From the
We studied the translocation of protein kinase C (PKC), the
endogenous phosphorylation and presence in the membrane fraction of
p47
Almost all tissues contain PKC,
The PKC
isozymes belong to conventional, non-conventional, or atypical PKCs,
so-called c-PKCs, n-PKC, and a-PKC, respectively. The c-PKCs require
Ca
It has been
shown that FAs themselves are able to modulate PKC activity in
reconstituted membrane systems (for review, see Ref. 8). Early
observations were made with unsaturated FAs and requirement for their
``cis'' configuration (9) in a monomeric
(non-micellar) solution (10) was recognized. It was also
established that (i) they activate PKC in the absence of the usual
cofactors PtdSer and Ca
We recently
found that the preincubation of HAMs in the presence of certain FAs
brought about a potentiating effect at nanomolar concentrations and a
depressing effect at micromolar concentrations on the NADPH
oxidase-mediated production of O(16) . This novel bimodal action
was assumed to be linked to PKC activity, and its magnitude was found
to be largely dependent on the FA chain structure. Moreover, it did not
occur in non-adherent monocytes and other leukocytes such as
neutrophils. In other words, this novel non-conventional action of FAs
seems to be a specific property of HAMs.
In this study, we
investigated how translocation of PKC, endogenous phosphorylation and
presence in membrane fraction of p47
Our results demonstrate the fact that, in intact cells, the
capacity of both iso15:0 and 20:4n-6 to potentiate or depress
the HAM O production is mediated by an action on the PKC translocation,
leading to a simultaneous endogenous phosphorylation and translocation
of p47
The evidence for FA modulation of the O production through a
strictly PKC-dependent route is not inconsistent with the conclusion of
several authors claiming a direct activating effect of arachidonate (28, 29, 30) or other cis-unsaturated
FAs (31) on the NADPH oxidase of neutrophils. It has more
recently been proposed that phosphorylation and translocation of p47,
although necessary, are insufficient for oxidase activity in these
cells(32) . FAs could specifically and differently act on
different leukocyte lines. We advance that FA modulation exclusively
mediated by PKC we describe may only take place in adherent
monocytes(16) .
The finding that both iso15:0 and
20:4n-6 alone were unable to trigger the PKC translocation
requires distinguishing between the cell-free PKC studies and those on
the intact cell PKC activity or translocation. The first ones have
demonstrated a direct FA action (33) or a synergistic FA
intervention with DAG (34) on the PKC activity. FA action more
strictly depends on the presence of DAG for the
In any case, however, these types of action took
place at micromolar FA concentrations. In addition to the FA action on
PKC translocation strictly depending on DAG or PMA, the present results
confirm both the FA stimulating action at nanomolar concentrations and
the depressing action at the micromolar concentrations (usually
considered as stimulating) upon O production. They also establish that
either action is mediated by phosphorylating and translocating
processes. Considering the opposite FA responses of circulating and
adherent monocytes (16) and that little is known on monocyte
PKC(36) , the question of whether and how the adherence plays a
role first in changing the distribution of PKC isotypes, and presumably
(i) in the properties of the protein components of the NADPH oxidase
(especially p47
An apparently contradictory finding is that R59022
did not potentiate (it did inhibit) the PKC translocation, whereas it
greatly enhanced the NADPH oxidase activity in the presence of DAG (the
activity was not enhanced in the presence of PMA). This can easily be
explained by the view that the increasingly available amounts of DAG
(due to the inhibition of DAG phosphorylation) are able to directly
activate NADPH oxidase(37, 38) . The fact that this
effect was totally canceled in the presence of GF109203X emphasizes the
crucial role of a preliminary phosphorylation of translocatable
subunit(s) in the oxidase activation by DAG, but also the priming
effect of large amounts of DAG probably through their action on the
membrane transfer of p47
It is
worth noting that preincubation in the presence of FAs was needed for
FA potentiation of PMA-stimulated, but not DAG-stimulated O production.
We previously established that FA modulation is due to an action
located at the membrane-interacting PMA-binding site of PKC and is
probably mediated by the FA membrane-perturbing properties(16) .
It can now be advanced that PMA and DAG PKC stimulations are not
equivalent and therefore probably lead to different lipid-protein
interactions and different lipid organization around the cysteine-rich
zinc-finger-like region of the regulatory domain of PKC. This is
consistent with recent results suggesting that PMA and DAG bind at
different sites on PKC and induce distinct activated conformational
forms of the protein(40) . The discrepancy (see Fig. 7)
between both the free FA incorporation time course and the comparative
capacity of these FAs to be incorporated, on the one hand, and the
preincubation time minimally needed for potentiation by these same FAs
(except for iso15:0, which is still not available as radiolabeled
molecule), on the other, supports the view that FAs act through their
free form rather than through their phospholipid-esterified form in
cell membranes. In this respect, it is striking that 20:4n-6
was slightly incorporated into lipids and that its free form declined
from the 100% value at zero time over the whole period studied,
although the maximal potentiating effect was reached for (less than) 5
min of preincubation.
