 |
INTRODUCTION |
The structure of the arterial wall and its vasoactive properties
are mainly supported by vascular smooth muscle cells, which represent a
prominent cell type in this tissue. These cells are involved in major
pathological conditions such as septic shock, neointima formation, and
restenosis after acute vascular injury or chronic pathological
processes such as atherosclerosis.
During severe infection, activation of inflammatory cells leads to the
massive release of proinflammatory cytokines, including interleukin-1
(IL-1)1
and tumor necrosis factor-
(1). During the onset of atherosclerosis, vascular smooth muscle cells undergo phenotypic changes switching from
a contractile phenotype to a proliferating and secretory phenotype.
Early atherosclerotic lesions have many characteristics in common with
the inflammatory reaction (2), and vascular smooth muscle cells
participate in the local inflammatory process involved in the
development of lesions and neointimal formation (3). IL-1 is thought
to play an important role in this process since vascular smooth muscle
cells express IL-1 receptors (4). Furthermore, in vascular smooth
muscle cells, IL-1 induces expression of intercellular adhesion
molecule-1 and vascular cell adhesion molecule-1 (5) and IL-1 itself
(6). IL-1 also stimulates extracellular matrix production by human
smooth muscle cells (7).
Type II secreted phospholipase A2 (type
II-sPLA2) has been implicated in the pathophysiological
processes of sepsis syndrome as well as atherosclerosis. High levels of
type II-sPLA2 have been found in the plasma of patients
with septic shock (8). In addition, high levels of this enzyme have
been demonstrated in the aorta of endotoxin shock rats (9). Type
II-sPLA2 has been evidenced in human atherosclerotic
plaques (10). A strong type II-sPLA2 immunoreactivity
colocalized with -actin-positive vascular smooth muscle cells in
both normal and atherosclerotic arteries (11). It has been suggested
that type II-sPLA2 could play an important role in early
atherosclerosis because it is present in the pre-atherosclerotic
arterial wall, where it may induce low density lipoprotein modification
(12) and foam cell formation (13).
The precise physiological substrate of type II-sPLA2 is
still conjectural. Although only indirect evidence has been published to indicate that type II-sPLA2 can hydrolyze mammalian cell
phospholipids in vivo to generate lipid mediators (14-16),
this enzyme seems involved in the specific synthesis of
lysophosphatidic acid, a potent growth-promoting factor (17), and is
inhibited by sphingomyelin, an important component of the outer leaflet
of plasma cell membranes (18). Vascular smooth muscle cell
proliferation is implicated in the pathogenesis of atherosclerosis and
restenosis following interventional revascularization procedures (19).
Several phospholipase A2-derived mediators in addition to
lysophosphatidic acid are thought to be involved in smooth muscle cell
proliferation, i.e. lysophosphatidylcholine (20),
cyclooxygenase, and lipoxygenase products (21) or arachidonic acid
itself (22) and might mediate the proliferative effect of IL-1
(23).
IL-1 induces the synthesis and secretion of type
II-sPLA2 in various cell types (14, 15), as well as in
vascular smooth muscle cells (24), but the precise signaling leading to
type II-sPLA2 gene expression remains to be elucidated. In
most cells, IL-1 triggers its action by at least two main pathways
involving specific receptors. IL-1 activates acid sphingomyelinase
to initiate the ceramide cascade (25). IL-1 also induces signaling
cascades which activate members of the nuclear transcription factor
B (NFB) family in smooth muscle cells (26). NFB activation has also been evidenced in vascular smooth muscle cells after rat arterial
injury (27) or in human arteriosclerosis (28). In arterial smooth
muscle cells, IL-1-induced type II-sPLA2 gene expression
can therefore be expected to involve either the ceramide pathway or the
NFB pathway. In most cells, NFB is retained in the cytoplasm by
inhibitory proteins called IBs. Signaling events that phosphorylate
IBs promote its degradation by the proteosome pathway and the
subsequent nuclear translocation and activation of NFB family
members (29). A cytokine-responsive IB kinase has been described
(30), but the actual pathway by which IL-1 might induce
phosphorylation of IBs is still unknown.
Recently, cytosolic phospholipase A2 (cPLA2),
an enzyme completely unrelated to type II-sPLA2 (31), has
been implicated in IL-1-mediated type II-sPLA2 gene
induction in rat fibroblasts (32). This raises the possibility of a
control of type II-sPLA2 gene by free fatty acids or their
derivatives via peroxisome proliferator-activated receptors (PPARs).
PPARs are key players in lipid metabolism and are members of the
nuclear receptor superfamily of transcription factors that regulate the
pattern of gene expression in response to the binding of low molecular
weight ligands (33). Three different subtypes have been described,
PPAR, PPAR/
, and 
. Although the identities of the ligands
that regulate in vivo activity remain to be established with
certainty, 15-deoxy-
12,14-prostaglandin J2
(15-dPGJ2) (34), 13- or 9-hydroxyoctadecadienoic acid
(HODE) (35), and certain polyunsaturated fatty acids (36) have been
demonstrated to stimulate PPAR-dependent transcription. Both PPAR and PPAR are present in smooth muscle cells (37, 38),
but there are conflicting views concerning their actual effect on the
development of atherosclerosis (37, 39, 40).
In the present paper, we demonstrate that, in rat vascular smooth
muscle cells, IL-1 induces type II-sPLA2 gene via the
NFB pathway but not via the ceramide pathway. It can be mimicked by arachidonic acid as well as PPAR agonists and blocked by
cPLA2 inhibitors and therefore involves the
cPLA2-PPAR pathway. In addition, nuclear translocation
of NFB is required for the stimulation by PPAR agonists,
suggesting that both NFB and PPAR are required to mediate the
IL-1 effect.
| |
MATERIALS AND METHODS |
Reagents--
Type I collagen from calf skin, glutamine,
penicillin, streptomycin, free fatty acid bovine serum albumin, and
Naja mossambica type II-sPLA2 were purchased
from Sigma, Saint Quentin Fallavier, France. Fetal calf serum was from
Roche Molecular Biochemicals. Antibodies against smooth muscle cell
-actin from hybridoma cells, clone 1A4, were from Dako S.A.,
Copenhagen, Denmark. Murine mammary lentivirus reverse
transcriptase and Random Primers were from Life Technologies, Inc., and
oligonucleotides were from Oligo Express; France. N+ nylon membranes,
ECL direct nucleic acid labeling system, and ECL reagents kit for
horseradish peroxidase were from Amersham Pharmacia Biotech.
