Modulation of cyclin D1 and early growth response factor-1 gene
expression in interleukin-1b-treated rat smooth muscle cells
by n-6 and n-3 polyunsaturated fatty acids
Souad Bousserouel, Michel Raymondjean, Arthur Brouillet, Gilbert Be
´
re
´
ziat and Marise Andre
´
ani
UMR 7079 Physiologie et Physiopathologie, Universite
´
Pierre et Marie Curie, Paris, France
The proliferation of smooth muscle cells (SMC) is a key
event in the development o f atherosclerosis. In addition to
growth factors or cytokines, we have shown previously that
n-3 polyunsaturated fatty acids (PUFAs) act in opposition
to n-6 PUFAs by modulating various steps of the inflam-
matory process. We have investigated the molecular mech-
anisms by which the incorporation of the n-6 PUFA,
arachidonic acid, increases the proliferation of r at SMC
treated with interleukin-1b, while the n-3 PUFAs eicosa-
pentaenoic acid (EPA) and docosahexaenoic acid ( DHA),
elicit no mitogenic response. Incorporation of EPA or DHA
into SMC, which a re then activated by interleukin-1b to
mimic inflammation, decreas e s promoter activity o f t he
cyclin D1 gene and phosphorylation of the retinoblastoma
protein. Together, our data demonstrate that n-3 effects are
dependent on the Ras/Raf-1/extracellular signal regulated
kinase (ERK)/mitogen-activated protein kinase pathway,
and that down-regulation of the cyclin D1 promoter activity

rotic effects by inhibiting the proliferation of SMC [7]. The
molecular mechanisms underlying these opposing effects of
n-3 and n-6 P UFAs are s till not clear. MAPK plays a central
role by transducing extracellular signals, including growth
factors and cytokines [8]. R ecent evidence i ndicates that
phosphatidylinositol 3-kinase (PI3-K) is i nvolved i n t he
regulation of MAPK in various cell systems, including SMC
[9]. Studies have demonstrated that the classical Ras/
extracellular signal regulated kinase (ERK) p athway regu-
lates G1 progression by directly controlling cyclin D1
production via phosphorylation o f v arious transcription
factors that bind t o defined elements within the cyclin D1
promoter [10,11]. Sequential cyclin activation leads to the
progressive phosphorylation of the retinoblatoma protein
(Rb) that is essential for entry into the S-phase.
The transcription factor Egr-1 (early growth response
factor-1) also seems t o be involved in t he control of cell
proliferation initiated by the MAPK cascade [12,13].
Above-normal concentrations of Egr-1 are found in
atherosclerotic lesions and these are associated with
increased activity o f the Egr-1 t arget genes implicate d in
the proliferative a nd chem otactic r esponses of SMC to
injury [14]. EPA and DHA also seem to reduce the
Correspondence to M. Raymondjean, UMR 7079 Physiologie et
Physiopathologie, Universite
´
Pierre et Marie Curie, Case Courrier
256, Baˆ timent A, 5e
`
me e

n-3 and n-6 PUFAs on the proliferation of SMC stimulated
via the activation of MAPK and PI3-K after treatment with
IL-1b, which mimics inflammation.
Materials and methods
Reagents
Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s
NaCl/P
i
(phosphate-buffered saline), trypsin, type I collagen
from calf skin, g lutamine, penicillin, s treptomycin, fatty
acid-free BSA, leupeptine, pepstatin, and phenyl-
methanesulfonyl fluoride were a ll purchased from Sigma
Chemical Co. (Sigma-Aldrich Corp., St. Louis, MO, USA).
Fetal bovine serum was from Life Technologies, Inc.
(Rockville, M D, USA). Murine-mammary lentivirus re-
verse transcriptase, lipofectAMINE and random primers
were from Life Technologies, Inc. Oligonucleotides were
from Oligo Express (Montreuil C edex, France). Hybond
N+ nylon membranes, t he enhanced chemiluminescence
(ECL) direct nucleic ac id labeling system, and t he ECL
reagent kit for horseradish peroxidase were from Amersham
Pharmacia B iotech (Amersham Biosciences UK Limited,
Little Chalfont, Buckinghamshire, UK). I L-1b was pur-
chased from Peprotech Inc. (Rocky Hill, NJ, USA). DHA,
peroxide-free AA and EPA were from Cayman C hemical
(Ann Arbor, MI, USA), and are protected from oxidation
by BHT (2,6-di-ter-butyl-4-methylphenol) in 0.1% (v/v)
ethanol. The luciferase reporter assay kit and pSV-bgal
plasmid were from Promega Inc. (San Luis Obispo, CA,
USA). LY294002, U0126 and PD98059 were from CalBi-