Such opposite FA effects can be considered as
a priming or a desensibilizing effect on HAMs, rendering cells more or
less reactive to mediators. They may be of utmost importance, given the
nanomolar and micromolar plasma concentrations of minor (typically
iso15:0) and major (typically 20:4n-6) free FAs, respectively,
in healthy and pathological conditions.
We thank Dr. M.-C. Billoud-Dagher and Professor V.
Vignais for the rabbit anti-peptide SPLEEERQTQRSK (residues
348-360 of p47
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(the 47-kDa cytosolic component of the
phagocyte NADPH oxidase), and the O production in human adherent
monocytes (HAMs). This was performed under phorbol myristate acetate
(PMA) or diacylglycerol stimulation after cell preincubation in the
presence of either 13-methyltetradecanoate or arachidonate. At 3 nM and 30 µM, both fatty acids had enhancing and
depressing effects, respectively, on PKC translocation and O production
strictly depending on the PMA- or diacylglycerol-stimulated state of
the cell. Endogenous phosphorylation and membrane presence of
p47
were markedly reinforced in PMA-stimulated
HAMs in the presence as compared to the absence of
13-methyltetradecanoate. These results emphasize the fact that in
intact cells the capacity of both FAs to potentiate or depress the HAM
O production is mediated by a direct action on the PKC membrane
translocation leading to a simultaneous endogenous phosphorylation and
membrane translocation of p47
. They confirm the
recent findings (Kadri-Hassani, N., Léger, C. L., and Descomps,
B.(1995) J. Lipid Med. Cell Signal. 11, 159-173) on the
PKC-mediated, adherent monocyte-specific capacity of these fatty acids
and others (with the exception of linear saturated fatty acids) to
enhance the PMA-stimulated O production at nanomolar concentrations and
to depress it at micromolar concentrations.
(
)a
phosphorylating and membrane-translocatable enzyme which plays a very
important role in cell functions, i.e. secretion,
proliferation and differentiation(1) . There are several
isozymes of PKC having four conserved and five variable regions in
their single polypeptide chain (C
-C
and
V
-V
, respectively). The N-terminal part
of the polypeptide designated as the regulatory domain is composed of
two conserved sequences functionally characterized. The C
region binds activatory lipids (DAG) or tumor promoters (PMA, for
example), whereas the C
region binds Ca
and PtdSer. Other Ca
binding sites exist and
probably depend on PKC-phospholipid interactions(2) . It is now
well established that the C
region contains two
cysteine-rich zinc-finger-like sequences probably needed for a strong
association of PKC with particular lipid-phase conformational forms of
biomembranes(3) . Moreover, the presence of at least one
cysteine-rich sequence is required for the phorbol-ester
binding(4, 5) , an assumption that is generally thought
to be extended to the binding of DAG. The PMA activation of
membrane-associated PKC does not imply an increasing penetration of
protein further into a lipid monolayer system(6) , suggesting
that activation may affect the lipid-protein interactions at the
surface of the lipid bilayer rather in the depth of it, as proposed by
the unfolding of PKC after its DAG or PMA activation at the surface of
the cytosolic leaflet of plasma membranes(3) .
and DAG (or PMA) to translocate from the cytoplasm
to the cell plasma membrane and become activated, the n-PKCs are
independent of Ca
and the a-PKCs are dependent only
on PtdSer and independent of DAG, PMA and Ca
for
activation (for exhaustive description, see Ref. 7).
, these latter being needed,
however, in the case of DAG or PMA activation, and (ii) they are able
in particular to activate the cytosolic form of PKC(11) . In a
cell-free system and in the presence of phospholipids and
Ca
, evidence for a synergistic action of DAG and
unsaturated FAs was provided(12) . The potential action of free
FAs on the different PKC isozymes was established (for review, see Ref.
7). In vivo, Lester et al.(13) demonstrated
that unsaturated FAs regulate a PKC-dependent function. Despite these
findings, free FA action remains unclear and controversial (14, 15) in physiological conditions. It is of utmost
importance to obtain more information on the ability of FAs to
translocate and therefore activate PKC in intact cells.
