Eur J Biochem 271, 4462–4473 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04385.x 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 Bereziat and Marise Andreani 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 inflammatory process We have investigated the molecular mechanisms 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 eicosapentaenoic 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 response factor-1 Finally, we have shown that the incorporation of EPA and DHA also increased the concentration 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 evidence indicating that, in contrast to n-6 PUFAs, n-3 PUFAs exert antiproliferative effects on SMC through the mitogenactivated protein kinase/ERK pathway 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 polyunsaturated 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 docosahexaenoic acid (DHA, 22:6 n-3) from fish oil lipids, also known as anti-inflammatory precursors, exert their antiatherosclerotic 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 regulates 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, Batiment A, 5eme etage, Quai Saint Bernard, 75252 Paris Cedex ˆ 5, France Fax: +33 44 27 51 40, Tel.: +33 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 May 2004, revised September 2004, accepted 29 September 2004) Keywords: caveolae; cyclin D1 gene expression; interleukin1b; n-3 PUFA; vascular smooth muscle cells Ó FEBS 2004 Anti-mitogenic action of EPA and DHA (Eur J Biochem 271) 4463 proliferation 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 characteristics 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 concentrations 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 phenylmethanesulfonyl 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 reverse 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 purchased 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 CalBiochem (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, antiactin Ig, and monoclonal anti-(cyclin D1) Ig (HD11) were from Santa-Cruz Biotechnology (Santa Cruz, CA, USA) Peroxidase-conjugated rabbit anti-mouse IgG and peroxidase-conjugated goat anti-rabbit IgG were from BioSys ` (Compiegne, France) R Muller (Institut of Molecular ă 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 dominantnegative 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, Innsbruck, Austria) M Braddock (Astraă Zenica, Leicestershire, UK) provided the 697 bp human Egr-1 promoter/reporter gene construct (pGLE) B Derijard (Laboratory of Cellular and Molecular Physiology, University of Nice-Sophia Antipolis, Nice, France) provided 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 minimal 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, mM glutamine, 100 unitsỈmL)1 of penicillin and 100 lgỈmL)1 of streptomycin SMC were subcultured every 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 for EPA or DHA and for AA The SMC were then washed with NaCl/Pi and stimulated with IL-1b (10 ngỈmL)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 semiquantitative PCR with glyceraldehyde-3-phosphate dehydrogenase (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 conditions: denaturation at 95 °C for min, oligonucleotide annealing for at 60 °C, and primary extension at 72 °C for 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 (Amersham Pharmacia Biotech) The bands on the autoradiogra- Ó FEBS 2004 4464 S Bousserouel et al (Eur J Biochem 271) 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 ng of of the first-strand cDNA and 300 nM primers Amplification was performed on an iCycler (Bio-Rad), according to the manufacturer’s instructions, and cycle parameters were: 50 °C (2 min) and 95 °C (10 min), followed by 40 cycles of 95 °C (15 s), 60 °C (30 s) and 72 °C (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 doublestranded oligonucleotides were 5¢ end-labelled by using the T4 polynucleotide kinase Binding reactions were carried out in 20 lL of binding reaction mixture [10 mM HEPES, pH 7.9, 50 mM NaCl, mM dithiothreitol, 10% (v/v) glycerol, 0.2% (v/v) Nonidet P-40, 0.5 mM EDTA] containing lg of nuclear protein and 50 000 counts per minute of labelled probe Samples were incubated at room temperature for 15 and fractionated by electrophoresis on 5% (w/v) polyacrylamide gels in 0.25· TBE (45 mM Tris borate, mM EDTA) All gels were pre-electrophoresed for 30 at 150 V without samples Samples were then added and separated at 150 V for 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 80 °C, 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) oligonucleotide, 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), constitutively active Ras K12, Raf-1 BXB and p38 MAPK (pCDNA-wt-p38) – were varied The transfection mixtures were incubated for 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 incorporation of PUFA, the cells were washed twice with NaCl/Pi and stimulated (or not stimulated) with IL-1b in serumfree 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 cedex, France) Caveolae-enriched membranes were isolated by Optiprep gradient ultracentrifugation, as described previously [25] Western blotting Cells were lysed in lysis buffer [20 mM Tris, pH 7.5, containing 0.5% (v/v) Nonidet P-40 plus lgỈmL)1 of leupeptine, lgỈmL)1 of pepstatin, mM phenylmethanesulfonyl fluoride, and 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 °C in NaCl/Pi containing 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 Immunoblots 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-bromo2¢-deoxyuridine (BrdU) was measured to determine the effects of PUFA on DNA synthesis SMC were plated in 96well plates at 10 000 cells per 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 incubated with PUFA for 24 h and stimulated (or not, untreated) 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 commercial detection kit (Roche Molecular Biochemicals) Ó FEBS 2004 Anti-mitogenic action of EPA and DHA (Eur J Biochem 271) 4465 Statistical 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 (n ¼ 6) P-values of < 0.05 were considered to be significant Other data represent the mean ± SEM of three independent experiments (n ¼ 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 day and then incubated with 50 lM AA, EPA or DHA for 24 h, incorporated significant amounts of each PUFA into their membrane phospholipids 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 incorporation into phospholipids [18] Under our culture conditions, 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) Fig Modulation of smooth muscle cell (SMC) proliferation and retinoblastoma 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 lM PUFA [arachidonic acid (AA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA)] for 24 h and then stimulated with 10 ngỈmL)1 IL-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 phosphorylation 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 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 