Fluorescent substrate
1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphoglycerol was from Interchim, Montluçon, France. IL-1 was
purchased from Immugenex Corp. Carbaprostacyclin,
9-hydroxyoctadecadienoic acid (9-HODE),
15-deoxy-12,14-prostaglandin J2
(15-dPGJ2) were from Cayman Chemical. Bromoenol lactone
(BEL), arachidonyltrifluoromethyl ketone (AACOCF3) and Z-Ile-Glu-(Ot-Bu)-Ala-leucinal (Z-AL) were from Calbiochem. Arachidonic acid, N-acetyl-Leu-Leu-norleucinal (ALLN),
9-cis-retinoic acid, indomethacin,
5,8,11,14-eicosatetraynoic acid (ETYA), nordihydroguaiaretic acid
(NDGA), and cycloheximide were from Sigma. The protein kinase C
inhibitor GF109203X was a gift from Glaxo-Wellcome, Les Ulis, France.
LipofectAMINE was from Life Technologies, Inc.; the
pCMV--galactosidase plasmid was from CLONTECH
Laboratories, and the luciferase reporter assay kit was from Promega, France.
Isolation and Culture of Rat Aortic Smooth Muscle
Cells--
Vascular smooth muscle cells were isolated by enzymatic
digestion of thoracic aortic media from male Wistar rats (300 g,
Elevage Janvier) according to the method of Michel and co-workers (41). The cells were seeded on dishes coated with type I collagen from calf
skin and were cultured in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal calf serum, 4 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin. The purity of the
smooth muscle cell preparation was evaluated by staining the cells with
monoclonal antibodies to smooth muscle cell -actin. More than 96%
of cells revealed immunoreactivity. Smooth muscle cells were
subcultured every 7 days, and experiments were performed on cells at
three to six passages after primary culture.
After confluence of the cells, quiescent mode was induced by incubation
for 24 h in serum-free medium and then incubated with the same
medium containing 0.2% free fatty acid bovine serum albumin and
appropriate agents as described in the figure legends. At the end of
incubation, the medium was removed for use in the phospholipase A2 assay, and the cells were treated for RT-PCR analysis,
protein determinations, or nuclear extract preparations.
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)--
Total RNA was extracted according to Chomczynski and
Sacchi (42), and 1.5 µg was used as template for the reverse
transcription (RT) reaction. First strand cDNA was synthesized
using reverse transcription reaction with murine mammary lentivirus
reverse transcriptase and random primers. For semi-quantitative
polymerase chain reaction (PCR) determinations, sPLA2
cDNA was coamplified with GAPDH cDNA as an internal control,
and the linear amplification was determined for each experiment
(currently 20-24 cycles). The primers were synthesized according to
published sequences for rat sPLA2 cDNA (43) corresponding to
nucleotides 155-438 from start codon and rat GAPDH cDNA (44)
corresponding to nucleotides 305-499 from start codon. The primers
used for type II-sPLA2 were GTG GCA GAG GAT CCC CCA AGG (CS
10, forward) and GCA ACT GGG CGT GTT CCC TCT GCA (CS 11, reverse), and
those used for GAPDH were CCA TGG AGA AGG CTG GGG (GS, forward) and CAA
AGT TGT CAT GGA TGA CC (GAS, reverse).
The following conditions were chosen as standard conditions for PCR
reactions in a volume of 25 µl: 2.5 µl of cDNA template generated from RT reactions, 1.25 units of Taq DNA
polymerase, 20-24 cycles of amplification in the presence of 160 nM CS 10 and CS 11 primers, 120 nM GS and GAS
primers. PCR amplifications were performed using a thermocycler (Hybaid
Omnigene) as follows: a denaturation step 3 min at 95 °C and then
subsequent cycles of PCR using the following conditions: denaturation 1 min at 95 °C, annealing 1 min at 64 °C, extension 1 min at
72 °C. A final extension for 4 min at 72 °C was then performed.
The PCR products (5 µl each sample) were electrophoresed using 2%
agarose gel, blotted, and fixed onto a Hybond N+ nylon membrane. The
identity of amplified cDNA products was confirmed by hybridization with 5'-CAA CCG TCT GGA GAA ACG TGG ATG TGG CAC-3' (nucleotides 216-245 from ATG) for sPLA2 and 5'-GTG AAC CAC GAG AAA TAT
GAC AAC TCC CTC-3' (nucleotides 397-426 from ATG) for GAPDH. The
oligonucleotide probes were labeled using ECL direct nucleic acid
labeling system. After hybridization, membranes were washed, revealed
using ECL reagents kit for horseradish peroxidase, and autoradiographed.
Phospholipase A2 Assay--
Phospholipase
A2 activity was measured using a selective fluorometric
assay as described by Pernas et al. (45). The activity secreted into the medium was assayed on 400-µl samples using 4 nmol
of fluorescent substrate
1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphoglycerol. Total hydrolysis of the substrate obtained by 0.1 unit of phospholipase A2 from N. mossambica was used as a reference to
calculate phospholipase A2 activity of the samples.
Spontaneous hydrolysis of the substrate was assayed in the presence of
fresh culture medium and subtracted.
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assay (EMSA)--
Nuclear extracts were prepared from smooth
muscle cells by the method of Dignam et al. (46) with minor
modifications. After washing in 5 ml of ice-cold phosphate-buffered
saline, cells were harvested and centrifuged at 1000 × g for 5 min. The cell pellet was resuspended in 500 µl of
buffer A (5 mM Hepes, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5% Nonidet P-40, 50 mM NaF, 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin).
Cell lysates were maintained for 10 min on ice and centrifuged at
3000 × g for 10 min. The nuclear pellet was then
resuspended in 100 µl of buffer B (20 mM Hepes, pH 7.9, 25% glycerol, 0.5 M NaCl, 1.5 mM
MgCl2, 0.5 mM EDTA, 50 mM NaF, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin). After 30 min at 4 °C, nuclear debris
was removed by centrifugation at 45,000 × g for 30 min, and the supernatant (nuclear extract) was distributed into 15-µl
aliquots that were stored at 
80 °C until analysis by EMSA. Protein
concentration was determined by the method of Lowry modified by
Peterson (47).
