent PLD activity from human PBMC were associated with the detergent-insoluble microdomains (Fig. 7C). The highest specific activity was
present in fraction 7 (3 nmol of PC hydrolyzed per h/mg of proteins). It was more than 60-fold higher than the specific activity detected in
the high density fractions (fractions 1-4). Immunoblot
analyses of the material precipitated with ice-cold acetone showed the presence of immunoreactive PLD1 (115 kDa) in both the high and low
density fractions of the gradient (Fig. 7C,
inset), which suggests a close correlation between PLD1
protein and GTPS plus ARF-dependent PLD activity.
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Fig. 7.
Characterization of the low density sucrose
gradient fractions prepared from control PBMC and association of PLD1
activity to detergent-insoluble microdomains. Triton X-100 lysates
were prepared from control human PBMC and fractionated by floatation in
a discontinuous sucrose density gradient, and the fractions were
analyzed for protein concentration, 5'-nucleotidase activity, GM1,
PIP2, cholesterol, phospholipid content, and ARF plus
GTPS-responsive and unresponsive
PIP2-dependent PLD activities as described
under "Experimental Procedures." For PLD1 Western blotting,
proteins from 400 µl of each fraction were precipitated with ice-cold
acetone, and pellets were dissolved in 40 µl of Laemmli buffer
containing 4 M urea. PLD2 proteins were immunoprecipitated
from 400 µl of gradient fractions prior to Western blotting using a
PLD2 antiserum. A, distribution of total protein
concentration, 5'-nucleotidase activity, and cholera toxin-reactive GM1
(inset). B, distribution of total
phospholipid, cholesterol, and immunoreactive PIP2
(inset). C, distribution of GTPS plus
ARF-dependent PLD activity and PLD1 protein
(inset). D, sucrose content, basal PLD
activity, and PLD2 protein distribution (inset). Results are
means ± S.E. of six separate experiments. Each blot
shown in the insets is representative of three.
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DHA Enrichment of PBMC Displaces Signaling Proteins Out of the
DIMs--
When PBMC were first enriched with 15 µM DHA
prior to Triton X-100 treatment and fractionation on sucrose gradient,
the pattern of GTPS plus ARF-dependent PLD activity
recovered in the gradient fractions was strongly modified. Less than
30% of total activity remained associated to DIMs, and most of the
activity appeared in the intermediate fractions from 30 to 25% sucrose
(Fig. 8, A and C).
This pattern was paralleled by changes in the distribution of PLD1
protein. The inset of Fig. 8A shows that only a
faint immunoreactive band remained associated with the low density
fraction 6, whereas most part of the immunoreactive PLD1 was recovered in the higher density fractions of the gradient. In contrast, none of
the other measured parameters (5'-nucleotidase and basal PLD
activities, GM1, cholesterol, total phospholipids) exhibited a modified
pattern with respect to control cells (not shown). Although in some
experiments PIP2 appeared to be delocalized toward the high
density detergent-soluble fractions, such a delocalization could not be
reproducibly obtained (not shown). When DHA-enriched PBMC were
stimulated with ConA prior to fractionation, GTPS plus ARF-dependent PLD activity was almost totally excluded from
DIM, with only 7% remaining associated to the low density fractions 6-8 (Fig. 8, B and C). Western blot analyses
confirmed that almost the whole PLD1 protein was delocalized to the
non-DIM fractions of the gradient (Fig. 8B,
inset). As observed for unstimulated DHA-enriched cells,
neither the distribution of 5'-nucleotidase nor that of basal PLD
activities along the gradient fractions was changed as compared with
controls (not shown).
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Fig. 8.
DHA enrichment of human PBMC displaces PLD1
activity from detergent-insoluble microdomains. A,
human PBMC were enriched with 15 µM DHA before lysis and
fractionation of the Triton X-100-treated lysates on a discontinuous
sucrose density gradient. The fractions were analyzed for GTPS plus
ARF-dependent PLD activity and PLD1 protein distribution
(inset) as described under "Experimental Procedures."
