The activity of the cyclin D1 gene promoter was unchanged after incubation for 24 h with IL-1b, but greatly increased threefold by incubation with fetal bovine serum Fig.. However, pretr
Trang 1Modulation 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 of 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 rat 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 are then activated by interleukin-1b to
mimic inflammation, decreases promoter activity of the
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
is mediated by the specific binding of the early growth re-sponse factor-1 Finally, we have shown that the incorpor-ation of EPA and DHA also increased the concentrincorpor-ation of caveolin-1 and caveolin-3 in caveolae, which correlated with n-3 PUFA inhibition of SMC proliferation through the mitogen-activated protein kinase pathway We provide evi-dence indicating that, in contrast to n-6 PUFAs, n-3 PUFAs exert antiproliferative effects on SMC through the mitogen-activated protein kinase/ERK pathway
Keywords: caveolae; cyclin D1 gene expression; interleukin-1b; n-3 PUFA; vascular smooth muscle cells
Early atherosclerosis lesions have many features of an
inflammatory process, and the proliferation of smooth
muscle cells (SMC) from the medial layer of the vessel to the
intima is a key event in the development of this disorder [1]
As interleukin-1 b (IL-1b) seems to act (probably in
association with other growth factors) as a mitogen in
cultured SMC, its release in large quantities by activated
endothelial cells and macrophages may contribute to
atherosclerotic lesions [2] Long-chain n-3 and n-6
poly-unsaturated fatty acids (PUFAs) may also modulate the
mitogenic response Arachidonic acid (AA, 20:4 n-6)
and its metabolites stimulate growth by activating the
mitogen-activated protein kinase (MAPK) pathway in many cell types, including SMC [3,4] There is also good evidence that growth factors and cytokines stimulate AA release [5,6]
Eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexa-enoic acid (DHA, 22:6 n-3) from fish oil lipids, also known
as anti-inflammatory precursors, exert their antiatheroscle-rotic effects by inhibiting the proliferation of SMC [7] The molecular mechanisms underlying these opposing effects of n-3 and n-6 PUFAs are still not clear MAPK plays a central role by transducing extracellular signals, including growth factors and cytokines [8] Recent evidence indicates that phosphatidylinositol 3-kinase (PI3-K) is involved in the regulation of MAPK in various cell systems, including SMC [9] Studies have demonstrated that the classical Ras/ extracellular signal regulated kinase (ERK) pathway regu-lates G1 progression by directly controlling cyclin D1 production via phosphorylation of various transcription factors that bind to 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 to be involved in the 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 of the Egr-1 target genes implicated in the proliferative and chemotactic responses 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, Baˆtiment A, 5e`me e´tage, 7 Quai Saint Bernard, 75252 Paris Cedex
5, France Fax: +33 1 44 27 51 40, Tel.: +33 1 44 27 32 06,
E-mail: michel.raymondjean@snv.jussieu.fr
Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; DHA,
docosahexaenoic acid; Egr-1, early growth response factor-1; EMSA,
electrophoretic mobility shift assay; EPA, eicosapentaenoic acid;
ERK, extracellular signal regulated kinase; IL-1b, interleukin-1b;
MAPK, mitogen-activated protein kinase; MEK, MAPK kinase;
NF-jB, nuclear factor-jB; PDGF, platelet-derived growth factor;
PI3-K, phosphatidylinositol 3-kinase; PGE2, prostaglandin E2;
PUFA, polyunsaturated fatty acid; Rb, retinoblastoma protein;
SMC, smooth muscle cells.