ded the p38 MAPK expression vectors. The wild-type (Wt
Egr-1) and the mutated construct (DEgr-1) of the ()137 to
)99 bp) cyclin D1 promoter fragment, subcloned into the
heterologous herpes simplex v irus-thymidine kinase min-
imal promoter, fused to the firefly luciferase reporter gene,
were obtained from A. K . Rustgi (Gastrointestinal Unit,
University of Pennsylvania, Philadelphia, PA, USA).
Isolation and culture of rat aorta SMC
Vascular SMC were isolated by e nzymatic digestion of the
media of thoracic aortas removed from male W istar rats
(weight 3 00 g; Elevage J anvier, L eGenest S t Isle, France)
[22]. Cells were seeded on dishes coated with type I collagen
from calf skin and were cultured in DMEM supplemented
with 10% (v/v) fetal bovine serum, 4 m
M
glutamine, 100
unitsÆmL
)1
of penicillin and 100 lgÆmL
)1
of streptomycin.
SMC w ere s ubcultured every 7 d ays, and experiments were
performed on cells at three to nine passages a fter primary
culture. Confluent cells were maintained in quiescent mode
by incubation for 24 h in serum-free m edium containing
0.2% (w/v) fatty acid-free BSA. Then, SMC were incubated
or not incubated (control) with PUFA complexed with fatty
acid-free BSA. Different lipid delivery parameters have been
tested [18] and the best results have been obtained with
PUFA concentrations of 50 l

oligonucleotide probes were labelled and detected by using
the ECL direct nucleic acid labelling detection kit (Amer-
sham Pharmacia Biotech). The b ands on the autoradiogra-
Ó FEBS 2004 Anti-mitogenic action of EPA and DHA (Eur. J. Biochem. 271) 4463
phy films were scanned and quantified by densitometry by
using
QUANTITY ONE
software (Bio-Rad, Hercules, CA,
USA).
Real-time quantitative RT-PCR
Quantitative RT-PCR was performed by using the qPCR
core kit for SYBR Green I-No ROX (Eurogentec, Liege
Science Park, Seraing, Belgium). Reactions were carried out
in a total volume of 25 lL containing SYBR Green PCR
core reagents with 8 n g of of the first-strand cDNA and
300 n
M
primers. Amplification was performed on an iCycler
(Bio-Rad), according to the manufacturer’s instructions,
and cycle parameters were: 50 °C(2min)and95°C
(10 min), followed by 40 c ycles of 95 °C(15s),60°C
(30 s ) and 72 °C (30 s). V ariations between the levels of
the total cDNA templates in different samples were
normalized by measuring G APDH gene expression. The
oligonucleotide primers used to quantify Egr-1 and
GAPDH mRNAs were identical to those used in the
RT-PCR (see above).
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from SMC [22]. The do uble-
stranded oligonucleotides were 5¢ end-labelled by using the