Results are means ± S.E. of five separate experiments. The blot
shown in the inset is representative of three. B,
DHA-enriched (15 µM) PBMC were stimulated with ConA (5 µg/106 cells) prior to lysis and fractionation. PLD assay
and Western blotting were performed as in A. Results are
means ± S.E. of five separate experiments. The blot
shown in the inset is representative of three.
C, the percentage of GTPS plus
ARF-dependent PLD activity remaining associated to
detergent-insoluble microdomains in the experiments of Fig.
7C, and A and B of this figure is
shown. Data were analyzed by ANOVA, and means were compared by a
protected t test. *, significantly different from control
unstimulated cells, p < 0.05.
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It is noteworthy that the extent of DHA enrichment was higher in
phospholipids of DIM (+50%) than non-DIM (+20%) membranes (Table I). On the whole, DHA
treatment of the cells increased the relative amount of polyunsaturated
fatty acids present in phospholipids of the DIM fractions at the
expense of the saturated palmitate, which is very likely to selectively
increase the fluidity of these domains.
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Table I
Fatty acid (FA) composition of total phospholipids of non-DIM and DIM
membranes isolated from control and DHA-enriched PBMC
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Because Src family tyrosine kinases are known to be associated with the
cytoplasmic layer of rafts (41) and because PUFA enrichment of Jurkat
cells has been shown to modify the cytoplasmic leaflet of these
structures (22), we compared the distribution of the Src family kinase
Lck in sucrose gradients prepared from DHA-enriched (Fig.
9B) and control non-enriched
PBMC (Fig. 9A) stimulated with ConA. Western blot
experiments showed that DHA treatment of the cells markedly displaced
Lck protein out of the DIMs (fractions 6-8) toward the high density
detergent-soluble fractions. In parallel experiments, we also compared
the distribution of ARF in the gradient fractions. Although most ARF
proteins were found in the high density detergent-soluble fractions, a
notable part was also recovered in the DIM fractions of non-enriched
PBMC (Fig. 9C). Interestingly, in DHA-enriched cells, very
little ARF remained associated to rafts, the bulk of the protein being
present in the detergent-soluble non-raft fractions (Fig.
9D).
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Fig. 9.
DHA enrichment of human PBMC displaces Lck
and ARF proteins from detergent-insoluble microdomains. Control or
DHA-enriched (15 µM) PBMC were stimulated with ConA (5 µg/106 cells) before lysis and fractionation of the
Triton X-100-treated lysates on a discontinuous sucrose density
gradient. A and B, 20 µl of
detergent-soluble (non-DIM) and detergent-insoluble (DIM) fractions
were analyzed for Lck immunoreactivity. C and
D, 400 µl of gradient fractions were precipitated in
ice-cold acetone, and pellets were dissolved in 40 µl of Laemmli
buffer and analyzed for ARF immunoreactivity. 5 (lanes 1 and
2) and 20 µl (lanes 6-8) of total Laemmli
extracts were layered.
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DISCUSSION |
The present study was undertaken to investigate the potential
mechanisms involved in the activation of PLD by the n-3
polyunsaturated fatty acid DHA that we have previously observed in
mitogen-activated PBMC (7). We confirm here that PLD activation is
proportional to DHA enrichment of cell membranes and that it can be
observed whatever the mitogenic effector used, concanavalin A or the
anti-CD3 monoclonal antibody OKT3 (Fig. 1). The whole results of the
present study suggest that DHA activates PLD1 enzyme by an indirect
mechanism involving lipid modifications of detergent-insoluble domains
within PBMC membranes.