The B double-stranded oligonucleotides corresponded to either a,
NFB-binding site consensus sequence (48) 5'-GGG ACA GAG GGG ACT TTC
CGA GAG G-3' (NFB consensus) or the sequence 5'-GTA TGA GGG CTT TTC
CCT CGC CCT-3' (NFB sPLA2) corresponding to the region (194,
174) of the rat type II-sPLA2 promoter described by
Walker et al. (49). The PPRE double-stranded
oligonucleotides corresponded to a PPAR-binding site consensus sequence
from acyl-CoA oxidase gene 5'-GGG AAC GTG ACC TTT GTC CTG GTC CC-3'
(PPRE consensus) (50) and to the sequence (160, 133) of the rat
type II-sPLA2 promoter (51) 5'-C AGG CCT GTT GGG GGG AAA
AGG GGA AAT T-3' (PPRE sPLA2) presenting extensive homology
with the DR1 element of the PPAR-binding site. They were annealed and
end-labeled using the T4 polynucleotide kinase in the presence of 50 µCi of [-32P]dATP. Unincorporated nucleotides were
removed using G-50 filtration. Binding reactions were carried out in a
20-µl binding reaction mixture (10 mM Hepes, pH 7.9, 50 mM NaCl, 1 mM dithiothreitol, 10% glycerol,
0.2% Nonidet P-40, 0.5 mM EDTA) containing 7 µg of
nuclear proteins and 50,000 cpm labeled (approximately 1 ng) with
either the NFB probe or the PPRE probe. Samples were incubated at
room temperature for 15 min and fractionated by electrophoresis on 5%
denaturing polyacrylamide gel in 0.25× TBE (45 mM Tris
borate, 1 mM EDTA), after pre-electrophoresis for 30 min at
180 V. Gels were run at 180 V for 3 h and then transferred to 3MM
paper (Whatman), dried in a gel dryer under vaccum at 80 °C, and
then exposed to Amersham Pharmacia Biotech x-ray film. To determine the
specificity of the DNA protein complexes, competition assays were
performed using a 100-fold molar excess of an unlabeled double-stranded oligonucleotide corresponding to each of the probes (specific inhibitors) or a 100-fold molar excess of a double-stranded
oligonucleotide corresponding to an AP1-binding site consensus sequence
(52), 5'-GGG AGC CGC AAG TGA GTC AGC GCG GGG CTG GTG CA-3' (nonspecific competitor).
Transfection and Luciferase Assays--
Twenty four hours before
transfection, cultured rat smooth muscle cells were seeded at a
concentration achieving 70% confluence in 6-well dishes. Smooth muscle
cells were cotransfected using 4 µl of LipofectAMINE, 800 ng of a
plasmid construct containing the (PPRE consensus)×2 thymidine kinase
promoter region fused to luciferase reporter gene (kindly provided by
Walter Wahli) and 200 ng of pCMV--galactosidase per well (as a
control of transfection efficiency). The reaction was carried out as
recommended by the manufacturer (Life Technologies, Inc.). On the day
after transfection, cells were washed twice with phosphate-buffered
saline and subsequently cultured for 24 h in serum-free medium and
incubated for 24 h in the same medium containing appropriate
agents, as indicated in the figure legends.
The -galactosidase activity in cell extracts was determined by an
enzymatic assay with orthonitrophenyl phosphate as substrate. The
luciferase activity was measured with a luciferase reporter assay kit,
with detection of the signal for 12 s by a luminometer (Berthold,
Inc.)
GC/MS Measurements--
The non-esterified fatty acids were
extracted and analyzed after methylation with diazomethane and
separated by gas chromatography on a capillary column of Supelcowax-10
(Supelco)-bonded phase (diameter 0.32 mm, length 30 m) in a
Hewlett-Packard 5890 Series II gas chromatograph as already described
(18). Fatty acids were detected by mass spectrometry (Nermag 10-10C)
in the chemical ionization mode with ammonia (10 kPa) as the reagent
gas. The positive quasi-molecular ions were monitored and
time-integrated. Quantification was performed with an internal standard
of heptadecanoic methyl ester with response factors calculated for the
various fatty acid methyl esters with calibrators.
Statistical Analysis--
The data illustrated in Table I were
analyzed statistically using a Student's t test.
| |
RESULTS |
Time Course and Dose-dependent Effect of IL-1 on
Type II-sPLA2 Gene Expression in Vascular Smooth Muscle
Cells--
In rat aortic smooth muscle cells, as in other cells in
primary culture, type II-sPLA2 gene is not expressed under
resting conditions. Interleukin 1 has been shown to induce type
II-sPLA2 gene expression in these cells (24). In order to
identify the signal transduction events involved in IL-1-mediated
type II-sPLA2 gene induction, we first characterized the
kinetic parameters of its induction. Induction, which was nil at the
onset of IL-1 addition, progressively increased with incubation
time, as evidenced by RT-PCR analysis of type II-sPLA2
mRNA and by measurement of sPLA2 activity in the
supernatant (Fig. 1). After addition of 10 ng/ml IL-1, the presence of type II-sPLA2 mRNA
became obvious after 2 h and reached a maximum at 24 h (Fig.
1B). In contrast, the enzymatic activity was barely detected
in the supernatant during the first 6 h and then increased
linearly until 24 h (Fig. 1A). This result suggests
that, in smooth muscle cells, as in chondrocytes (15), the secretion of
type II-sPLA2 is delayed after mRNA and protein
synthesis. Type II-sPLA2 mRNA can be evidenced 6 h
after addition of as little as 0.1 ng/ml IL-1, but the enzymatic activity is only detectable with 10 ng/ml IL-1 at this incubation time (Fig. 2). Stimulation by 10 ng/ml
IL-1 was nearly maximal, and maximal stimulation was reached for 100 ng/ml. Most of the following experiments were therefore performed using
10 ng/ml IL-1 for 6 or 24 h of stimulation.
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Fig. 1.
Time course of type II-sPLA2 gene
induction by IL-1 in VSMC. Serum-starved
cells were incubated in DMEM in the presence ( , +) or absence ( ,
) of 10 ng/ml IL-1 for various times. A, phospholipase
A2 activity was determined in the extracellular medium by
spectrofluorimetric assay (see "Materials and Methods"). Maximal
activity obtained after 24 h was 2.7 ± 0.5 mmol/min/mg
protein and is expressed as 100% in the figure. Results are means ± S.D. from three independent experiments performed in triplicate.