CCCAACGGA-3¢ wasusedasacompetitor(at100-fold
molar e xcess) in all t he EMSA experiments to eliminate
Sp1 complexes [13]. The n uclear factor-Y (NFY) oligo-
nucleotide, 5¢-GGGGTAGGAACCAATGAAATGAAA
CGTTA-3¢, corresponded to the binding site of the r at
albumin promoter [24].
Transfection and luciferase assays
Cultured rat SMC were seeded in 12-well dishes at a
concentration that gave 70% c onfluence 24 h later. The
SMC were transfected with 1.5 lL o f LipofectAMINE
Plus (Life T echnologies), 300 ng of plasmids containing a
firefly luciferase reporter gene plus the 973 bp human
cyclin D1 promoter ()973 to +29 bp), the full-length (Wt
Egr-1) or the mutated (mutEgr-1) ()137 to )99 bp) cyclin
D1 promoter, or the 697-bp human Egr-1 (pGLE), and
100 ng of pSV-bgal p lasmid (Promega). The amounts of
the r elevant e xpression vectors – the dominant-negative
Ras N17, Raf-1 C4 and Dp85a subunit (a deletion mutant
of the regulatory subunit of PI3-K lacking 102 amino
acids from residues 466–567 of the i nter-SH2 domain that
confers binding to the catalytic subunit p 110), constitu-
tively active Ras K12, Raf-1 BXB and p38 MAPK
(pCDNA-wt-p38) – were varied. The transfection mix-
tures were incubated for 3 h, as recommended by the
manufacturer. Transfected cells were cultured for 24 h in
serum-free medium and incubated for 24 h i n the same
medium containing EPA, DHA or AA. After incorpor-
ation of P UFA, the cells were washed twice with NaCl/P
i
and s timulated (or not stimulated) with IL-1b in serum-

containing 0.5% (v/v) Nonidet P -40 p lus 1 lgÆmL
)1
of
leupeptine, 1 lgÆmL
)1
of pepstatin, 1 m
M
phenyl-
methanesulfonyl fluoride, and 1 m
M
EDTA]. Nuclear
extracts were prepared as described above. Equal amounts
of protein (20 lg) were fractionated b y SDS/PAGE and
transferred to poly(vinylidene difluoride) membranes. Free
binding sites were blocked by incubation overnight at 4 °C
in NaCl/P
i
containing 5% (v/v) nonfat milk and 0.1% ( v/v)
Tween-20. Blots were washed in NaCl/P
i
/Tween and then
incubated with the indicated primary antibodies. Immuno-
blots were developed by using appropriate secondary
horseradish peroxidase-coupled antibodies and t he ECL
Western blotting kit (Amersham Pharmacia Biotech).
Measurement of DNA synthesis
Incorporation of t he thymidine analogue 5 -bromo-
2¢-deoxyuridine (BrdU) was measured to determine the
effects of PUFA on DNA synthesis. SMC were plated in 96-
well plates at 10 000 cells per well and left to adhere for

AA, EPA or DHA for 24 h, incorporated significant
amounts of each PUFA into t heir membrane phospholi-
pids. Incubation with PUFAs at higher concentrations (up
to 100 l
M
) for a longer time-period (up to 48 h), or with
different a lbumin : PUFA r atios, did not enhance incor-
poration into phospholipids [18]. Under our culture condi-
tions, the triglyceride content did not significantly change
after supplementation with any PUFA. DNA synthesis by
quiescent cells treated w ith IL-1b alone for 2 4 h [i.e. the
control (–)] was not greater than in untreated cells (Fig. 1A).
However, the incorporation of BrdU into SMC was
strongly stimulated by incubation in medium containing
10% ( v/v) fetal bovine s erum. A lthough the incorporation
of AA alone did not affect BrdU incorporation, incubating
these AA-enriched cells with IL-1b for 24 h resulted in a
fourfold increase of DNA synthesis. In contrast, the n-3
PUFAs – EPA or DHA – did not increase SMC prolifer-
ation in response to IL-1b. I ncorporating EPA and DHA
alone, without stimulation with IL-1b, did not alter BrdU
incorporation. In contrast to conditions in the presence of
IL-1b , AA incorporated into membranes did not stimulate
the proliferation of SMC incubated with fetal bovine s erum
(Fig. 1B). Moreover, EPA and DHA reduced the increased
BrdU incorporation in response to serum by 50 or 60%,
respectively (Fig. 1B). Altogether, these results confirm that
AA, or AA metabolites, stimulates SMC proliferation and
that this mitogenic effect requires t reatment with IL- 1b.
There was also no mitogenic response by IL-1b alone under