(Received 6 May 2004, revised 9 September 2004,
accepted 29 September 2004)
Trang 2proliferation of SMC by modulating the mitogenic signal
transduction induced by platelet-derived growth factor
(PDGF) [15], serotonin and thromboxane A2 [16,17] We
have shown previously that the incorporation of n-3 PUFAs
into the SMC membrane modulates various steps of the
inflammatory process induced by IL-1b [18] Several lines of
evidence suggest that the change in membrane
characteris-tics following the incorporation of EPA or DHA alters the
signal transduction elicited by IL-1b [19–21]
The present study was therefore carried out to examine
molecular events affecting the production and
concentra-tions of cyclin D1, Rb and Egr-1, in order to determine how
the incorporation of EPA and DHA modulate SMC
proliferation This article describes the differing effects of
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/Pi(phosphate-buffered saline), trypsin, type I collagen
from calf skin, glutamine, penicillin, streptomycin, fatty
acid-free BSA, leupeptine, pepstatin, and
phenyl-methanesulfonyl fluoride were all purchased from Sigma
Chemical Co (Sigma-Aldrich Corp., St Louis, MO, USA)
Fetal bovine serum was from Life Technologies, Inc
(Rockville, MD, USA) Murine-mammary lentivirus
re-verse transcriptase, lipofectAMINE and random primers
were from Life Technologies, Inc Oligonucleotides were
from Oligo Express (Montreuil Cedex, France) Hybond
N+ nylon membranes, the enhanced chemiluminescence
(ECL) direct nucleic acid labeling system, and the ECL
reagent kit for horseradish peroxidase were from Amersham
Pharmacia Biotech (Amersham Biosciences UK Limited,
Little Chalfont, Buckinghamshire, UK) IL-1b was
pur-chased from Peprotech Inc (Rocky Hill, NJ, USA) DHA,
peroxide-free AA and EPA were from Cayman Chemical
(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-ochem (San Diego, CA, USA) The polyclonal anti-Rb and
p-Rb Igs were from Cell Signaling Technology (Beverly,
MA, USA) Anti-caveolin-1 Ig and anti-caveolin-3 Ig were
from Transduction Laboratory (BD) (LC Laboratories,
Woburn, MA, USA) Anti-ERK1/2MAP kinase Ig,
anti-actin Ig, and monoclonal anti-(cyclin D1) Ig (HD11) were
from Santa-Cruz Biotechnology (Santa Cruz, CA, USA)
Peroxidase-conjugated rabbit anti-mouse IgG and
peroxi-dase-conjugated goat anti-rabbit IgG were from BioSys
(Compie`gne, France) R Mu¨ller (Institut of Molecular
Biology, Baldinger Strasse, Marburg, Germany) provided
the 973 bp human cyclin D1 promoter fragment linked to a
luciferase reporter gene The dominant-negative Ras N17
and constitutive-active Ras K12 mutants were provided by
F Schweighoffer (ExonHit Therapeutics SA, Paris,
France) Constitutive-active Raf-1 BXB and
dominant-negative Raf-1 C4 mutants were gifts from Z Luo (Diabetes
Research Laboratory, Boston, MA, USA) The plasmid Dp85a was obtained from A Eder (Institut fur Biochemie Pharmacology, Innsbru¨ck, Austria) M Braddock (Astra-Zenica, Leicestershire, UK) provided the 697 bp human Egr-1 promoter/reporter gene construct (pGLE) B Derij-ard (Laboratory of Cellular and Molecular Physiology, University of Nice-Sophia Antipolis, Nice, France) provi-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 virus-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 enzymatic digestion of the media of thoracic aortas removed from male Wistar rats (weight 300 g; Elevage Janvier, LeGenest St 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 mM glutamine, 100 unitsÆmL)1of penicillin and 100 lgÆmL)1of streptomycin SMC were subcultured every 7 days, and experiments were performed on cells at three to nine passages after primary culture Confluent cells were maintained in quiescent mode
by incubation for 24 h in serum-free medium 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 lM with albumin : PUFA ratios of 3 for EPA or DHA and 4 for AA The SMC were then washed with NaCl/Pi and stimulated with IL-1b (10 ngÆmL)1) or 10% (v/v) fetal bovine serum for 24 h Appropriate inhibitors were added in some experiments RT-PCR
Total RNA (1.5 lg) was used as a template for reverse transcription First-strand cDNA synthesis and semiquan-titative PCR with glyceraldehyde-3-phosphate dehydro-genase (GAPDH) cDNA amplification, as an internal control, were carried out as described previously [22] The primers used for Egr-1 were 5¢-CAGCAGTCCC ATTTACTCAG-3¢ (forward) and 5¢-GACTGGTAGCTG GTATTG-3¢ (reverse) [23] PCR was performed in a Hybaid Omnigene thermocycler under the following con-ditions: denaturation at 95C for 1 min, oligonucleotide annealing for 1 min at 60C, and primary extension at