B, the cells were washed at the end of each incubation time,
and total RNA was extracted for mRNA analysis by RT-PCR as
indicated under "Materials and Methods." A representative
autoradiogram of three independent experiments is shown.
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Fig. 2.
Dose-response of type II-sPLA2
gene induction by IL-1 in VSMC.
Serum-starved cells were incubated for 6 h in DMEM containing
increasing concentrations of IL-1 (0.1 to 200 ng/ml). A,
phospholipase A2 activity was measured as in Fig. 1, and
results are expressed in pmol/min/ml and represent the mean of two
independent experiments. B, RT-PCR analysis of mRNA as
indicated in Fig. 1. A representative autoradiogram of three
independent experiments is shown.
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Effect of Actinomycin D and Cycloheximide on IL-1-induced Type
II-sPLA2 Gene Expression in Vascular Smooth Muscle
Cells--
To investigate the mechanism by which IL-1 elicits type
II-sPLA2 mRNA in aortic smooth muscle cells, these
cells were treated for 6 h with 10 ng/ml IL-1 in the presence
or absence of the protein synthesis inhibitor cycloheximide or the
transcriptional inhibitor actinomycin D. IL-1-induced type
II-sPLA2 gene expression was inhibited by 5 µg/ml
actinomycin D, as measured by either RT-PCR analysis of cellular RNA or
enzymatic activity in the supernatant (result not shown). This confirms
that synthesis of type II-sPLA2 mRNA is a prerequisite
to secretion of the enzyme. In contrast, cycloheximide alone induced a
type II-sPLA2 mRNA increase (Fig. 3), as observed for the expression of
other genes (53). Furthermore, when IL-1 was coincubated for 6 h with cycloheximide, we observed a dramatic increase of type
II-sPLA2 mRNA as compared with IL-1 alone. This is
in contrast to the situation observed in chondrocytes (15), in which
cycloheximide inhibited the IL-1 effect on type II-sPLA2
gene expression. This result indicated that the action of IL-1 does
not need ongoing protein synthesis in smooth muscle cells. On the
contrary, cycloheximide could inhibit a labile repressor molecule whose
disappearance allows either a greater efficiency of IL-1 on gene
transcription or an increased stability of type II-sPLA2
mRNA.
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Fig. 3.
Cycloheximide effect on type
II-sPLA2 mRNA in VSMC.
Serum-starved cells were incubated for 6 h in DMEM in the presence
(lanes 3 and 4) or absence (lanes 1 and 2) of 10 ng/ml IL-1 and in the presence (lanes
2 and 4) or absence (lanes 1 and
3) of 10 µg/ml CHX. CHX was added 30 min before the
addition of IL-1. RT-PCR analysis of mRNA is as indicated in
Fig. 1. A representative autoradiogram of three independent experiments
is shown.
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Exploration of the Involvement of NFB in IL-1-induced Type
II-sPLA2 Gene Expression--
In rat mesangial cells,
Walker et al. (49) have previously found that NFB is an
essential component of the IL-1-dependent up-regulation
of type II-sPLA2 gene transcription. We therefore used
electrophoretic mobility shift assay (EMSA) to assess whether NFB is
involved in IL-1 responses in rat aortic smooth muscle cells. The
nuclear extracts from untreated cells gave one major complex with
labeled oligonucleotides bearing the B site. This was obtained using
oligonucleotides bearing either NFB consensus or NFB site of type
II-sPLA2 promoter (NFB sPLA2) (Fig.
4). Nuclear extracts from smooth muscle
cells treated by 10 ng/ml IL-1 for 24 h gave much more intense
complexes than those from control cells. These complexes were competed
out by an excess of the corresponding unlabeled oligonucleotide but not
by an excess of oligonucleotide bearing the AP1 site as nonspecific
control (result not shown). Translocation of NFB to the nucleus
results from degradation of IB inhibitory proteins by the proteasome machinery (54). We therefore tested the effect of proteinase inhibitors
known to block proteasome activity. The specific proteasome inhibitor
Z-IE(O-t-butyl)A-leucinal (ZAL) (Fig. 4) or the calpain inhibitor I N-acetyl-Leu-Leu-norleucinal (ALLN) (not shown)
completely blocked the IL-1-induced increase of NFB binding.
These two inhibitors also induced dose-dependent inhibition
of the induction of type II-sPLA2 gene expression by
IL-1 (Fig. 5, A and
B). Nuclear NFB translocation and type
II-sPLA2 gene induction were completely blocked by 500 nM ZAL and 12 µM ALLN, concentrations in the
range of those used to inhibit proteasome activities.
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Fig. 4.
Effect of IL-1 and
proteinase inhibitors on nuclear translocation of
NFB in VSMC. Nuclear extracts were
prepared from untreated cells and from cells treated for 24 h by
10 ng/ml IL-1 as indicated under "Materials and Methods." When
required, proteinase inhibitors were added 1 h before IL-1.
Electromobility shift assays were performed to evidence NFB binding
using probes bearing either an NFB consensus sequence (lanes
1-5) or an NFB-binding site described on the type
II-sPLA2 promoter (lanes 6-8) as indicated
under "Materials and Methods." Labeled probes were incubated with
nuclear extracts from untreated cells (lanes 1 and
6) or from IL-1-treated cells (lanes 2-5 and
7-8) in the presence of 1 µM ZAL (lane
4) or 5 µM quinacrine (lane 5). A large excess
(×100) of unlabeled B probe was used to validate the specificity of
binding (lanes 3 and 8).
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Fig. 5.
Effect of proteinase inhibitors on type
II-sPLA2 gene induction by IL-1 in
VSMC. Cells were preincubated for 1 h with increasing
concentrations of ALLN (A) or ZAL (B) and then
stimulated with 10 ng/ml IL-1 for 6 h. Cellular mRNA was
assessed by RT-PCR as indicated in Fig. 1. Representative results of
three (A) and two (B) independent experiments are
shown. Numbers given in the frame are arbitrary units
indicating inhibitory effect of each dose calculated as the percentage
of each sPLA2 mRNA/GAPDH mRNA ratio obtained by densitometer
scanning relative to the ratio obtained by IL-1 stimulation without
inhibitors.
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Arachidonic acid has been suggested to mediate nuclear NFB
translocation under certain experimental conditions (55). To examine
whether this mechanism occurs in smooth muscle cells, we tested the
ability of quinacrine, a broad phospholipase A2 inhibitor,
to suppress the IL-1-induced nuclear translocation of NFB.