One of the first questions we have addressed concerns the identity of
the PLD isoforms present in human PBMC. Information on the expression
levels, subcellular localization, and function of PLD in leukocytes is
limited. It is usually accepted that human peripheral leukocytes
express little or no PLD (13). Variable results have been reported for
PLD mRNA and proteins in different leukemic cell lines. Thus, human
myeloid leukemic HL60 have been shown to express only PLD1 and mouse
lymphocytic leukemic L1210 only PLD2, whereas neither isoform seems to
be present in mouse thymoma EL4 (42, 43). Although some authors have
only detected PLD2 in Jurkat cells, studies showing that PLD activation
by the chemokine RANTES (regulated on activation normal T cell
expressed and secreted) requires ARF and RhoA G proteins strongly
suggest the presence of PLD1 in these cells (12, 43). In addition, they
also contain an oleate-dependent PLD activity, which is
increased during actinomycin D-induced apoptosis (11). By using
anti-PLD1- and PLD2-specific antibodies, we showed that human PBMC
express both isoforms. RT-PCR using specific primers for PLD1 and PLD2 indicated that both 1a and 1b splice variants of PLD1 are expressed together with PLD2 at the mRNA level. The assay of PLD activity in
cell lysates confirmed the PIP2 requirement for activity
(the hallmark of PLD1 and PLD2) and its partial GTPS dependence
(suggesting the presence of PLD1). Furthermore, it clearly showed the
absence of an oleate-dependent activity (Fig. 2).
Because PLD1 activity is known to be regulated by conventional PKC, at
least in some types of cells (9, 10), we hypothesized that DHA might
favor the recruitment of some PKC isoforms in the vicinity of PLD1.
However, our whole results clearly indicate that the DHA-induced PLD
activation observed in stimulated PBMC was PKC-independent. Indeed, it
was affected neither by the PKC inhibitors calphostin C and BIM nor by
PKC down-regulation (Fig. 4). In addition, although DHA decreased the
amount of immunoreactive PKC present in the cytosolic fractions of
unstimulated cells with a concomitant increase in the proportion of the
particulate protein, this effect was entirely reversed after ConA
stimulation (Fig. 3). We observed a similar behavior of the PKC and
atypical PKC
isoforms (not shown). Thus, in conditions of maximal
PLD activation (DHA-enriched and ConA-stimulated cells), most part of
PKC proteins (at least the , , and isoforms) were present in
the cytosolic compartment, which further supports the lack of PKC
involvement in this activation.
The observation that PLD activation was lost when DHA-enriched PBMC
were depleted in GTP and small G proteins by permeabilization prior to
ConA activation prompted us to investigate the potential involvement of
ARF in this mechanism. Our results showing that the fungal metabolite
BFA markedly inhibited the DHA-induced PLD activation (Fig. 5) suggest
a role for active ARF proteins. Indeed, BFA has been shown to inhibit
the exchange activity of several ARF guanine nucleotide-exchange
factors, thus preventing the formation of active GTP-bound ARF (44,
45). By using an anti-ARF1-3 antibody in Western blotting experiments,
we showed that DHA and ConA synergize to recruit ARF proteins to the
particulate fractions of PBMC (Fig. 6). Furthermore, this increase of
particulate ARF was dose-dependently related to the DHA
enrichment of cell membranes, which further supports the role of ARF in
DHA-induced PLD activation. The lack of any oleate-stimulated PLD
activity in PBMC lysates together with the present results showing the
involvement of ARF proteins strongly suggest that PLD1 was the main
target of DHA in mitogen-activated PBMC. However, we cannot totally
rule out PLD2 because human PLD2 has also been described as
ARF-activable in some cells, although to a lesser extent than PLD1
(46). The present results are at variance with the commonly accepted
inhibition of PIP2-dependent PLD activity by
fatty acids (9) and with the results of Kim et al. (42)
showing that recombinant hPLD2, but not hPLD1, was activated by fatty
acids in vitro.