72C for 1 min Amplifications were carried out for 22 cycles The PCR products of Egr-1 (345 bp), and GAPDH (195 bp) were electrophoresed in a 2% (w/v) agarose gel, blotted, and transferred to a hybond N+ nylon membrane The identity of the amplified cDNA products was confirmed
by hybridization with 5¢-CCCGCCTCCTGCCTACCC TGCCGCC-3¢ for Egr-1 and with 5¢-GTGAACCACGA GAAATATGACAACTCCCTC-3¢ for GAPDH The oligonucleotide probes were labelled and detected by using the ECL direct nucleic acid labelling detection kit (Amer-sham Pharmacia Biotech) The bands on the
Trang 3autoradiogra-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 ng of of the first-strand cDNA and
300 nMprimers Amplification was performed on an iCycler
(Bio-Rad), according to the manufacturer’s instructions,
and cycle parameters were: 50C (2 min) and 95 C
(10 min), followed by 40 cycles of 95C (15 s), 60 C
(30 s) and 72C (30 s) Variations between the levels of
the total cDNA templates in different samples were
normalized by measuring GAPDH 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
double-stranded oligonucleotides were 5¢ end-labelled by using the
T4 polynucleotide kinase Binding reactions were carried
out in 20 lL of binding reaction mixture [10 mMHEPES,
pH 7.9, 50 mM NaCl, 1 mM dithiothreitol, 10% (v/v)
glycerol, 0.2% (v/v) Nonidet P-40, 0.5 mM EDTA]
con-taining 7 lg of nuclear protein and 50 000 counts per
minute of labelled probe Samples were incubated at room
temperature for 15 min and fractionated by electrophoresis
on 5% (w/v) polyacrylamide gels in 0.25· TBE (45 mMTris
borate, 1 mMEDTA) All gels were pre-electrophoresed for
30 min at 150 V without samples Samples were then added
and separated at 150 V for 3 h The separated products
were transferred to Whatman 3MM paper (Whatman Ltd,
Clifton, NJ, USA), which was dried in a gel dryer under
vacuum at 80C, and then exposed to Hyperfilm MP
(Amersham Pharmacia Biotech) The Egr-1 oligonucleotide
5¢-GCGCCCGCCCCCGCCCCCC-3¢ corresponded to the
region ()117 to )99 bp) of the human cyclin D1 promoter
[13] Consensus Sp1 oligonucleotide 5¢-TGAAGCCCCGC
CCCAACGGA-3¢ was used as a competitor (at 100-fold
molar excess) in all the EMSA experiments to eliminate
Sp1 complexes [13] The nuclear factor-Y (NFY)
oligo-nucleotide, 5¢-GGGGTAGGAACCAATGAAATGAAA
CGTTA-3¢, corresponded to the binding site of the rat
albumin promoter [24]
Transfection and luciferase assays
Cultured rat SMC were seeded in 12-well dishes at a
concentration that gave 70% confluence 24 h later The
SMC were transfected with 1.5 lL of LipofectAMINE
Plus (Life Technologies), 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 plasmid (Promega) The amounts of
the relevant expression 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 inter-SH2 domain that confers binding to the catalytic subunit p110), 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 in the same medium containing EPA, DHA or AA After incorpor-ation of PUFA, the cells were washed twice with NaCl/Pi and stimulated (or not stimulated) with IL-1b in serum-free medium The luciferase activity was determined by using a luciferase reporter assay kit (Promega), and signals were detected for 12 s by using a luminometer (Lumat LB9507; Berthold Technologies, Bad Wildbad, Germany) The b-galactosidase activities were measured to normalize variations in transfection
Isolation of caveolae SMC (50· 106) were washed with ice-cold NaCl/Pi and scraped off into lysis buffer containing 1% (v/v) Triton X-100, 25 mM Tris/HCl pH 7.5, 150 mM NaCl, 2.5 mM EDTA and protease inhibitors (Roche Molecular Bioche-micals, Roche Diagnostics, France S.A., Meylan ce´dex, France) Caveolae-enriched membranes were isolated by Optiprep gradient ultracentrifugation, as described previ-ously [25]
Western blotting Cells were lysed in lysis buffer [20 mM Tris, pH 7.5, containing 0.5% (v/v) Nonidet P-40 plus 1 lgÆmL)1 of leupeptine, 1 lgÆmL)1 of pepstatin, 1 mM phenyl-methanesulfonyl fluoride, and 1 mM EDTA] Nuclear extracts were prepared as described above Equal amounts