(phosphorylated Rb) (p-Rb) Ig (lower part) and the quality of the
preparation and the amount of protein loaded were evaluated by using
anti-Rb I g (upper part). Each blot is representative of two independent
experiments.
Ó FEBS 2004 Anti-mitogenic action of EPA and DHA (Eur. J. Biochem. 271) 4465
Modulation of cyclin D1 synthesis and
hyperphosphorylation of Rb by n-3 and n-6 PUFAs
Induction of cyclin D1 is one of the e arliest effects of
mitogenic factors lea ding to cell c ycle re-entry, G1-phase
progression, and transition to t he DNA synthetic S phase.
We examined the effects of PUFA on cyclin D1 by measuring
its concentration in whole cell extracts by using a monoclonal
anti-(cyclin D1) Ig. There was little cyclin D1 protein in SMC
stimulated with IL-1b for 24 h (Fig. 1C). Incorporating AA
before stimulation by IL-1b increased the cyclin D1 concen-
tration, whereas EPA and DHA reduced the conce ntration
of cyclin D1 it until it was barely detectable.
As the retinoblastoma p rotein, Rb, i s a key target of
cyclin D1/cyclin-depe ndent kinase complexes, we investi-
gated whether inhibiting cyclin D1 with EPA or DHA also
affected the phosphorylation status of R b. Western blot
analysis showed that hyperphosphorylated Rb (pRb) accu-
mulatedinIL-1b-treated cells supplemented with AA, and
this was more marked in cells treated with serum (Fig. 1D).
In contrast, EPA and DHA completely inhibited the
phosphorylation of Rb, although the total amount of Rb
protein was not affected (Fig. 1D, upper part). The
phosphorylation status of Rb s eems to be correlated with
the cyclin D1 promoter activity.
Effects of n-3 and n-6 PUFAs on the cyclin D1 gene

p85 subunit from PI3-K, to examine t he signalling path-
ways. We probed the role of p42/p44 MAPK (ERK1/2) in
AA-induced proliferation using cells incubated w ith t he
MAPK kinase (MEK) inhibitor, U0126. U0126 completely
inhibited the cyclin D1 promoter activity in AA-pretreated
SMC incubated with IL-1b and reduced it in cells incubated
with fetal bovine serum (Fig. 2B). Similar results were
obtained with 20 l
M
PD98059, another specific MEK
inhibitor (data not shown). Transfected SM Cs were also
treated with the specific inhibitor, LY294002, to determine
whether PI3-K was activated in AA-mediated proliferation.
This inhibitor blocked the a ctivation of the cyclin D1 gene
promoter in AA-treated cells stimulated with IL-1 b,and
strongly reduced the activation by fetal bovine serum.
Comparable effects were obtained with 200 n
M
wortman-
nin, another PI3-K inhibitor (data not shown). We
confirmed the implication of PI3-K in this signalling
pathway by using a d ominant-negative mutant of the p85
subunit of P I3-K (Dp85a) coexpressed in SMC stimulated
with IL-1b: AA-induced cyclin D1 promoter activity was
strongly repressed. The IL-1b-stimulated promoter activity
of AA-treated cells was completely i nhibited by
LY294002 + U0126. Similarly, both inhibitors strongly
reduced promoter activation by fetal bovine serum
(Fig. 2B). These results indicate that the MAPK/ERK
and PI3-K signalling pathways are both required for the

Inhibitory effects of n-3 PUFAs on cyclin D1 gene
expression and inhibition of IL-1b-induced Egr-1 gene
activation
Egr-1 is the product of an immediate-early gen e that
regulates SMC proliferation [12]. We demonstrated recently
4466 S. Bousserouel et al.(Eur. J. Biochem. 271) Ó FEBS 2004
that the activation of cyclin D1 gene transcription is
mediated mainly by the transcription factor, Egr-1, via the
cis-regulatory element of the cyclin D1 promoter located
between )112 and )105 bp [13]. We decided to investigate
the effect of PUFAs on the Egr-1-binding activity by using
EMSAs with a
32
P-labeled double-stranded oligonucleotide
spanning )117 to )99 bp of the human cyclin D1 promoter
(Fig. 4A). This oligonucleotide bears the Egr-1-binding site
which overlaps a Sp1 recognition motif that has been
previously characterized [13]. In order to reveal clearly the
Egr-1-binding activity, a 100-fold molar excess of the
unlabeled consensus Sp1 o ligonucleotide was systemically
added to the EMSA experiment. Under these conditions,
the formation of Sp1 complexes was totally abrogated, as
shown previously [13], and the nuclear extracts from IL-1b-
treated cells (c ontrol) gave one complex, wh ich was le ss
intense than that p roduced by stimulation with 10% (v/v)
fetal bovine serum. The specificity of the binding was
confirmed after incubation with a 100-fold excess of
unlabeled oligonucleotide of the cyclin D1 promoter.
Nuclear e xtracts from c ells pretreated with EPA showed
less IL-1b-induced Egr-1 binding activity than AA-enriched

