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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:771.)
© 2001 American Heart Association, Inc.

Vascular Biology

Lysophosphatidylcholine Induces Early Growth Response Factor-1 Expression and Activates the Core Promoter of PDGF-A Chain in Vascular Endothelial Cells

Masafumi Morimoto; Noriaki Kume; Susumu Miyamoto; Yasushi Ueno; Hiroharu Kataoka; Manabu Minami; Kazutaka Hayashida; Nobuo Hashimoto; Toru Kita

From the Departments of Geriatric Medicine (N.K., M. Minami, K.H., T.K.) and Neurosurgery (M. Morimoto, S.M., Y.U., H.K., N.H.), Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Correspondence to Noriaki Kume, MD, PhD, Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail nkume{at}kuhp.kyoto-u.ac.jp

	Abstract

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Abstract—Lysophosphatidylcholine (lyso-PC), a polar phospholipid that is increased in atherogenic lipoproteins and atherosclerotic lesions, has been shown to transcriptionally induce the expression of endothelial genes relevant to atherogenesis. In cultured bovine aortic endothelial cells (BAECs), we show that lyso-PC induces the expression of early growth response factor (Egr)-1 and thereby activates the proximal promoter of the platelet-derived growth factor (PDGF)-A chain located 55 to 71 bp upstream from the transcription start site, which has been shown to be crucial for PDGF-A chain expression induced by fluid shear stress and fibroblast growth factor-1. Northern blot analyses showed that lyso-PC (10 to 20 µmol/L) transiently (30 minutes to 1 hour) induced expression of Egr-1 mRNA. Induced expression of Egr-1 mRNA, which was associated with increased amounts of Egr-1 protein in nuclei, preceded PDGF-A chain mRNA induction in lyso-PC–activated BAECs. Nuclear runoff assay revealed that lyso-PC stimulates transcription of the Egr-1 gene. Transient transfection of the oligonucleotide corresponding to the proximal promoter of the PDGF-A chain (oligo A) linked to the luciferase reporter gene revealed that lyso-PC can activate the core promoter of the PDGF-A chain by 5-fold. Insertion of a guanine at 3 sites in the oligo A abolished the lyso-PC–induced increases in luciferase activities. Electrophoretic mobility shift assay with use of radiolabeled oligo A showed a lyso-PC–inducible shift band, which was suppressed by excess amounts of unlabeled oligo A or an anti–Egr-1 antibody. In addition, lyso-PC–induced Egr-1 expression was inhibited by PD98059, a specific inhibitor of mitogen-activated protein kinase kinase-1 (MEK1), suggesting that lyso–PC-induced expression of Egr-1 depends on the MEK1/extracellular signal–regulated kinase pathway. Taken together, transcriptional activation of Egr-1–dependent genes by this atherogenic lipid may be a key regulator of atherogenesis.

Key Words: early growth response factor • lysophosphatidylcholine • platelet-derived growth factor • ERK signaling pathway

	Introduction

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Lysophosphatidylcholine (lyso-PC) is a prominent phospholipid component of atherogenic lipoproteins and atherosclerotic lesions.^{1 2 3} Lyso-PC is also generated in wounds and inflammatory lesions through the actions of extracellularly secreted phospholipase A₂.⁴ Previous reports have shown that lyso-PC can induce gene expression of platelet-derived growth factor (PDGF)-A and -B chains, heparin-binding epidermal growth factor,^{5 6 7} vascular cell adhesion molecule-1, intercellular adhesion molecule (ICAM)-1,⁸ endothelial cell NO synthase (ecNOS),^{9 10} and cyclooxygenase-2¹¹ in cultured vascular endothelial cells, in addition to the induction of apoptosis¹² as well as the inhibition of endothelium-dependent vasorelaxation¹³ and endothelial cell migration.¹⁴