of protein (20 lg) were fractionated by SDS/PAGE and transferred to poly(vinylidene difluoride) membranes Free binding sites were blocked by incubation overnight at 4C
in NaCl/Picontaining 5% (v/v) nonfat milk and 0.1% (v/v) Tween-20 Blots were washed in NaCl/Pi/Tween and then incubated with the indicated primary antibodies Immuno-blots were developed by using appropriate secondary horseradish peroxidase-coupled antibodies and the ECL Western blotting kit (Amersham Pharmacia Biotech) Measurement of DNA synthesis
Incorporation of the 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 96-well and left to adhere for
24 h Cell growth was then stopped by placing them in serum-free medium for 48 h These cells were then incuba-ted with PUFA for 24 h and stimulaincuba-ted (or not, untreaincuba-ted) with IL-1b or 10% (v/v) fetal bovine serum for a further
24 h Finally, 10 lM BrdU was added to each well and incubation continued for 16 h The cells were then fixed and the BrdU incorporated was quantified by using a commer-cial detection kit (Roche Molecular Biochemicals)
Trang 4Statistical analysis
The measured values are expressed as mean ± SEM
Statistical analysis was performed by one-way analysis of
variance (ANOVA) followed by the Bonferroni in
experiments where the results represent the mean ±
SEM of six independent experiments (n¼ 6) P-values of
< 0.05 were considered to be significant Other data
represent the mean ± SEM of three independent
experi-ments (n¼ 3), in which different conditions are tested in
duplicate
Results
Modulation of SMC proliferation by n-3 and n-6 PUFAs
First, we evaluated the effect of PUFA on IL-1b-induced
SMC proliferation by measuring BrdU incorporation into
DNA A previous study, using rat aorta SMC in primary
culture, showed that SMC, synchronized in quiescence by
depriving them of serum for 1 day and then incubated with
50 lMAA, EPA or DHA for 24 h, incorporated significant
amounts of each PUFA into their membrane
phospholi-pids Incubation with PUFAs at higher concentrations (up
to 100 lM) for a longer time-period (up to 48 h), or with
different albumin : PUFA ratios, 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 with IL-1b alone for 24 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 serum Although 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 Incorporating 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 serum (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 treatment with IL-1b There was also no mitogenic response by IL-1b alone under our cell culture conditions We therefore used fetal bovine serum as a positive control of SMC proliferation
Fig 1 Modulation of smooth muscle cell (SMC) proliferation and
ret-inoblastoma protein (Rb) phosphorylation after supplementation with n-3
and n-6 polyunsaturated fatty acids (PUFAs) (A,B) Effects of n-3 and
n-6 PUFAs in cells stimulated by interleukin-b (IL-1b) and fetal
bovine serum, respectively Serum-starved cells were enriched or not
(untreated) with 50 l M PUFA [arachidonic acid (AA),
eicosapentae-noic acid (EPA) or docosahexaeeicosapentae-noic acid (DHA)] for 24 h and then
stimulated with 10 ngÆmL)1IL-1b or with 10% fetal bovine serum for
24 h Incorporation of 5-bromo-2¢-deoxyuridine (BrdU) was measured
by using a detection kit, as described in the Materials and methods.
The results are expressed as the percentage of stimulation relative to
the value obtained with IL-1b alone (100%) which is the control
experiment shown (–) in (A) The results are expressed as the
mean ± SEM (bars) of six independent experiments with IL-1b
treatment and of three independent experiments with fetal bovine
serum, in which different conditions were tested in duplicate ** ¼
P < 0.01, significantly different compared with AA in IL-1b-treated
cells (C,D) n-3 PUFAs inhibit cyclin D1 expression and
phosphory-lation of Rb Lysates of whole cells were prepared from cells treated as
described above and 20 lg aliquots of protein were separated by SDS/
PAGE [10% (w/v) gels] (C) The gel was blotted with a monoclonal
anti-(cyclin D1) Ig (lower part) and the quality of the preparation and
the amount of protein loaded were evaluated with anti-actin Ig (upper
part) (D) The gel was blotted and incubated with specific
anti-(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 Ig (upper part) Each blot is representative of two independent
experiments.