proliferation 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 4466 S Bousserouel et al (Eur J Biochem 271) Modulation 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 concentration, 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 investigated whether inhibiting cyclin D1 with EPA or DHA also affected the phosphorylation status of Rb Western blot analysis showed that hyperphosphorylated Rb (pRb) accumulated 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 pathways We probed the role of p42/p44 MAPK (ERK1/2) in Ó FEBS 2004 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 wortmannin, 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 antiproliferative 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 Ó FEBS 2004 Anti-mitogenic action of EPA and DHA (Eur J Biochem 271) 4467 Fig 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 lM PUFA [arachidonic acid (AA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA)] for 24 h, and stimulated with 10 ngỈmL)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 lM U0126; U) and the phosphatidylinositol 3-kinase (PI3-K) inhibitor (50 lM 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 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 32P-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-1btreated 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 AAenriched cells In order to verify these results, we used semiquantitative and real-time RT-PCR analysis to determine the effects of PUFAs upon egr-1 gene expression 4468 S Bousserouel et al (Eur J Biochem 271) Ó FEBS 2004 Fig 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 dominantpositive 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 (Fig 4B) Quiescent SMC were treated with IL-1b and harvested after h, because egr-1 gene expression is transiently stimulated by cytokine [12,26] A similar timedependent 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 expression 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 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-1 promoter 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 modulation of egr-1 gene expression (Fig 4B) Influence of n-3 PUFAs on egr-1 promoter activity, dependent on the Ras/Raf/MEK/ERK pathway 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 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 Caveolins are scaffolding proteins that interact and negatively regulate many components through the p42/44 MAP kinase pathway [28] Interestingly, the activity of the cyclin Ó FEBS 2004 Anti-mitogenic action of EPA and DHA (Eur J Biochem 271) 4469 D1 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 incorporation (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 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 (IL1b)-induced DNA-binding activity of Egr-1 Serum-starved cells were enriched with 50 lM PUFA [arachidonic acid (AA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA)] for 24 h and then stimulated with 10 ngỈmL)1 of IL-1b for h Fetal bovine serum [10% (v/v)] was added to the cells for h as an activation control Nuclear extracts were analyzed by electrophoretic mobility shift assay (EMSA) using a 32P-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 representative 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 transcription 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 histogram 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 dehydrogenase (GAPDH) amplification Serum-starved smooth muscle cells (SMC) were treated or not treated (–) with 50 lM PUFA (AA, EPA or DHA) for 24 h and then stimulated with IL-1b or 10% (v/v) fetal bovine serum for 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 4470 S Bousserouel et al (Eur J Biochem 271) Ó FEBS 2004 Fig 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 lM polyunsaturated fatty acid (PUFA) (AA, EPA or DHA) for 24 h and then incubated with interleukin-b (IL-1b) (10 ngỈmL)1, 24 h) with or without the mitogen-activated protein kinase kinase (MEK) inhibitor, U0126 (U, 50 lM), and the PI3-K inhibitor, LY294002 (LY, 50 lM) 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 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 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 phospholipids 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, 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 Ó FEBS 2004 Anti-mitogenic action of EPA and DHA (Eur J Biochem 271) 4471 Fig Effect of n-3 and n-6 polyunsaturated fatty acid (PUFA) supplementation 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 isolated 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 experiments eicosanoids 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 Moreover, 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 ciselements, 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 atherosclerosis, 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 vitro and 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-1 gene expression and increases the binding of Egr-1, as observed for Swiss 3T3 fibroblasts [42] In addition, our functional approach demonstrates that activation of the cyclin D1 gene through the ()137 to )99 bp) region of the promoter is mediated by Egr-1 in a Ras/Raf/MAPK-dependent Ó FEBS 2004 4472 S Bousserouel et al (Eur J Biochem 271) manner We conclude, in agreement with our previous report, that EPA and DHA act in opposition to AA, to reduce cyclin D1 promoter activity by inhibiting the binding of the transcription factors Egr-1, as for NF-jB and Ets-1, probably by reducing MAPK activity [18] As NF-jB, Ets-1 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 which 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 role in the regulation of cell proliferation, including SMC, by inhibiting signalling molecules [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 proproliferative, oncogenic and antiapoptotic proteins such as EGF-R, PDGF-R, Ras, Src and PI3-K [28] In this respect, signal transduction via the MAPK proliferation cascade is inhibited [31] and overproduction of caveolin-1 also represses cyclin D1 gene promoter activity [29] and regulates 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 ERK1/2 [32] We postulate that the antiproliferative effect of EPA and DHA is a result of the negative regulation of the MAPK pathway, caused by increasing ERK1/2 in isolated caveolae which may be redistributed from the cell surface to the perinuclear cytoplasm Further investigation is needed to provide direct evidence of the influence of n-3 PUFAs on caveolin synthesis in SMC, and extensive research is required to determine how a change in membrane characteristics, following the incorporation of EPA or DHA, alters the caveolin-dependent signal transduction by inhibiting MAPK In conclusion, we show that the cellular uptake 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the incorporation of EPA and DHA modulate SMC proliferation This article describes the differing effects of n-3 and n-6 PUFAs