AA increased, by fivefold, the amount of Egr-1 mRNA
accumulatedinresponsetoIL-1b. In contrast, EPA and
DHA completely impeded IL-1b-induced egr-1 gene expres-
sion. In the light of these findings, we wished to determine
the ability of the region ( )137 to )99 bp ) of the human
cyclin D1 promoter, encompassing the Egr-1-binding site,
to activate transcription. We tested two constructs fused to
the HSV-tk minimal promoter: one contained the wild-type
region (Wt Egr-1) and the other contained a m utation
affecting the Egr-1-binding site (mut Egr-1) [27]. Quiescent
cells incubated or not incubated w ith different PUFAs were
transiently transfected with these constructs and then
stimulated with IL-1b.The()137 to )99 bp) Wt Egr-1
construct was regulated by PUFAs in the same manner as
the ()973 to +29 bp) cyclin D1 gene promoter (Fig. 4C).
The twofold stimulation caused by AA p retreatment was
still seen, whereas EPA and DHA incorporation did not
increase cyclin D1 promoter activity in response to I L-1b.
The basal activity of the mut Egr-1 was severely diminished,
and AA did not stimulate the promoter. Our results show
that the Egr-1-binding site motif located between ()137 and
)99 bp) is responsible for cyclin D1 activation induced by
AA, a nd the d iffering effects of n-6 and n-3 PUFAs on
Egr-1 mRNA accumulation are consistent with their a ction
upon the activation of cyclin D1 transcription.
Influence of n-3 PUFAs on egr-1 promoter activity,
dependent on the Ras/Raf/MEK/ERK pathway
We next attempted to study the action o f PUFAs on
transiently t ransfected egr-1 human gene promoter
()697 bp Egr-1/LUC) activity to confirm t he impact of

lation of egr-1 gene expression (Fig. 4B).
Effects of n-3 and n-6 PUFAs on the synthesis
of caveolin-1 and caveolin-3 and on the location
of p42/44 MAPK caveolae
Caveolins are scaffolding proteins that interact and negat-
ively regulate m any components through the p42/44 MAP
kinase pathway [28]. Interestingly, the activity of the cyclin
Fig. 3. Influence of n-3 polyunsaturated fatty acids (PUFAs) on cyclin D1 promoter activity induced by the Ras/Raf pathways. Smooth muscle cells
(SMC) were transfected with the ()973 to +29) hum an cyclin D 1 promot er luciferase/reporter plasmid (alone; c ontrol), with either the dominant-
positive Ras K12, Raf BXB, or the dominant-negative Ras N17, Raf C14 expression vectors, and treated as described previously (Fig. 2). Relative
luciferase activities represent the percentage stimulation relativetothevalueobtainedwithIL-1b-treated cells transfected with the ()973 to +29)
human cyclin D 1 p romoter luciferase/reporter plasmid alone (control; –) an d are expressed as the me an value ± SEM (bars) of three indepen dent
experiments in which different conditions were tested in duplicate.
4468 S. Bousserouel et al.(Eur. J. Biochem. 271) Ó FEBS 2004
D1 gene promoter is repressed by caveolin-1 [29]. We
decided to measure (by Western blotting) t he concentrations
of the caveolin-1 and caveolin-3 proteins in the caveolae of
cells stimulated with IL-1b in order to determine whether
caveolin synthesis is also modulated by PUFA incorpor-
ation (Fig. 6A). Pretreatment of SMC with A A slightly
decreased, in comparison with IL-1b-stimulated cells, the
amount of caveolin-1 protein, whereas both the n-3 PUFAs
(EPA, DHA) increased the intracellular caveolin-1 to a level
above that of control cells. As expected, SMC stimulated
with 10% (v/v) fetal bovine serum also had a reduced total
content of caveolin-1. The concentration of the caveolin-3
isoform, mainly found in muscle, was also measured [30].
Western blot analysis showed that EPA and DHA increased
the amount of intracellular caveolin-3, whereas AA had
no effect. A s caveolin-1 and caveolin-3 may f unction as