Treatment of the cells by 5 µM quinacrine for 24 h,
inducing inhibition of IL-1-stimulated type II-sPLA2 gene
expression, did not prevent the increase in NFB-DNA binding elicited
by IL-1 in smooth muscle cells (Fig. 4).
Exploration of the Sphingomylinase/Ceramide Pathway and the
Cytosolic PLA2 Pathway in IL-1-induced Type
II-sPLA2 Gene Induction--
Several authors (25) have
implicated the sphingomyelinase/ceramide pathway in the transcriptional
effect of IL-1. To determine whether this pathway is relevant in
IL-1-induced type II-sPLA2 gene induction, aortic smooth
muscle cells were treated with bacterial sphingomyelinase and
cell-permeable ceramide, N-acetylsphingosine (C2-ceramide). RT-PCR analysis of cellular mRNA did not
evidence any increase in type II-sPLA2 mRNA in cells
treated for 6 h by 10 units/ml sphingomyelinase or either 1 or 10 µM C2-ceramide (results not shown).
Recently, the specific cytosolic-PLA2 inhibitor,
arachidonyltrifluoromethyl ketone (AACOCF3), was shown to
block IL-1-induced type II sPLA2 gene induction in rat
fibroblasts (32). We therefore tested the ability of IL-1 to
mobilize fatty acids from phospholipids in aortic smooth muscle cells
by gas chromatography/mass spectrometry (GC/MS) measurement of cellular
free fatty acids (Table I). After 6 h, IL-1 induced a marked increase in cellular unsaturated free fatty
acids as follows: free oleic acid was increased 1.7-fold, free linoleic
acid was increased 10-fold, and free arachidonic acid was increased
2.5-fold. This increase was completely blocked by AACOCF3
(Table I). We then investigated the effect of several phospholipase
A2 inhibitors on IL-1-induced type II-sPLA2
gene expression. The nonspecific phospholipase A2
inhibitor, quinacrine, as well as AACOCF3
dose-dependently blocked the IL-1-induced increase in
type II-sPLA2 mRNA (Fig.
6, A and B).
Complete inhibition was achieved for concentrations (5 and 20 µM, respectively) known to inhibit phospholipase
A2 activities and smooth muscle cell proliferation (22).
Conversely, bromoenol lactone (BEL), a specific inhibitor of the
calcium-independent phospholipase A2, only slightly inhibited IL-1-induced type II-sPLA2 mRNA increase
at a very high concentration (Fig. 6, lane 9).
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Table I
cPLA2-mediated stimulation by IL-1 of intracellular free
unsaturated fatty acids
Cells were incubated in serum-free DMEM for 24 h with or without
10 ng/ml IL-1 in the presence or in the absence of 20 µM AACOCF3. AACOCF3 was added 1 h
before stimulation with IL-1. Free fatty acids were extracted and
quantified by GC/MS as described under "Materials and Methods."
Values are means ± S.D. for three determinations. Results are
expressed as nnmol/dish.
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Fig. 6.
Inhibition of IL-1
induction of type II-sPLA2 by cPLA2
inhibitors and MEK inhibitor in VSMC. Serum-starved cells were
preincubated for 1 h with increasing concentrations of
AACOCF3 (lanes 3-5), quinacrine (lanes
6-8), BEL (lane 9), and PD98059 (lane 10)
and then stimulated for 24 h with 10 ng/ml IL-1 (lanes
2-10). Control experiment in lane 1. Cellular mRNA
was analyzed by RT-PCR as indicated in Fig. 1. A,
autoradiogram displaying a representative experiment of the three
experiments performed. B, quantification of the inhibitory
effect by densitometer scanning of the sPLA2 mRNA/GAPDH mRNA
ratio. Results are given as a percentage of the stimulatory effect
obtained by IL-1 stimulation without inhibitors and are the
mean ± S.E. from two experiments performed in triplicate.
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Activation of cPLA2 was found to associate membrane
translocation of the enzyme and phosphorylation at serine 505 by a MAP kinase (MAPK)-mediated process, and alternative pathways might also
involve protein kinase C (56). We therefore tested the effects of
inhibition of these two pathways on IL-1-induced type II-sPLA2 gene induction. Treatment of the cells by various
concentrations (10, 100, and 500 nM) of the protein kinase
C inhibitor GF109203X had no effect on IL-1-induced type
II-sPLA2 mRNA increase (result not shown). This result
ruled out any effect of the main protein kinase C species on
IL-1-induced type II-sPLA2 gene induction. In most
cells, MAPK is activated by a protein kinase cascade including MEK, a
MAP kinase kinase and Raf-1, a MAP kinase kinase kinase, in which MAPK
is directly phosphorylated and activated by MEK. Treatment of smooth
muscle cells by the specific MEK inhibitor PD98059 leads to marked
inhibition of IL-1-induced increase in type II sPLA2
mRNA (Fig. 6, lane 10), suggesting that the MAPK cascade
may well be involved in IL-1-induced type II-sPLA2 gene transcription.
Effects of Ligands of Peroxisome Proliferator-activated Receptors
(PPARs) and Retinoid X Receptor (RXR) on Type II-sPLA2
mRNA Gene Expression--
Among the products of
cPLA2-induced hydrolysis of cellular phospholipids,
polyunsaturated free fatty acids have been shown to regulate the
transcriptional activity of several genes. It has also been suggested
that lysophosphatidylcholine (LPC) might regulate type
II-sPLA2 gene in rat fibroblasts (20). This LPC effect was
never observed in rat aortic smooth muscle cells under our experimental
conditions (result not shown). Since IL-1 increased free arachidonic
acid in smooth muscle cells (Table I), we tested the ability of this
fatty acid to induce the type II-sPLA2 gene. We found a
marked dose-dependent increase in type II-sPLA2
mRNA when cells were treated with free arachidonic acid (Fig.
7A). This effect was
reproduced by the non-metabolizable analogue, eicosatetraynoic acid
(ETYA), at the same concentrations (Fig. 7A). However, the
actual physiological inducer produced by IL-1-stimulated cytosolic-PLA2 activity might not be arachidonic acid
itself, since nordihydroguaiaretic acid (NDGA), a well known
lipoxygenase inhibitor, was also able to inhibit the IL-1 effect in
smooth muscle cells (Fig. 7B). In contrast, the
cyclooxygenase inhibitor, indomethacin, did not affect the
IL-1-induced response.