Several recent reports (10, 16, 17) have described the presence of PLD
activity and/or proteins associated with caveolins in the caveolae of
different cell lines. But whether PLD is present in the lipid rafts of
cells devoid of caveolins was not known. Although lymphocytes do not
express any detectable caveolin, detergent-insoluble rafts closely
related to caveolae do exist in these cells where they play a crucial
role in the transduction of the mitogenic signals (20, 21). Upon
ligation of the T cell antigen receptor, a signaling cascade is
initiated via activation of the Src family kinases present in lipid
rafts. This, in turn, allows the recruitment of the Syk family kinases
and the downstream activation of phospholipase C and the
extracellular signal-regulated kinase/mitogen-activated protein
kinase cascade, while phosphatases such as CD45 become excluded from
the rafts (20, 21). Most studies dealing with the role of lipid rafts
in lymphocyte activation have been performed with murine thymocytes and
cell lines (31, 33) or with human Jurkat cells (22, 47), and those
using human peripheral lymphocytes are very scarce (48). After
fractionation of the detergent-treated membranes from PBMC on sucrose
density gradient, we used several biochemical markers to characterize
glycolipid-enriched complexes floating at a low density during
centrifugation. Besides GM1 which is widely used as a raft marker in
hematopoietic cells (49), we chose the cell surface antigen CD73 as a
glycosylphosphatidylinositol-anchored protein. This cell surface marker
is widely distributed within the various B and T cell subpopulations
and possesses an intrinsic 5'-nucleotidase activity that can be easily
measured (39, 50). In addition, we also determined the localization of
PIP2 in the gradient fractions using a specific
PIP2 antiserum, because this signaling phospholipid is
known to be present in detergent-insoluble, cholesterol-rich membranes
(51, 52). The fractions of gradient recovered at a low density, between
15 and 20% sucrose, contained most of GM1, PIP2, and
5'-nucleotidase activity, had a low phospholipid to cholesterol ratio
and a very low protein content (Fig. 7) and thus fulfilled the main
criteria for lipid rafts (49). An important result of the present study
is that these low density fractions originating from cells devoid of
caveolae also contain a large proportion of the PLD activity stimulated
by rARF in the presence of GTPS and a substantial part of PLD1
protein. In contrast, the basal PLD activity and PLD2 protein were
mainly recovered in the high density fractions containing
detergent-solubilized membranes. This allows us to conclude that in
human PBMC, PLD1 is largely associated to rafts, as has been observed
for caveolae-rich cells such as rat fibroblasts (15) and mouse skeletal
myotubes (16), whereas PLD2 is mainly present in non-raft membranes. Because detergent-insoluble membranes isolated from cell lysates most
probably originate from the plasma membrane (53), this suggests that a
substantial part of PLD1 is located in this compartment. However some
detergent-insoluble materials may also derive from intracellular membranes.
DHA enrichment of PBMC before raft isolation did not affect the
distribution of GM1, PIP2, or 5'-nucleotidase activity in the gradient fractions, while part of both GTPS plus
ARF-dependent PLD activity and PLD1 protein was delocalized
to the high density fractions. The delocalization was almost total when
DHA-enriched cells were also stimulated by ConA (Fig. 8). We also
observed such a delocalization for Lck and ARF (Fig. 9). It is
noteworthy that DHA enrichment of phospholipids was higher in DIM than
in non-DIM membranes (Table I). This increase in polyunsaturated fatty
acids at the expense of saturated palmitate is very likely to affect
the physicochemical properties and spatial organization of lipid rafts.
The fact that alteration of the rafts by DHA had no effect on GM1 and
5'-nucleotidase distribution points out the specificity of DHA effect
on PLD1. It is consistent with the results of Stulnig et al.
(22, 54) showing that n-3 PUFAs affect the cytoplasmic face
of the lipid leaflet in rafts.