(the
47-kDa cytosolic phagocyte oxidase factor) are modified by iso15:0 and
20:4n-6 in order to explain the bimodal action of FAs in HAMs.
These two FAs were chosen because of their marked bimodal action and
their particular physiological significance, respectively. We concluded
that FAs act on PKC and p47
translocation and
endogenous p47
phosphorylation in a manner that
accounts for their concentration-dependent bimodal action on the
PMA-stimulated O production.
Materials
PtdSer, DAG
(1,2-didecanoyl-sn-glycerol), oleate (18:1n-9),
arachidonate (20:4n-6), histone III-S, EGTA, EDTA,
dithiothreitol, antipain, E-64, leupeptin, pepstatin, PMA, superoxide
dismutase, and the luminescent probe, lucigenin
(10,10`dimethyl-9,9`-bisacridinium dinitrate), were purchased from
Sigma (St. Quentin Fallavier, France) and 13-methyltetradecanoate
(iso15:0) from Larodan Fine Chemicals (Malmö, Sweden). Percoll was
from Pharmacia (St. Quentin en Yvelines, France); the nutritive mediums
and the fetal calf serum were from Life Technologies, Inc. (Glasgow,
Scotland). R59022 was from TEBU (Le Parray en Yvelines, France), and
GF109203X was kindly provided by Dr. J. Kirilovsky.
[-
P]ATP,
P
,
ECL-Western blotting detection reagents, anti-rabbit IgGs,
nitrocellulose membranes for hybridization transfer, and Hyper MP film
were from Amersham (Les Ulis, France). The molecular weight markers for
electrophoresis were purchased from Bio-Rad S.A. (Ivry sur Seine,
France). The rabbit polyclonal anti-peptide SPLEEERQTQRSK corresponding
to residues 348-360 of p47
was kindly
provided by M.-C. Billoud-Dagher from the Laboratoire de Biochimie
(Centre d'Etudes Nucléaires, Grenoble, France).
Preparation of Human Adherent Monocytes
Human
monocytes were isolated as reported previously(17) . Briefly,
peripheral blood was recovered from healthy volunteers by venipuncture
using heparin. Mononuclear cells were obtained by centrifugation of
blood samples over a discontinuous isotonic Percoll gradient
(1.086/1.097 gml
) in order to separate
neutrophils from monocytes. Erythrocytes were discarded by lysis, and
the mononuclear cell suspension was then washed with RPMI 1640 medium.
After evaluating monocyte number, cells (15
10
)
were incubated for 2 h at 37 °C in a humid atmosphere with 5%
CO
in Petri dishes (35
10 mm) with 10 ml of RPMI
1640 containing 20% of fetal calf serum. Dishes were washed three times
with the same medium to eliminate non-adherent cells. The purity of the
remaining HAMs was >95% as assessed by May-Grunwald-Giemsa staining.
Incubation Conditions of Adherent Monocytes
HAMs
(15 10
) were preincubated with FAs added in ethanol
(final concentration inferior to 0.1%) for 30 min (unless otherwise
indicated), and PMA or DAG was added for activation. For the
translocation assessments, supernatants were discarded after a 10- or
30-min incubation time for PMA or DAG, respectively, and HAMs were
washed twice with 5 ml of RPMI 1640 and then once with 5 ml of PBS. The
viability (>95%) was determined by trypan blue exclusion. For the
assessment of NADPH oxidase activity through the lucigenin-detected
superoxide anion production, cells (2
10
in RPMI
1640) were preincubated as above. A first measurement of
chemiluminescence (CL) was performed immediately after adding
10
M lucigenin in order to obtain the level
of O production in the absence of PMA or DAG. PMA or DAG was then added
for PKC stimulation (i.e. 30 min after adding FAs) and
incubations were routinely stopped after 60 min, the time needed for CL
peak to be reached in the presence of PMA. For a more exhaustive study
of PMA and DAG stimulation, the incubation was stopped after 4 h. When
used, the inhibitors of PKC activity were added just before PMA or DAG.