Trang 5Modulation of cyclin D1 synthesis and
hyperphosphorylation of Rb by n-3 and n-6 PUFAs
Induction of cyclin D1 is one of the earliest effects of
mitogenic factors leading to cell cycle re-entry, G1-phase
progression, and transition to the 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 concentration
of cyclin D1 it until it was barely detectable
As the retinoblastoma protein, Rb, is a key target of
cyclin D1/cyclin-dependent kinase complexes, we
investi-gated whether inhibiting cyclin D1 with EPA or DHA also
affected the phosphorylation status of Rb Western blot
analysis showed that hyperphosphorylated Rb (pRb)
accu-mulated in IL-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 seems to be correlated with
the cyclin D1 promoter activity
Effects of n-3 and n-6 PUFAs on the cyclin D1 gene
promoter
We studied the mechanism(s) underlying the inhibition of
cyclin D1 gene expression by EPA and DHA by examining
the effects of n-3 and n-6 PUFAs incorporation on cyclin
D1 promoter activity Quiescent SMC were transiently
transfected with the )973 bp human cyclin D1 promoter
fragment linked to the luciferase reporter gene These cells
were then incubated or not incubated (–) with different
PUFAs and either IL-1b or 10% (v/v) fetal bovine serum
(positive control) The activity of the cyclin D1 gene
promoter was unchanged after incubation for 24 h with
IL-1b, but greatly increased (threefold) by incubation with
fetal bovine serum (Fig 2A) Whatever the pretreatment of
SMC with n-6 or n-3 PUFAs, the basal cyclin D1 gene
promoter activity was not altered in the absence of IL-1b
(data not shown) However, pretreatment of SMC with AA
increased the cyclin D1 gene promoter activity in response
to IL-1b by twofold and by fourfold in response to fetal
bovine serum In contrast, the incorporation of n-3 PUFAs
(EPA or DHA) did not stimulate the basal cyclin D1 gene
promoter activity EPA and DHA also reduced the
increased cyclin D1 activity in response to serum
IL-1b-induced cyclin D1 promoter activity
in AA-pretreated SMC is dependent on ERK
and PI3-K activation
Numerous studies have shown that cell proliferation and
cyclin D1 gene activation is dependent upon the activation
of MAPK and PI3-K We therefore carried out transient
transfection studies with the)973 bp cyclin D1 promoter,
chemical inhibitors and a dominant-negative mutant of the
p85 subunit from PI3-K, to examine the signalling
path-ways We probed the role of p42/p44 MAPK (ERK1/2) in
AA-induced proliferation using cells incubated with the 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 lM PD98059, another specific MEK inhibitor (data not shown) Transfected SMCs were also treated with the specific inhibitor, LY294002, to determine whether PI3-K was activated in AA-mediated proliferation This inhibitor blocked the activation of the cyclin D1 gene promoter in AA-treated cells stimulated with IL-1b, and strongly reduced the activation by fetal bovine serum Comparable effects were obtained with 200 nM wortman-nin, another PI3-K inhibitor (data not shown) We confirmed the implication of PI3-K in this signalling pathway by using a dominant-negative mutant of the p85 subunit of PI3-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 inhibited 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 mitogenic action of AA in the presence of IL-1b In contrast, the p38 pathway is probably not involved in this activation, because the cotransfection of the p38-MAPK expression vector did not significantly affect the cyclin D1 promoter activity Similar results were obtained with 10 lM SB203580, a specific p38 inhibitor (data not shown)
Action of EPA and DHA on cyclin D1 promoter activity induced by the Ras/Raf pathways
Stimulation of Ras activity is known to promote cell cycle progression and a concomitant activation of cyclin D1 gene expression In order to assess the implication of the Ras/Raf pathway, SMC were cotransfected with the)973 bp cyclin D1 gene promoter and either the dominant-positive or the dominant-negative Ras and Raf mutants, and the effects of incorporated n-6 and n-3 PUFAs were explored in cells treated with IL-1b or 10% (v/v) fetal bovine serum (Fig 3) Ras K12 or Raf BXB (dominant-positive mutants) greatly increased (up to sixfold) the cyclin D1 gene promoter activity
in AA-enriched cells Moreover, the IL-1b-induced cyclin D1 promoter activity remained very sensitive to the action of the dominant-negative Ras N17 and Raf-1 C4 mutants These results indicate that AA-induced cyclin D1 promoter activity is mediated mainly via the Ras/Raf pathway, and that incorporation of n-3 PUFAs interferes with these signalling molecules The cyclin D1 promoter activity induced by Ras or Raf dominant-positive expression vectors
is lower for n-3 PUFA-treated cells than after AA treatment
We estimate that EPA and DHA could exert antiprolifer-ative effects by interfering with Ras/Raf pathways
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 gene that regulates SMC proliferation [12] We demonstrated recently