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Fig. 7.
Effect of arachidonic acid, ETYA, and
inhibitors of lipoxygenase and cyclooxygenase on type
II-sPLA2 gene expression in VSMC. mRNA was
analyzed by RT-PCR as indicated in Fig. 1. The autoradiogram displays a
representative experiment of the two experiments performed in
triplicate. A, cells were incubated in DMEM for 24 h in
the absence (lane 1) or in the presence of IL-1
(lane 2), ETYA (lanes 3 and 4), or
arachidonic acid (lanes 5 and 6).
Numbers given in the frame are arbitrary units indicating
stimulatory effect of the various agonists calculated as the increase
of each sPLA2 mRNA/GAPDH mRNA ratio obtained by densitometer
scanning relative to the ratio obtained without stimulation.
B, cells were preincubated for 1 h with NDGA
(lanes 2 and 3) or indomethacin (lanes
4 and 5) or vehicle alone (lane 1) and were
stimulated by 10 ng/ml IL-1 (lanes 1-5).
Numbers given in the frame are arbitrary units indicating
inhibitory effect of each inhibitor calculated as the percentage of
each sPLA2 mRNA/GAPDH mRNA ratio obtained by densitometer
scanning relative to the ratio obtained by IL-1 stimulation without
inhibitors.
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The transcriptional effect of fatty acids or their derivatives was
thought to be mediated by peroxisome proliferator-activated receptors
(PPARs) (34). We therefore investigated the ability of several putative
PPAR ligands to reproduce the transcriptional effect of IL-1. We
tested the effect of
15-deoxy-12,14-dehydro-prostaglandin J2
(15-dPGJ2) and 9-hydroxy-octadecadienoic acid (9-HODE),
which are activators of PPAR, clofibric acid, and oleic acid and
which are activators of PPAR and carbaprostacyclin, a stable
analogue of prostaglandin I2, which is an activator of PPAR and PPAR / (Fig. 8).
Treatment of smooth muscle cells with both 15-dPGJ2 and
9-HODE induced a dose-dependent increase in cellular type
II-sPLA2 mRNA (Fig. 8, lanes 3-7). The
concentrations used are in the range of those known to stimulate
PPAR activation (34, 35). Neither clofibric acid nor
oleic acid induced type II-sPLA2 gene expression (Fig. 8,
lanes 10 and 11). In contrast, carbaprostacyclin
induced the gene (Fig. 8, lanes 8 and 9)
suggesting that PPAR/ but not PPAR may also transactivate.
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Fig. 8.
Effect of various PPAR ligands and
9-cis-retinoic acid on type II-sPLA2 gene
expression. Cells were incubated in serum-free DMEM for 24 h
in the absence (lane 1) or in the presence of IL-1
(lane 2), 15-dPGJ2 (lanes 3 and
4), 9-HODE (lanes 5-7), carbaprostacyclin
(lanes 8 and 9), clofibric acid (lane
10), oleic acid (lane 11), and
9-cis-retinoic acid (lane 12). Cellular mRNA
was analyzed by RT-PCR as indicated in Fig. 1. The autoradiogram
displays a representative example of the two experiments performed in
triplicate. Numbers given in the frame are arbitrary units
indicating the stimulatory effect of the various agonists calculated as
the increase of each sPLA2 mRNA/GAPDH mRNA ratio obtained by
densitometer scanning relative to the ratio obtained without
stimulation.
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To examine whether the effect of PPAR ligands can be mediated by
pathways independent of the IL-1 pathway, we costimulated smooth
muscle cells using IL-1 and various PPAR ligands. Neither additive nor synergistic effects were observed (results not shown).
PPAR are a class of nuclear receptors that exert their action via
heterodimerization with another nuclear receptor RXR. It has been shown
that genes that respond to PPAR/RXR heterodimers are also stimulated
by the RXR ligand 9-cis-retinoic acid. We observed a marked
increase in type II-sPLA2 mRNA when rat aortic smooth
muscle cells were stimulated for 24 h by 9-cis-retinoic acid (Fig. 8, lane 12).
IL-1 Stimulates PPAR Activity in Smooth Muscle Cells, which
Requires NFB Nuclear Translocation--
To demonstrate formally the
involvement of PPAR in the IL-1 effect, we first performed
electrophoretic mobility shift assay (EMSA) to assess whether IL-1
modifies the binding of PPAR to type II-sPLA2 promoter in
rat aortic smooth muscle cells. The nuclear extracts from untreated
cells gave one major complex with labeled oligonucleotides bearing
either the PPAR-binding site consensus sequence from acyl-CoA oxidase
gene (50) or the sequence of the rat type II-sPLA2 promoter
presenting extensive homology with the DR1 element of PPAR-binding
sites (51) (Fig. 9A). Nuclear extracts from smooth muscle cells treated with 10 ng/ml IL-1 for
24 h gave more intense complexes than those from control cells (Fig. 9A). These complexes were competed out by an excess of
the corresponding unlabeled oligonucleotide.
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Fig. 9.
IL-1-induced
activation of PPAR in smooth muscle
cells. A, vascular smooth muscle cell nuclear extracts
were prepared as indicated under "Materials and Methods" from cells
incubated for 24 h in the absence (lanes 1 and
4) or in the presence of 10 ng/ml IL-1 (lanes 2, 3, 5, and 6). EMSA experiments were performed as in Fig. 4
and as described under "Materials and Methods" with
oligonucleotides bearing either the PPAR-binding site consensus
sequence from acyl-CoA oxidase gene (lanes 1-3) or a
related PPAR-binding sequence of the type II-sPLA2 promoter
(lanes 4-6). B, vascular smooth muscle cells
were transiently cotransfected by a plasmid construct containing the
(PPRE consensus) ×2 thymidine kinase promoter region fused to
luciferase reporter gene and pCMV--galactosidase as a control using
the LipofectAMINE technique as described under "Materials and
Methods." One day later, the cells were incubated for 24 h in
the absence or in the presence of 10 ng/ml IL-1 or 5 or 10 µM 15-dPGJ2, and luciferase activity was
measured as indicated under "Materials and Methods." All luciferase
activity values were reported to -galactosidase activity and
represent three independent experiments in which different conditions
were tested in duplicate.