Purification of Soluble and Particulate PKC
Fractions
HAMs (15 10
) were scraped and
resuspended in 5 ml of the ice-cold buffer A (20 mM Tris/HCl,
pH 7.5, 2 mM EDTA, 10 mM EGTA, 0.3 M sucrose, 2 mM dithiothreithol, and 2 mM phenylmethylsulfonyl fluoride) containing leupeptin, E-64,
antipain, and pepstatin (50 µg/ml each) and sonicated by two 30-s
pulse treatments with 1-min interval by means of a cell disrupter
(Bronson B30 Sonic Power OSI, Paris, France). Soluble (cytosolic) and
particulate (membrane) fractions were separated by ultracentrifugation
for 40 min at 4 °C, using a Ti70 rotor at 40,000 rpm (Beckman
Instruments). The supernatant (cytosolic fraction) was kept at 4
°C, whereas the pellet (membrane fraction) was suspended in 5 ml of
buffer A containing leupeptin, E-64, antipain, and pepstatin plus 1%
Triton X-100, sonicated, and then kept for 1 h at 4 °C. Each
fraction was partially purified through a DE52 column (a siliconized
Pasteur's pipette), first using buffer B (20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, and
1 mM dithiothreithol) and then the same solution with a
100-350 mM NaCl elution gradient in order to obtain PKC
without its partially proteolyzed form PKM.
Assay of PKC
PKC was assessed through the histone
kinase activity, i.e. by measuring the incorporation of
[-
P]ATP into 0.5 mg of histone type
III-S/ml. Fractions of elution (50 µl) were simultaneously assayed
in a 96 Multiwell plate 25860B (Corning Glass Works, Poly Labo,
Strasbourg, France) for 5 min at room temperature in the presence or
absence of 1 mM CaCl
, 80 µg of PtdSer, and 8
µg of DAG/ml. The reaction was stopped after 5 min of incubation by
adding 100 µl of 40% trichloroacetic acid and 20 µl of a
solution containing 50 mM ATP and 5 mg of bovine serum
albumin/ml. The precipitated proteins were then collected on glass
fiber filters using a Skatron cell harvester (Lier, Norway).
Radioactivity retained on the filters was determined by counting in the
presence of 2 ml of scintillation fluid. PKC activity was assessed by
subtracting the results obtained in the absence of
Ca
, PtdSer and DAG from those obtained in their
presence. It was expressed as pmol of
P phosphorylating
histone III-S/min/10
cells. Total PKC activity was the sum
of those of cytosol plus membrane.
Chemiluminescence Assays
O production was
specifically measured using lucigenin as an enhancer of CL as described
previously(18, 19) . This procedure was chosen because
experiments clearly show results similar to those obtained by the
superoxide dismutase-inhibitable reduction of ferricytochrome c(17) .
Monocytes (2 P Labeling of Monocytes and Measurement
of Endogenous Phosphorylation
10
cell in 1 ml) were suspended in RPMI 1640 in the presence of 500
µCi of
P
(370 MBq/ml) and incubated at 37
°C for 30 min. They were then incubated in the same conditions but
in the presence of the tested FA. Finally, cells were stimulated by
adding 30 nM PMA in the incubation medium. After 5 min the
cells were scraped, transferred into a microcentrifugation tube
containing ice-cold 50% trichloracetic acid, and rapidly mixed. Pellets
obtained after centrifugation (12,000
g, 5 min) of
trichloroacetic acid-precipitated material were rapidly washed with 1
ml of distilled water and solubilized in 30 µl of Tris-HCl buffer
(0.01 M, pH 6.8) containing 0.01% bromphenol blue, 3% urea, 1%
dithiothreitol, and 1% SDS. After immersion in a boiling water bath for
5 min, solubilized proteins were subjected to SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) with 0.1% SDS and 12% acrylamide gel using
the Laemmli solution as buffer(20) . After staining with
Coomassie Blue, gels were destained, dried, and exposed to Hyper MP
film for 40-80 h. The films were finally scanned by means of a
Chromoscan 3 densitometer (Joyce Loebl, Gateshead, United Kingdom).
p47
Aliquots (20 µg of protein) of the membrane
fraction provided by activated HAMs were submitted to SDS-PAGE (0.1%
SDS, 10% acrylamide gel) according to the Laemmli method. Proteins were
transferred to nitrocellulose membranes using a Mini Trans-Blot
electrophoretic transfer cell apparatus (Bio-Rad) in 25 mM Tris-HCl, 192 mM glycine, and 20% (v/v) methanol. Blots
were incubated for 60 min in TBS (50 mM Tris-HCl, 130
mM NaCl, pH 7.4) containing 15% milk powder and then for 120
min in the same medium containing the rabbit anti-peptide SPLEEERQTQRSK
corresponding to residues 348-360 of p47Translocation
Assessment
.
The antipeptide antibody was used with a 1:1000 dilution. Blots were
rinsed several times in TTBS (TBS + 0.1% Tween 20) and then
incubated for 60 min at room temperature in the same medium containing
the peroxidase-labeled anti-rabbit IgGs with a 1:5000 dilution and 0.1%
milk powder. After washing, bound antibodies were revealed by enhanced
CL detection reagents. Blots were dried and exposed to Hyper MP film
for 5 min. Films were finally scanned as reported above.