real-time quantitative RT-PCR analysis. Semiquantitative RT-PCR
and real-time quantitative RT-PCR analyses of Egr-1 mRNA were
normalized with ubiquitous glycerald ehyde-3-phosp hate dehydro-
genase ( GAPDH) amplification. Seru m-starved smooth muscle cells
(SMC) were treated or not treated (–) with 50 l
M
PUFA (AA, EPA or
DHA) for 24 h and then stimulated w ith IL-1 b o r 10% (v/v) fetal
bovine serum for 1 h. (C) Analysis, by mutagenesis, of the function of
Egr-1 in the cyclin D1 promoter. SMC were transiently transfected
with the ()973 to +29) construct of the human cyclin D1 luciferase
reporter plasmid (control) or with the ()137 to )99)regionofthe
cyclin D1 promoter containing the Egr-1 motif (Wt Egr-1) or the
mutant of th is region (mut Egr-1) fused to th e herpes simplex virus
thymidine k inase m inimal promoter. Then, the transfected cells were
enriched with PUFA fo r 24 h and stimulated w ith IL-1 b for 24 h. The
results are expressed as the percentage stimulation relative to the value
obtained with cells transfected with ( )973 to +29) cyclin D1 construct
and treated with IL-1b alone (–). Luciferase activity w as assayed a s
described previously. Relative luciferase activities are expressed as the
mean value ± SEM (bars) of three independent experiments in which
different conditions were tested in duplicate.
Ó FEBS 2004 Anti-mitogenic action of EPA and DHA (Eur. J. Biochem. 271) 4469
determine whether the incorporation of PUFA also affected
the concentration of the p42/44 MAPK in caveolae.
Pretreatment with EPA or DHA increased the amount of
ERK1/2 protein, whereas the incorporation of AA did not
(Fig. 6 B).
Discussion
Several lines of evidence suggest that the incorporation of

with 50 l
M
polyunsaturated fatty acid (PUFA) (AA, EPA or DHA) for 24 h and then incubated with interleukin-b (IL-1b)(10 ngÆmL
)1
,24 h)with
or withou t the mitogen-activated protein kinase kinase (MEK) i nhibitor, U0126 (U, 50 l
M
), and the PI3-K inhibitor, LY294002 (LY, 50 l
M
).
Luciferase activity was assayed as described previously. The results of six independent experiments are expressed as the percentage stimulation
relative to t he value obtained from cells treated with IL-1b alone (–). * ¼ P<0.05, significantly differen t from AA in IL-1b-treated cells. (B)
Quiescent SMC were transiently t ransfected with pGLE (alone; contro l) and the dominant-negative Ras N17 or Raf-1 C4 mutants, or the
dominant-positive Ras K12 mutant, following which the transfected cells were cultured as indicated a bove. Relative luciferase activities are
expressed as the mean value ± SEM (bars) of th ree independent experiments in which different conditions were tested in duplicate.
4470 S. Bousserouel et al.(Eur. J. Biochem. 271) Ó FEBS 2004
eicosanoids that have much lower inflammatory and
mitogenic properties than AA-derived lipid mediators [7].
The n -3 PUFAs are also competiti ve i nhibitors of AA
metabolism, especially for the cyclooxygenase (COX)
pathway [37]. In addition, we have demonstrated that
EPA and DHA block the formation of prostaglandin E2
(PGE2) by inhibiting the IL-1b-stimulated production of
COX-2 mRNA expression [18]. PUFAs modulate SMC
activation similarly for endothelial cells (i.e. proliferation,
synthesis of adhesion molecules and expression of
inflammatory genes) when they are added to cultured
SMC o r Human Umb elical Vascular Endothelium cells
(HUVEC) days before stimulation with cytokines [38]. All
of these data indicate that the mitogenic effect of IL-1b on