Trang 6that 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 a32P-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 oligonucleotide 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 (control) gave one complex, which was less intense than that produced 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 extracts from cells pretreated with EPA showed less IL-1b-induced Egr-1 binding activity than AA-enriched cells, although ubiquitous NFY-binding activities were very similar For cells treated with EPA and DHA, the Egr-1-binding activity seems to be less intense than in AA-enriched cells In order to verify these results, we used semiquantitative and real-time RT-PCR analysis to deter-mine the effects of PUFAs upon egr-1 gene expression
Fig 2 Modulation of cyclin D1 promoter activity by supplementation with n-3 and n-6 polyunsaturated fatty acids (PUFAs) (A) Serum-starved smooth muscle cells (SMC) were transiently transfected with the ( )973 to +29) cyclin D1 human promoter/luciferase reporter plasmid, incubated with 50 l M PUFA [arachidonic acid (AA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA)] for 24 h, and stimulated with 10 ngÆmL)1 interleukin-b (IL-1b) or 10% (v/v) fetal bovine serum for 24 h The results, normalized to the b-galactosidase activity, are expressed as the percentage of stimulation relative to the value obtained with IL-1b alone (–) Relative luciferase activities are expressed as the mean ± SEM (bars)
of six independent experiments * ¼ P < 0.05, significantly different from AA in IL-1b-treated cells and # ¼ P < 0.05, significantly different from
AA in fetal bovine serum-treated cells (B) Cells were cotransfected with the ( )973 to +29) cyclin D1 human promoter/luciferase reporter plasmid and the dominant-negative Dp85a mutants, p38 mitogen-activated protein kinase (MAPK) expression vector or preincubated with the MAPK kinase (MEK) inhibitor (50 l M U0126; U) and the phosphatidylinositol 3-kinase (PI3-K) inhibitor (50 l M LY294002; LY), and treated as described above Relative luciferase activities are expressed as the mean ± SEM (bars) of six independent experiments for the treatment with IL-1b and of three independent experiments with fetal bovine serum ** ¼ P < 0.01, significantly different from AA in IL-1b-treated cells.
Trang 7(Fig 4B) Quiescent SMC were treated with IL-1b and
harvested after 1 h, because egr-1 gene expression is
transiently stimulated by cytokine [12,26] A similar
time-dependent regulation is observed after stimulation with fetal
bovine serum (data not shown) Pretreatment of SMC with
AA increased, by fivefold, the amount of Egr-1 mRNA
accumulated in response to IL-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 mutation
affecting the Egr-1-binding site (mut Egr-1) [27] Quiescent
cells incubated or not incubated with 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 pretreatment was
still seen, whereas EPA and DHA incorporation did not
increase cyclin D1 promoter activity in response to IL-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, and the differing effects of n-6 and n-3 PUFAs on
Egr-1 mRNA accumulation are consistent with their action
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 of PUFAs on
transiently transfected egr-1 human gene promoter
()697 bp Egr-1/LUC) activity to confirm the impact of
n-6 and n-3 PUFAs on IL-1b-induced Egr-1 mRNA
accumulation [13] Pretreatment of SMC with AA increased the egr-1 promoter activity twofold in response to IL-1b, whereas EPA and DHA incorporation had no stimulatory effect (Fig 5A) We then investigated the contribution of the MAP kinase/ERK and PI3-K pathways to AA-induced egr-1promoter activation by using the inhibitors U0126 and LY294002 Both drugs severely inhibited the activity of the )697 bp Egr-1/LUC The activity of the egr-1 gene promoter in IL-1b-stimulated cells was not significantly reduced by U0126 or by LY294002 (data not shown), and it was strongly stimulated (up to threefold) by incubation in medium containing fetal bovine serum In parallel to their effects on the cyclin D1 gene promoter, EPA and DHA also reduced the egr-1 gene promoter activity stimulated by the coexpression of the dominant-positive Ras K12 mutant (Fig 5B) The egr-1 promoter activity of cells cotransfected with the dominant-negative Ras N17 and Raf C4 mutants was strongly inhibited, indicating that AA-induced egr-1 gene promoter activity may be mediated via the Ras/Raf pathway Thus, egr-1 gene promoter activity is dependent upon the Ras/Raf-1/MEK/ERK and PI3-K pathways, and EPA and DHA act in opposition to AA by preventing egr-1 gene expression in IL-1b-treated cells However, transient transfection experiments, using the )697 bp human egr-1 promoter fragment, did not completely account for the regulation of egr-1 gene expression in the chromatin context The drastic down-regulation of Egr-1 mRNA levels, which is observed in n-3 treated cells, also indicates that other binding elements may contribute to the modu-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 many 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) human cyclin D1 promoter luciferase/reporter plasmid (alone; control), 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 relative to the value obtained with IL-1b-treated cells transfected with the ( )973 to +29) human cyclin D1 promoter luciferase/reporter plasmid alone (control; –) and are expressed as the mean value ± SEM (bars) of three independent experiments in which different conditions were tested in duplicate.