Fatty Acid Incorporation in Monocyte and Lipid
Extraction
HAMs (2 10
) were incubated in 2
ml of RPMI with [
C]20:4n-6 or
[
C] 18:1n-9 (50 µCi/ml, specific
activity 55 mCi/mmol) for 30, 60, and 90 min. The incubating medium was
discarded for measuring the non-incorporated radioactivity. The
remaining cells were scraped, suspended in 1 ml of 0.9% NaCl (pH 2.8),
thoroughly agitated in 5 ml of chloroform/methanol (2:1,
v/v)(21) , and centrifuged at 3000
g for 20
min. The lower phase (total lipids) was recovered, dried under
nitrogen, and resuspended in chloroform. One part of the chloroform
solution was used for assessing the total lipid-incorporated
radioactivity. The other part was submitted to lipid class TLC
separation on plates (2.5
20 cm, with concentrating zone)
coated with Silica Gel 60 previously activated at 115 °C for 60
min. The solvent of migration was petroleum ether/diethyl ether/acetic
acid (90:10:1, v/v/v). The determination of radioactivity under the
spots representing phospholipids, free FAs, and triglycerides was
carried out by using a Chromolec 101 Numelec scanner (La
Verrière, France).
Statistical Analysis
Values were reported as mean
± S.E.. Data were analyzed using ANOVA-repeated measures.
Effect of PMA on the Translocation of
PKC
Fig. 1A shows the PKC activity of membrane
and cytosol fractions of human monocytes after different incubation
times. Before adding PMA, more than 90% of the activity was localized
in the cytosol, whereas adding 100 nM PMA brought about a
redistribution of the PKC activity resulting in a substantial increase
in the proportion of the activity recovered in the membrane fraction
(up to 80% for a 10-min incubation). This is indicative of a rapid
translocation of PKC. Fig. 1B illustrates that the
percentage of PKC activity recovered in the membrane fraction after a
10-min incubation time was only multiplied by 1.5 from 30 nM to 100 nM PMA, whereas it was multiplied by 7.9 between 0
and 30 nM, showing a saturation phenomenon near the 100
nM PMA concentration. The half-translocation was obtained for
about 30 nM. Therefore, given these results, the PMA
concentrations of 30 and 100 nM were chosen for the respective
FA potentiation and inhibition of the 10-min incubated PMA stimulating
effect on translocation.
Figure 1:
Effect
of PMA on the translocation of PKC in human adherent monocytes. The
Ca/PtdSer-dependent PKC activity was measured in
cytosolic and particulate (membrane) fractions and expressed as pmol of
P-phosphorylating histone III-S/min/10
cells.
We only show here the percent distribution of recovered activity found
in either fractions. A, distribution versus the
incubation time after adding 100 nM PMA. B,
distribution versus PMA concentration after 10-min incubation.
The total (cytosolic plus particulate) activity expressed as indicated
above was 8.0 ± 0.2 for A and 8.7 ± 0.3 for B (means ± S.E. from two separate preparations).
,
cytosolic fraction;
, particulate (membrane)
fraction.
Effect of Nanomolar and Micromolar Concentrations of
Iso15:0 or 20:4n-6 in the Presence or Absence of
PMA
Fig. 2A shows that 3 nM iso15:0
alone did not produce changes in the distribution of PKC between
membrane and cytosol compared to the control without FA. On the other
hand, adding 30 nM PMA to the preincubated iso15:0 clearly led
to an increased translocation compared to that obtained with PMA alone.
It is noteworthy that translocation with 30 nM PMA + 3
nM iso15:0 was similar to that with 100 nM PMA alone.
These data provide evidence for a 3 nM iso15:0-potentiating
action on the PKC-translocating effect of PMA, whereas iso15:0 alone
was not able to promote any translocation. Contrasting with that, 30
µM iso15:0 totally blocked the translocation provided by
100 nM PMA. Exposure of HAMs to 20:4n-6 in the same
conditions (Fig. 2B) brought about similar results.
However, 20:4n-6 appears to be less effective at enhancing
translocation at 3 nM and depressing translocation at 30
µM compared to iso15:0.
Figure 2:
Effect of nanomolar and micromolar
concentrations of either iso15:0 or 20:4n-6 on the
translocation of PMA-stimulated PKC. The PKC activity was measured and
expressed as indicated in Fig. 1. A and B, results
obtained with 30-min preincubation in the presence of iso15:0 and
20:4n-6, respectively, before adding PMA. The total (cytosolic
plus particulate) PKC activity expressed as indicated in Fig. 1 was 8.7
± 0.2 for A and 7.5 ± 0.1 for B (mean
± S.E. from three separate preparations). All conditions were
statistically (p < 0.01) different from the control, except
for the micromolar concentration of iso15:0. For more details, see the p values above the different bars.