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To confirm that the increased binding of nuclear factors to PPRE
oligonucleotides induced by IL-1 reflects functional activation of
PPAR, we tested the effect of IL-1 on the transcriptional activity of a construct containing the (PPRE consensus) ×2 thymidine kinase promoter region fused to a luciferase reporter gene (57). LipofectAMINE-transfected vascular smooth muscle cells presented a
3-fold increase in luciferase activity when stimulated with 10 ng/ml
IL-1 (Fig. 9B). This stimulatory effect is in the same range as the effect observed with 5 and 10 µM
15-dPGJ2, a well known agonist of PPAR.
Recent concepts concerning the activation of transcriptional machinery
have emphasized the role of cooperation between nuclear receptors to
achieve a complete transcriptional effect via coactivator molecules
(58). We therefore wondered whether NFB, which is a ubiquitous
nuclear factor activated by various inflammatory cytokines, is required
to obtain the transcriptional response to PPAR ligands. We found
that the proteinase inhibitors ZAL and ALLN, at a concentration that
completely blocks both NFB nuclear translocation and IL-1-induced
type II-sPLA2 gene activation, strongly inhibited
9-HODE-induced, arachidonic acid-induced and 15-dPGJ2-induced type II-sPLA2 gene
transcription (Fig. 10). This effect is
not mediated by inhibition of the binding of activated PPAR to its
nuclear target, since ZAL was unable to block the formation of PPAR
oligonucleotide complexes (data not shown). This suggests that the
binding of both NFB and PPAR to type II-sPLA2
promoter is required to stimulate gene transcription.
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Fig. 10.
Effect of proteinase inhibitor on type
II-sPLA2 gene induction by PPAR
ligands. Cells were incubated in serum-free DMEM for 24 h in the absence (lanes 1 and 2) or in the
presence of 10 µM 9-HODE (lanes 3 and
4), 50 µM arachidonic acid (lanes 5 and 6), 5 µM 15-dPGJ2 (lanes
7 and 8). 2 µM ZAL was added (lanes
4, 6, and 8) or not (lanes 1-3, 5, and
7) 1 h before stimulation by effectors. Positive
control was obtained with 10 ng/ml IL-1 (lane 2).
Cellular mRNA was analyzed by RT-PCR as indicated in Fig. 1. A
representative autoradiogram of two independent experiments is
shown.
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DISCUSSION |
Recent studies have shown that calcium-sensitive and
calcium-insensitive cellular phospholipases A2 are involved
in regulating immediate eicosanoid gene generation (59). However,
several pieces of evidence have shown that the secreted enzymes are
also involved in the synthesis of lipid mediators such as eicosanoids or lyso-derivatives (14-17). Type II-sPLA2 is a major
secreted phospholipase A2 found in many cell types when
tissues are triggered by proinflammatory agents and are thought to
participate in the development of the inflammatory process by
generating delayed production of various lipid mediators (31). Our
results confirm that type II-sPLA2 gene, which is silent in
rat aortic smooth muscle cells under resting conditions, is markedly
induced by IL-1 in a time-dependent and
dose-dependent manner (Figs. 1 and 2). This is an intense
and sustained process that is maintained for 48 h (results not
shown). In the present study, we explored the signaling pathways which
lead from IL-1 receptor to the transcriptional machinery of the type
II-sPLA2 gene in rat aortic smooth muscle cells.
Since transcriptional up-regulation of proinflammatory genes is
strongly dependent on NFB activation, we first investigated NFB
involvement in type II-sPLA2 gene induction in response to IL-1. The rat type II-sPLA2 promoters possess an
NFB-binding site (49). By using this site as well as consensus
NFB, we found a marked increase of active NFB in nuclear extracts
from IL-1-treated cells in EMSA experiments (Fig. 4). Proteinase
inhibitors (ZAL and ALLN), known to block proteasome and therefore the
degradation of the NFB inhibitors IB and , decreased
IL-1-induced NFB nuclear translocation and induction of type
II-sPLA2 gene (Figs. 4 and 5). We concluded that in rat
aortic smooth muscle cells, as in rat mesangial cells (49), IL-1
induces type II-sPLA2 gene expression via an NFB-mediated process.
In unstimulated smooth muscle nuclear extracts, we found a basal NFB
binding activity (Fig. 4) that varied from one experiment to another
(data not shown). The existence of such a basal activity has been
challenged by Bourcier et al. (28) and might be reminiscent
of the presence of growth factors from fetal calf serum added to the
culture medium before starving the cells for 24 h. Stimulation of
NFB binding by IL-1 is observed after only 1 h and persists
for at least 24 h (data not shown). This rather unusual sustained
stimulation of NFB binding might be related to a prolonged
degradation of IBs. In human smooth muscle cells, Bourcier et
al. (28) demonstrated that IL-1 triggers a transient IB
decrease and a sustained IB decrease. The involvement of IB
turnover in the duration of IL-1 effects is supported by the results
obtained with the protein synthesis inhibitor, cycloheximide, which
superinduced both basal and IL-1- induced type II-sPLA2
gene expression (Fig. 3). This suggests that cycloheximide blocks the
resynthesis of IBs. Under unstimulated conditions, type
II-sPLA2 mRNA is poorly detectable in RT-PCR assays
(Fig. 3). Although we cannot exclude that cycloheximide induces lasting
stabilization of a weakly expressed mRNA, it more probably inhibits
the synthesis of a labile repressor of type II-sPLA2 gene transcription.
In several cell systems, it has been suggested that IL-1 exerts its
effects on gene activation via activation of NFB with or without
involvement of the sphingomyelinase-ceramide pathway (25, 60). In rat
aortic smooth muscle cells, neither sphingomyelinase nor permeable
C2-ceramide was able to stimulate type II-sPLA2 gene expression, excluding the participation of the
sphingomyelinase-ceramide pathway in the IL-1 effect.
Kuwata et al. (32) recently demonstrated that IL-1
stimulates cPLA2 in rat fibroblasts, inducing increased
arachidonic acid release and synthesis of eicosanoids. IL-1-induced
cPLA2 activation was responsible for stimulation of type
II-sPLA2 gene expression in this cell type. This is in
agreement with our present results showing that, in smooth muscle
cells, IL-1-induced type II-sPLA2 gene expression is
blocked by both a broad phospholipase A2 inhibitor and a
specific cPLA2 inhibitor (Fig. 6). In addition, inhibition
of MEK by PD98059, which prevents MAPK activation necessary for the
cPLA2 stimulatory process (56), strongly inhibits the IL-1-induced type II-sPLA2 mRNA increase (Fig. 6).