Trang 8D1 gene promoter is repressed by caveolin-1 [29] We
decided to measure (by Western blotting) the 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 AA 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 As caveolin-1 and caveolin-3 may function as
negative regulators of the p42/44 MAPK [31,32], we used
Western blotting of detergent-resistant membrane fractions
isolated from PUFA-enriched SMC induced by IL-1b, to
Fig 4 Effects of polyunsaturated fatty acid (PUFA) supplementation
on the early growth response factor-1 (Egr-1)-mediated activity of the
cyclin D1 gene promoter (A) Effect of PUFA on the interleukin-b
(IL-1b)-induced DNA-binding activity of Egr-1 Serum-starved cells were
enriched with 50 l M PUFA [arachidonic acid (AA), eicosapentaenoic
acid (EPA) or docosahexaenoic acid (DHA)] for 24 h and then
sti-mulated with 10 ngÆmL)1of IL-1b for 1 h Fetal bovine serum [10%
(v/v)] was added to the cells for 1 h as an activation control Nuclear
extracts were analyzed by electrophoretic mobility shift assay (EMSA)
using a32P-labeled oligonucleotide probe containing Egr-1-binding
sites corresponding to the human cyclin D1 promoter region ( )117 to
)99) and a large excess of unlabelled consensus Sp1 oligonucleotide
[13] The specificity of the Egr-1 complex was assessed by using an
excess (· 100) of unlabeled probe (AA + competitor) A
representa-tive autoradiogram of three independent experiments is shown The
quality of the preparation and the amounts of protein loaded were
evaluated by comparison with the ubiquitously expressed NFY
tran-scription factor (B) Effect of PUFA on IL-1b-induced Egr-1 mRNA.
The upper part of the figure is a representative autoradiogram of three
independent semiquantitative RT-PCR experiments, whereas the
his-togram shown in the lower part of the figure represents data from
real-time quantitative RT-PCR analysis Semiquantitative RT-PCR
and real-time quantitative RT-PCR analyses of Egr-1 mRNA were
normalized with ubiquitous glyceraldehyde-3-phosphate
dehydro-genase (GAPDH) amplification Serum-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 with IL-1b or 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) region of the
cyclin D1 promoter containing the Egr-1 motif (Wt Egr-1) or the
mutant of this region (mut Egr-1) fused to the herpes simplex virus
thymidine kinase minimal promoter Then, the transfected cells were
enriched with PUFA for 24 h and stimulated with IL-1b 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 was assayed as
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.
Trang 9determine 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 6B)
Discussion
Several lines of evidence suggest that the incorporation of
n-3 PUFA causes changes in the SMC membrane that
modulate the mitogenic signal transduction induced by
PDGF [15], serotonin [16] and thromboxane [17] As Cdk2
activity is a key event of the G1-S transition, the inhibition
of Cdk2 phosphorylation and activity by EPA and DHA
may explain the inhibition of DNA synthesis, as cyclins E
and H are not modified [33] Numerous in vitro studies have
shown that the effect of IL-1b on proliferation depends on
the type of SMC, their preparation, and how long they are
exposed to this cytokine Consequently, IL-1b has been considered to be a mitogen and effective co-mitogen acting
in conjunction with other growth factors [2] The present study confirms that the incorporation of AA increases the proliferation of SMC [3,4], but only in response to IL-1b stimulation IL-1b alone does not stimulate the growth of SMC under our culture conditions We demonstrated previously that IL-1b can mobilize PUFA from phospho-lipids in rat SMC via a cytosolic phospholipase A2-dependent mechanism [22] Recent studies have also shown that growth factors cause the release of AA from cell membrane fatty acid pools via the activation of cytosolic PLA2 [34,35] Our working hypothesis is that EPA and DHA, released from the membrane by the IL-1b-induced activation of cytosolic PLA2, do not increase SMC proliferation, while AA does [36] EPA and DHA serve as alternative lipid precursors for all metabolic pathways hitherto recognized for AA, with the formation of trieonic
Fig 5 Effect of arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on egr-1 gene promoter activity induced by Ras/ Raf/ERK and phosphatidylinositol 3-kinase (PI3-K) pathways (A) Smooth muscle cells (SMC) were transiently transfected with the )697 bp human egr-1 promoter fragment fused to the luciferase reporter (pGLE) The cells were stimulated with 10% (v/v) fetal bovine serum for 24 h or enriched 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 without the mitogen-activated protein kinase kinase (MEK) inhibitor, 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 the value obtained from cells treated with IL-1b alone (–) * ¼ P < 0.05, significantly different from AA in IL-1b-treated cells (B) Quiescent SMC were transiently transfected with pGLE (alone; control) 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 above Relative luciferase activities are expressed as the mean value ± SEM (bars) of three independent experiments in which different conditions were tested in duplicate.