Effect of DAG on the Translocation of PKC
Fig. 3shows the effect of increasing concentrations of
DAG on the PKC translocation after a 30-min incubation time. The
following two points need to be made. First, the concentration of DAG
for a half-translocation was approximately 10 times higher
than that of PMA (25 µMversus 30 nM);
Second, it clearly appears that the concentrations of DAG higher than
25 µM were unable to translocate more than 50% of the PKC
activity, contrasting with the almost complete translocation brought
about by PMA. R59022, an inhibitor of the DAG kinase, was used to
determine whether this incomplete translocation was due to a rapid
conversion of DAG into phosphatidic acid. If so, we would have expected
R59022 to reinforce the DAG translocation of PKC. R59022 was found to
be ineffective for promoting translocation further. On the contrary, it
totally canceled the PKC translocation (not shown).
Figure 3:
Effect of DAG on the translocation of PKC
in human adherent monocytes. For legend, see Fig. 1, PMA being replaced
by DAG. The total (cytosolic plus particulate) PKC activity expressed
as indicated was 7.7 ± 0.2 (mean ± S.E. from two separate
preparations).
Effect of Nanomolar and Micromolar Concentrations of
Iso15:0 and 20:4n-6 in the Presence of DAG
Using the same
rationale as above, we chose two concentrations of DAG, i.e. 15 and 25 µM, for testing the respective potentiating
and depressing FA effects. Fig. 4(A and B)
illustrates very clearly that the responses to iso15:0 and
20:4n-6 were similar to those obtained in the presence of PMA.
It is particularly striking that the PKC translocation responses at
both nanomolar and micromolar concentrations of 20:4n-6 were
attenuated as compared to those obtained at the same concentrations of
iso15:0. This was the same phenomenon as that typically observed for
PMA.
Figure 4:
Effect of nanomolar and micromolar
concentrations of either iso15:0 or 20:4n-6 on the
translocation of DAG-stimulated PKC. For legend, see Fig. 2. The total
(cytosolic plus particulate) PKC activity expressed as indicate was 7.7
± 0.3 for A and 7.5 ± 0.2 for B (mean
± S.E. from three separate preparations). All conditions were
statistically (p < 0.01) different from the control, except
for 15 µM DAG (p < 0.05). For more details,
see the p values above the different
bars.
Effect of Iso15:0 and 20:4n-6 on the O Production in
the Presence or Absence of PMA or DAG
Fig. 5shows
that, in the absence of PMA or DAG, neither iso15:0 nor
20:4n-6 brought about a modification in the O production.
Conversely, in the presence of PMA both FAs were able to potentiate or
depress the production at 3 nM or 30 µM,
respectively. However, it clearly appears that both the potentiating
and depressing effects were much more pronounced in the case of
iso15:0. GF109203X, a selective and potent inhibitor of PKC, totally
abolished the PMA-stimulated O production enhanced by 3 nM iso15:0 or 20:4n-6. Thus, such results correlated with
the translocation data. As shown in Fig. 6A, the peak O
production was shifted from 60 min for 5 and 10 µM DAG to
120 min for 20 µM DAG. In spite of this, 3 nM iso15:0 was able to potentiate the O production stimulated by 10
and 20 µM DAG. It was without effect in the presence of 5
µM DAG. Similar results were obtained with
20:4n-6 in the presence of 10 µM or 20 µM DAG (Fig. 6, C and D). However, when
studied in greater detail, the results showed that 20:4n-6
produced a potentiation of the effect of 20 µM DAG at 60
min, and not at 120 min as was reported for iso15:0. In order to
determine whether the last peak could be attributed to a direct effect
of DAG on NADPH oxidase, i.e. to an effect non-mediated by
PKC, we successively examined the actions of R59022 and R59022 +
GF109203X on the 10 µM DAG-stimulated O production. Fig. 6E demonstrates that R59022 considerably
potentiated the stimulating effect of 10 µM DAG as
expected by its available-DAG enhancing effect. Moreover, this action
was totally inhibited by GF109203X, providing evidence for a strictly
PKC-mediated effect of DAG. Fig. 6F also shows that the
peak PMA-stimulated O production was obtained at the same time (60 min)
as the peak production stimulated by 5 or 10 µM DAG. We
also verified (Fig. 6F) that R59022 itself did not
possess a delaying effect on the peak O production.