IL-1 is able to induce the release of free arachidonic acid, but the
IL-1 effect is not restricted to this fatty acid, as a more marked release of free linoleic acid, also blocked by a specific
cPLA2 inhibitor, was observed (Table I). This might be
controversial since cPLA2 has been found to be very
specific for phospholipids containing arachidonic acid (61). However,
using the GC/MS methodology, we recently indicated that
cPLA2 is also able to hydrolyze phospholipids containing
linoleic acid which are much more abundant than those containing
arachidonic acid.2
In contrast with the results of Kuwata et al. (32),
lysophosphatidylcholine failed to stimulate type II-sPLA2
gene expression in smooth muscle cells, but we were able to induce this
expression by exogenous arachidonic acid in a concentration range known
to stimulate gene expression in other cell systems (Fig.
7A). As the lipoxygenase inhibitor, NGDA, but not the
cyclooxygenase inhibitor, indomethacin, was found to block the IL-1
effect (Fig. 7B), one might therefore speculate whether
arachidonic acid or its lipoxygenase metabolites are involved in the
IL-1 effects. This was suggested by Kuwata et al. (32),
who indicated preliminary results showing the stimulation of type
II-sPLA2 gene expression by 15-hydroxyeicosatetraenoic acid
(15-HETE).
One mechanism by which polyunsaturated fatty acids or their derivatives
activate gene expression is the binding of PPAR factors (36). Recently,
controversy has arisen concerning the presence of various PPAR isoforms
in smooth muscle cells (37, 38) and their actual effect on
proinflammatory genes (35, 37, 38). We demonstrated a PPRE on the type
II-sPLA2 promoter that binds nuclear factors as does an
oligonucleotide bearing a consensus PPRE sequence (Fig. 9A).
Furthermore, the PPAR ligand 15-dPGJ2 activates a
luciferase reporter gene whose promoter contains two consensus PPRE
binding domains (Fig. 9B). To investigate a putative involvement of PPARs in type II-sPLA2 gene expression in
rat aortic smooth muscle cells, we tested the ability of various PPAR
ligands to stimulate this gene. We found that clofibric acid and oleic acid, which are activators of PPAR (62), are unable to induce type
II-sPLA2 gene expression. Only PPAR and PPAR/
ligands, i.e. 15-dPGJ2, 9-HODE, and
carbaprostacyclin, were able to induce type II-sPLA2 gene
expression (Fig. 8) at concentrations described to activate PPAR
effectively (34, 35). It has been demonstrated that PPAR mediates
its translocating action via heterodimers with RXR. RXR/PPAR
heterodimers can activate the transcriptional response to ligands
specific for either subunit of the dimer (63). This is clearly the case
for induction of type II-sPLA2 in smooth muscle cells,
since 15-dPGJ2, 9-HODE, or 9-cis-retinoic acid
alone induced sustained stimulation of the gene (Fig. 8). Mutation of PPAR but not RXR in the hormone-dependent activation
domain inhibits the ability of RXR/PPAR heterodimers to respond to
ligands specific for either subunit (63). This indicates that the
presence of PPAR ligands is required to obtain a functional
heterodimer in the absence of 9-cis-retinoic acid.
The involvement of PPAR in IL-1 stimulation of the type
II-sPLA2 gene is also consistent with the stimulatory
effect of IL-1 on the binding of nuclear factors to a PPRE of the
type II-sPLA2 promoter (Fig. 9A) and with the
stimulatory effect of IL-1 on a luciferase reporter gene whose
promoter contains two consensus PPRE binding domains (Fig.
9B). This effect is of the same amplitude as those induced
by the PPAR ligand 15-dPGJ2. This is also in line with
the presence of PPAR in rat aortic smooth muscle cells (Fig.
9A) (38) and with the result reported by Tontonoz et
al. (64) indicating high levels of PPAR in atherosclerotic lesions, using sections of aorta from spontaneously atherosclerotic mice.
Since the IL-1 effect is not inhibited by indomethacin (Fig.
7B), 15-dPGJ2 cannot be the endogenous PPAR
ligand induced by cytosolic PLA2 activation in rat aortic
smooth muscle cells. On the contrary, 9-HODE, which strongly stimulates
the type II-sPLA2 gene (Fig. 8), may be produced from
linoleic acid via the lipoxygenase pathway (12). This may be related to
the marked increase in cellular free linoleic acid induced by IL-1
in smooth muscle cells (Table I). In preliminary experiments, we failed
to demonstrate HODE synthesis in rat aortic smooth muscle cells in
response to IL-1 stimulation (result not shown). However, type
II-sPLA2 has been recently shown to increase
lipoxygenase-induced HODE production from low density lipoproteins
(12). This raises the possibility that type
II-sPLA2-induced HODE synthesis constitutes a positive autocrine loop in the aorta during the inflammatory stage of
atherosclerosis progression.
IL-1 stimulation induced only a slight increase in free arachidonic
acid, but massive addition of exogenous arachidonic acid might drive
type II-sPLA2 gene expression either directly, since this
action is mimicked by the non-metabolizable analogue ETYA (Fig.
7A), or indirectly via the production of lipoxygenase
metabolites. Nagy et al. (35) have indeed indicated that
15-hydroxyeicosatetraenoic acid, a lipoxygenase derivative of
arachidonic acid, is able to stimulate PPAR-driven promoters albeit
with a lower efficiency than HODEs.
The organization of an enhanceosome in the promoter region able to
interact with the basic transcription machinery is an emerging concept
in the regulation of gene expression (65). This complex recruits
coactivator proteins, such as CREB-binding protein (CBP/p300), to
stimulate the transcription rate (66). PPAR was found to interact
with CBP/p300 (67), and components of the USA coactivator are involved
in transcriptional activation of the human immunodeficiency virus
promoter by NFB (68). NFB was found to down-regulate PPAR-driven promoters in human smooth muscle cells (37). Nuclear transl
Induces Type II-secreted Phospholipase
A2 Gene in Vascular Smooth Muscle Cells by a Nuclear Factor
B and Peroxisome Proliferator-activated Receptor-mediated
Process*
TOP
ABSTRACT
INTRODUCTION