Trang 10eicosanoids that have much lower inflammatory and
mitogenic properties than AA-derived lipid mediators [7]
The n-3 PUFAs are also competitive inhibitors 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 or Human Umbelical Vascular Endothelium cells
(HUVEC) days before stimulation with cytokines [38] All
of these data indicate that the mitogenic effect of IL-1b on
SMC is caused, at least in part, by the liberation of PUFA
from membrane phospholipids by cytosolic PLA2
More-over, IL-1b may maintain the synthesis of eicosanoids that
modulate long-term proliferation of SMC by activating the
secreted PLA2 and COX-2 In previous work we have
demonstrated that AA incorporation markedly increases
PGE2 synthesis, while n-3 PUFAs completely reduce the
production of PGE2 [18] The inhibition of PGE2 release
was the consequence of a reduction of COX-2 and secretory PLA2 mRNA expression Moreover, the IL-1b-induced expression of secretory PLA2 in AA-treated cells was inhibited by indomethacin, a pharmacological inhibitor of COX
AA, and probably its metabolites, stimulates SMC proliferation by activating the MAPK pathway [4] We find that the specific MEK1/2 inhibitor, U0126, blocks the IL-1b-induced cyclin D1 gene promoter activity in AA-enriched cells Overexpression of the dominant-negative mutants, Ras N17 and Raf C4, significantly suppresses cyclin D1 promoter activity, thus indicating that Ras and Raf are also required for activation by AA We conclude that the mitogenic effects of AA in SMC are mediated by activation of the Ras/Raf/MAPK/ERK pathways We also find that the incorporation of n-3 PUFAs – EPA and DHA – does not induce the cyclin D1 gene promoter activity in cells stimulated with IL-1b or 10% fetal bovine serum In addition, cyclin D1 promoter activity, induced by the overexpression of the dominant-positive mutants Ras K12 and Raf BXB, is much lower in cells with incorporated n-3 PUFAs than in cells containing AA These different effects of 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 increases 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 with the 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 be essential and 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 PI3-K pathways The promoter region of the cyclin D1 contains multiple cis-elements, all involved in activation of the transcription [40,41] We have shown previously that the coordinated activation of proinflammatory 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 factors In addition, the expression of genes, associated with athero-sclerosis, that contain overlapping GC-box expression are controlled at the transcriptional level by the transiently expressed Egr-1 [12,14] The model proposed involves the displacement of Sp1 factor by Egr-1 on promoter regions
In vitroand in vivo studies on SMC indicate that the latter factor is implicated in the control of cell proliferation [40] and the transcription of the cyclin D1 gene [41] This present study shows that, in contrast to EPA or DHA, AA pretreatment of SMC stimulated with IL-1b rapidly leads to egr-1gene expression and increases the binding of Egr-1, as observed for Swiss 3T3 fibroblasts [42] In addition, our func-tional approach demonstrates that activation of the cyclin D1 gene through the ()137 to )99 bp) region of the pro-moter is mediated by Egr-1 in a Ras/Raf/MAPK-dependent
Fig 6 Effect of n-3 and n-6 polyunsaturated fatty acid (PUFA)
sup-plementation on caveolin-1 and caveolin-3 expression and their impact on
p42/44 mitogen-actived protein kinase (MAPK) in caveolae (A) Lysates
of whole cells were prepared from cells treated as described previously,
and 20 lg aliquots of protein were separated by SDS/PAGE [12%
(w/v) gel] The gels were blotted and incubated with specific
anti-(caveolin-1) and anti-(caveolin-3) Ig (B) Caveolae fractions were
iso-lated from smooth muscle cells (SMC) as described in the Materials
and methods The subcellular enriched caveolae fraction, concentrated
by acetone precipitation, was separated by SDS/PAGE [10% (w/v) gel]
and immunoblotted with anti-MAP kinase ERK1/2 The quality of the
preparation and the amount of protein loaded were evaluated by using
anti-actin Ig Each blot is representative of three independent
experi-ments.