Figure 5:
Effect
of iso15:0 or 20:4n-6 on the lucigenin-detected superoxide
anion production in human adherent monocytes in the presence or absence
of 30 nM PMA. Data were recorded by means of an ultrasensitive
photon-counting imaging camera equipped with a computer-assisted image
processor (Argus 100; Hamamatsu Photonics, Japan). Lucigenin
(10M) chemiluminescence assays were
performed every minute in the dark at room temperature. The curve
obtained for each condition represents the number of photons/min/mg of
protein against the elapsed time. The peak of the lucigenin-CL was
reached and measured 60 min after adding 30 nM PMA, i.e. 90 min after adding FAs taking into account the 30-min
preincubation in the presence of FAs. Because each volunteer appeared
to be constitutively different for the PMA-stimulated adherent-monocyte
CL (139,200 ± 15,000, mean ± S.E., 52% of coefficient of
variation), we chose to compare the CL values obtained for a given
subject to the arbitrary value of 100 obtained in the presence of 30
nM PMA alone for the same subject. GFX, 100 nM GF109203X. The preparation number was 14 for monocytes alone,
monocytes plus PMA, and monocytes plus PMA plus 3 nM iso15:0,
and was 2 or 3 for the remaining conditions.
Figure 6:
Effect of iso15:0 or 20:4n-6 on
the lucigenin-detected superoxide anion production in human adherent
monocytes in the presence or absence of DAG. Recording, measures and
expression of results were the same as in Fig. 5. In this case,
however, the values were given for different times from 0 to 4 h after
adding DAG. A-D, the time course of the superoxide anion
production for different concentrations of DAG in the presence of 3
nM (A) and 30 µM (B) iso15:0;
of 3 nM (C) and 30 µM (D)
20:4n-6. E, the production time course for monocytes (MC) or monocytes plus 10 µM DAG in the presence
or absence of 5 µM R59022 (DAG+R) or 5
µM R59022 plus 100 nM GF109203X (DAG+R+G). F, the production time course
for monocytes (MC), monocytes plus R59022 (+R),
or monocytes plus 30 nM PMA (+PMA) in the
presence of R59022 (PMA+R), GF109203X (PMA+G), or R59022 plus GF109203X (PMA+R+G). The value of 100 on the vertical axes has
the same meaning as in Fig. 5 and corresponds to 167,500 ±
17,500 photons/min/mg of protein. Results were given from one typical
experiment in A-E and from 2 separate preparations in F.
Effect of Incubation Time on O Production and Lipid
FA Incorporation
It has already been demonstrated by our
group that the potentiating effect of certain FAs is specifically
obtained in adherent monocytes within the 3-300 nM concentration range. Therefore, in this study we used a FA
concentration of 60 nM to obtain information on the influence
of the FA preincubation time on both the O production and the FA cell
incorporation into phospholipids and triglycerides. Because
radiolabeled iso15:0 was not available for this experiment, we only
used 18:n-9 and 20:4n-6, as it can be estimated that
their physical and metabolic membrane properties are close to those of
iso15:0. Fig. 7A shows that the potentiating effect of
the FAs was not immediate since no activation took place when they were
added just before PMA. On the other hand, the potentiating effect was
about maximal from the 5th min of incubation. Such a time course
appears to be completely different from that of cell lipid
incorporation (Fig. 7B). It can also be noted (Fig. 7, C and D) that FA distribution into
phospholipid, triglyceride, and free FA fractions was not greatly
modified, except for the part of the FAs incorporated into the free FA
fraction that tended to steadily decline in relation to time, whereas
the triglyceride or phospholipid incorporation was enhanced for
20:4n-6 or 18:1n-9, respectively. Interestingly,
there was an immediately maximal FA potentiation when the O production
was stimulated by DAG (not shown), contrary to that observed for PMA
stimulation.
Figure 7:
Comparative influence of the fatty acid
preincubation time on their capacity to potentiate the
lucigenin-detected superoxide anion production and their cell
incorporation. A, superoxide anion production versus the FA preincubation time before adding 30 nM PMA; for
meaning of the vertical axes units, see Fig. 5. B, cell lipid
incorporation of 18:1n-9 and 20:4n-6 versus their preincubation time. HAMs (2 10
) were
incubated in the presence of [
C]
20:4n-6 or [
C] 18:1n-9 (50
µCi/ml, 55 mCi/mmol). An aliquot of the extracted lipids was used
for assessing the 20:4n-6 and 18:1n-9 lipid
incorporation. The other one was submitted to the TLC separation of
lipid classes. C and D, incorporation of
20:4n-6 and 18:1n-9, respectively, in phospholipid (PL) triglyceride (TG), and free fatty acid (FFA) fractions. Results were given as follows: A,
from 2 separate preparations for 18:1n-9 and 20:4n-6
and from one single preparation for iso15:0; B-D, from
one single preparation.