It has been shown that the association of PLD with the
caveolin-scaffolding domain inhibits PLD activity (15). On the basis of
the present results, it can be proposed that PLD1 association to rafts
also impairs its activity and that the DHA-induced PLD activation that
we have observed in ConA-stimulated cells results from the PLD1
exclusion from rafts. PLD1 activation might conceivably result either
from conformational changes of the enzyme switched to a different
membrane environment or from the possibility for PLD1 to interact with
ARF, which is present out of the rafts. In support of this hypothesis,
DHA enrichment increased the formation of membrane-bound ARF, and this
increase only concerns the detergent-soluble non-raft membrane
fractions. Regarding the above hypotheses, an apparently discrepant
result is that the DHA-induced increase in PLD activity observed in
intact cells was no more evident when considering the PLD activity
profiles of fractionation gradients. However, if PLD1 sequestration
into rafts entails its maintenance under an inactive state due to
interactions with the particular lipid environment or to interactions
with inhibitory factors present in these structures, it is likely that
raft isolation in the presence of detergent and sucrose disturbs their
organization. This may in turn relieve the inhibitory constraints and
allow the measurement of a PLD activity in DIM fractions of control
PBMC, whereas this activity is masked in intact cells. As an
alternative explanation, if PLD1 activation in intact cells results
from its ability to interact with ARF caused by the DHA-induced
displacement of the enzyme out of rafts and by the DHA-induced
translocation of cytosolic ARF to non-raft membranes, it cannot be
observed in broken cell assays where an excess of exogenous ARF is
added. Indeed, these assay conditions are expected to induce a maximal
activation of PLD1 trapped into DIMs, which should normally be inactive
due to an insufficient amount of activated ARF.
Although further studies aiming at fully delineating the relationships
between DHA-induced PLD activation and exclusion of PLD1 from raft
microdomains are required, the novel mode of PLD activation described
in this study is potentially important to explain the modulation of
cell signaling by polyunsaturated fatty acids. Because PLD activation
in lymphoid cells is known to transmit antiproliferative signals (55),
the partial disorganization of lipid microdomains induced by DHA,
leading to the exclusion of Src kinases and PLD1 from rafts, might
impair the transduction of the mitogenic signal in DHA-enriched
lymphocytes and participate in the immunodepressive effect of this
fatty acid.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Michel Record for kindly
providing hPLD1b and hPLD2 cDNA and Dr. Blandine Geny for the
generous gift of rARF.
| |
FOOTNOTES |
*
This work was supported by a CNR-INSERM joint program grant
(to C. S. and A. F. P.).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: Unité INSERM
352, Laboratoire de Biochimie et Pharmacologie, INSA de Lyon,
Bâtiment Louis Pasteur, 11 avenue Jean Capelle, 69621 Villeurbanne Cedex, France. Tel.: 33-4-72-43-85-71; Fax:
33-4-72-43-85-24; E-mail: prigent@insa-lyon.fr.
Published, JBC Papers in Press, July 24, 2002, DOI 10.1074/jbc.M202376200
| |
ABBREVIATIONS |
The abbreviations used are:
PUFA, polyunsaturated fatty acid;
PLD, phospholipase D;
DHA, docosahexaenoic
acid;
PBMC, peripheral blood mononuclear cells;
ARF, ADP-ribosylation
factor;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
DIM, detergent-insoluble membrane;
HSA, human serum albumin;
BIM, bisindolylmaleimide;
BFA, brefeldin A;
DPPC, dipalmitoylphosphatidylcholine;
PKC, protein kinase C;
PIP2, phosphatidylinositol bisphosphate;
AMPCP, adenosine 5'-(,-methylene)diphosphate;
PC, phosphatidylcholine;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
ConA, concanavalin A;
RT, reverse transcription;
ANOVA, analysis of variance;
rARF, recombinant ARF.
| |
REFERENCES |
| 1.
|
Calder, P. C.,
and Zurier, R. B.
(2001)
Curr. Opin. Clin. Nutr. Metab. Care
4,
115-121[Medline]
[Order&n |
TOP
ABSTRACT
INTRODUCTION