effects o f n-3 PUFAs and AA on cyclin D1 promoter
activity induced by the Ras/Raf/MAPK/ERK pathway are
in agreement with our previous finding that AA in creases
ERK1/2 activity in IL-1b-treated cells, whereas n-3 PUFAs
reduce it [18]. Although EPA and DHA inhibit the
activation of ERK1/2 by mitogens, n-3 PUFAs may still
interfere w ith t he MAPK pathway upstream of p42/44
MAPK phosphorylation. We find that the IL-1b-induced
response is also mediated by activation of the PI3-K
pathway, and a functional crosstalk between the PI3-K and
MAPK pathways, also well documented, may amplify the
response [39].
The activation of cyclin D1 gene expression by mitogenic
stimuli in cells appears to b e essential a nd rate limiting for
progression to the S phase. Expression of the cyclin D1 gene
seems to be regulated essentially at the transcription level. It
is blocked by inhibitors of the MAPK or P I3-K pathways.
The promoter re gion of t he cyclin D1 contains multiple cis-
elements, all involved in activation of the transcription
[40,41]. We have shown previously that the coordinated
activation o f p roinflammatory genes, which occurs in cells
incubated with AA, is correlated with increases in the
binding of nuclear factor-kappa-B (NF-jB), Ets-1 and
C/EBP transcription factors [18]. By contrast, n-3 PUFAs
decrease the IL-1b-induced binding of these f actors. In
addition, the expression of genes, associated with athero-
sclerosis, that contain overlapping GC-box expression are
controlled at the transcriptional l evel by the t ransiently
expressed Egr-1 [12,14]. The model proposed involves the
displacement of Sp1 factor by Egr-1 on promoter regions.

and Egr-1 are substrates of MAPK [12,43,44], the inhibition
of MAPK by the incorporation of n-3 PUFAs could alter
the phosphorylation of these transcription factors, affecting
their binding to DNA and trans-activation. As the egr-1
gene promoter is also a target for the MAPK pathway [12],
n-3 PUFAs may act through the Ras/Raf cascade to inhibit
egr-1 gene expression. These results therefore provide
evidence that membrane-incorporated EPA and DHA act
upon SMC proliferation by interfering with the Ras/Raf/
ERK pathway.
It was recently reported that n-3 PUFAs act by altering
membrane lipid microdomains, of w hich caveolae are a
subtype enriched with caveolin proteins [22]. Several studies
have shown that caveolin, a 21–24 kDa integral membrane
protein, plays a critical r ole in the regulation of cell
proliferation, including SMC, by inhibiting signalling mol-
ecules [ 45–47]. Caveolin-3 is the main isoform in muscle
cells, including SMC [30]. Caveolin-1 and caveolin-3 seem to
act as scaffolding proteins that negatively regulate the
activity or the release of many molecules, including pro-
proliferative, oncogenic and antiapoptotic proteins such as
EGF-R,PDGF-R,Ras,SrcandPI3-K[28].Inthisrespect,
signal transduction via the MAPK proliferation cascade is
inhibited [31] and overproduction of caveolin-1 also repres-
ses cyclin D 1 gene promoter activity [29] and regu lates the
shear stress-dependent activation of ERK in vascular
endothelial cells [48]. Interestingly, caveolin-3 knockout
mice develop a cardiomyopathy, probably resulting from
hyperactivation of E RK1/2 [ 32]. We postulate that the
antiproliferative e ffect o f EPA and DHA is a result of the

(ARC)Õ. We t han k Martine Glorian for h e r critical review of th e
manuscript and Claire Monne
´
for producing the smooth muscle cells.
Lastly, we thank Owen Parkes for ling uistic assistance.
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