Enhanced Phosphorylation and Membrane Translocation of
p47
Fig. 8shows the enhanced endogenous phosphorylating
activity of monocytes following PMA stimulation after iso15:0
preincubation as compared to monocytes stimulated by PMA in the absence
of iso15:0. It also shows that the phosphorylation of the band
corresponding to p47in the Presence of Iso15:0
was enhanced. This
enhancing effect was very low in the presence of iso15:0 and absence of
PMA. On the other hand, the anti-peptide antibody directed toward the
residues 348-360 of p47
highlighted that
3 nM and 30 µM iso15:0 were able to increase and
decrease, respectively, the p47
membrane
concentration after PMA cell treatment as compared to that obtained in
the presence of PMA alone (Fig. 9). This led to the conclusion
that iso15:0 is readily able to modulate NADPH oxidase activity through
the phosphorylation-dependent modulation of p47
translocation.
Figure 8:
Phosphorylation of the HAM proteins during
stimulation by iso15:0, PMA, or iso15:0 plus PMA. A, complete
autoradiograms of P-phosphoproteins from cells in the
following conditions: a, unstimulated; b, stimulated
by 3 nM iso15:0; c, stimulated by 30 nM PMA; d, stimulated by 3 nM iso15:0 plus 30 nM
PMA; e, stimulated by 30 µM iso15:0 plus 30
nM PMA. The visualized phosphoproteins correspond to 2
10
cells for each lane. FA preincubation and PMA
stimulation lasted 30 and 5 min, respectively. B,
densitometric scans of autoradiograms; inset shows the
calibration curve for molecular weight determinations obtained with
phosphorylase b (M
94,000), albumin (M
67,000), ovalbumin (M
43,000), carbonic anhydrase (M
30,000), and
trypsin inhibitor (M
20,000). Three other
experiments showed similar results.
Figure 9:
Presence of p47phox in the membrane
fraction of HAMs in unstimulated and diversely stimulated cells. A, Western blot analysis of p47 in the particulate fraction
obtained after utracentrifugation at 40,000 rpm for 40 min. Membrane
(particulate) proteins (20 µg) were submitted to SDS-PAGE,
transferred to the nitrocellulose membrane, immunoblotted with
anti-rabbit anti-peptide (residue 348-360 of p47) antibodies, and
revealed by peroxidase-labeled anti-rabbit IgGs. Cells were: a, unstimulated; b, stimulated by 30 nM PMA; c, stimulated by 30 nM PMA plus 30 µM
iso15:0; d, stimulated by 30 nM PMA plus 3 nM iso15:0. B, densitometric scans obtained for each
condition. Two other experiments showed similar
results.
. They fully account for the
concentration-dependent bimodal action of these two FAs and others
(with the exception of linear saturated ones) on the PKC-mediated O
production that we recently established in HAMs(16) . They are
in line with the literature reporting the implication of PKC in the
activation of the respiratory burst in neutrophils (22, 23, 24) and the capacity of the cytosolic
p47
to be an endogenous phosphorylation
substrate for PKC(25, 26) , this phosphorylation being
in its turn definitely involved in the p47
translocation from cytosol to plasma membrane and in the
resulting activation of the assembled multicomponent NADPH oxidase
complex(27) . The above explanation is also supported by the
fact that (i) we were unable to highlight any direct effect of either
iso15:0 or 20:4n-6 on the NADPH oxidase activity as shown by
the absence of O production without PMA or with both PMA and GF109203X,
and (ii) both FAs were totally unable to modulate the translocation of
PKC in the absence of PMA or DAG. In this respect, it is also worth
noting that iso15:0 alone did not produce any appreciable action on the
endogenous phosphorylation of p47
as compared
with the basal condition, contrary to what was found in the presence of
PMA.
- and
-isotypes and not for the
-isotype of c-PKC(34) . In
the intact cells studied until now, only a synergistic FA stimulation
of PKC activity has clearly been shown with
DAG(13, 35) . This is, to some extent, in line with the
present results.
), or (ii) in the lipid
environment of PKC and/or NADPH oxidase in the plasma membrane remains
to be addressed.
and p67
(another translocatable subunit of NADPH oxidase) as already
described in neutrophils on a cell-free system(39) .
) polyclonal antibody.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.