With regard to the signal transduction pathways, lyso-PC has been shown to mobilize intracellular calcium,¹⁵ stimulate the tyrosine phosphorylation of platelet and endothelial cell adhesion molecule-1,¹⁶ disrupt a receptor–G protein coupling,¹⁷ elevate intracellular cAMP,¹⁸ activate mitogen-activated protein (MAP) kinases such as extracellular signal–regulated kinase (ERK)¹⁹ and c-Jun N-terminal kinase (JNK),^{19 20} protein kinase C,²¹ activator protein (AP)-1,^{20 22} and nuclear factor (NF)-B²³ in cultured vascular endothelial cells. Furthermore, reagents (such as forskolin and dibutyryl cAMP) that increase intracellular cAMP suppress lyso-PC–induced expression of PDGF-B chain and ICAM-1²⁴ ; however, it remains unclear whether these signal transductions elicited by lyso-PC are directly linked to gene transcription induced by lyso-PC. Cieslik and colleagues^{25 26} have shown that lyso-PC can activate stimulatory protein (Sp)-1–dependent transcription of the ecNOS gene; however, it remains unclear which factors are involved in the transcriptional regulation of other genes, including PDGF-A chain.

Recent studies have revealed that the human PDGF-A chain gene contains a single TATA box 36 bp upstream from the transcription initiation site.^{27 28 29} The proximal promoter of the PDGF-A chain gene, which is located between -71 and -55 bp upstream from the transcription start site, contains overlapping recognition elements for the zinc-finger transcription factors, early growth response factor (Egr)-1 and Sp-1.^{30 31 32} This site is constitutively occupied by Sp-1; however, transcriptional activation of this gene involves the replacement of this binding site with Egr-1, whose expression is strongly elevated in atherosclerotic lesions³³ and can rapidly be induced by biological stimuli such as fibroblast growth factor and fluid shear stress.^{34 35 36 37 38} In the present study, therefore, we have tested the hypothesis that lyso-PC may induce expression of Egr-1 and that lyso-PC–induced PDGF-A chain expression may depend on transcriptional activation of this site by Egr-1.

	Methods

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Reagents
Lyso-PC (1-palmitoyl) was obtained from Avanti Polar Lipids. Antibodies directed to Egr-1 (rabbit polyclonal IgG), Sp-1 (goat polyclonal IgG), c-Jun (rabbit polyclonal IgG), and c-Fos (goat polyclonal IgG) were obtained from Santa Cruz Biotechnology Inc. PD98059 and SB202190 were purchased from Calbiochem Corp.

Cell Culture
Bovine aortic endothelial cells (BAECs) were harvested from bovine aortas by scraping with a sterile glass coverslip and were cultured in DMEM supplemented with 10% (vol/vol) FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin and grown in an atmosphere of 95% air/5% CO₂ at 37°C. Confluent BAECs with passage numbers between 5 and 20, which were serum-starved for 24 hours, were used for experiments.

Northern Blot Analysis
Total cellular RNA was isolated from BAECs by the acid-guanidinium phenol-chloroform method. Equal amounts of RNA were subjected to 1% agarose gel electrophoresis containing formaldehyde and subsequently transferred onto nitrocellulose membranes (Schleicher & Schuell, Inc). Northern blots were hybridized with cDNA probes for Egr-1 and PDGF-A chain labeled with [-³²P]dCTP with the use of random hexanucleotide primers (DNA Labeling Kit, Pharmacia), exposed to a phosphorimaging plate, and analyzed by Fujix Bioimage Analyzer BAS 2000 (Fuji Photo Film Co Ltd).

Nuclear Runoff Assay
A nuclear runoff assay was performed as previously described,⁵ with minor modification. Briefly, the cells were washed with ice-cold PBS and lysed with 0.5% Nonidet P-40 solution (10 mmol/L Tris HCl, 10 mmol/L NaCl, 3 mmol/L MgCl₂, and 0.5% NP-400 [vol/vol], pH 7.4). The nuclei were isolated by centrifugation and resuspended in a 40% glycerol buffer (50 mmol/L Tris HCl, 40% [vol/vol] glycerol, 5 mmol/L MgCl₂, and 0.1 mmol/L EDTA, pH 8.3). Nascent transcription in vitro was performed with [³²P]UTP and other unlabeled nucleotides at 30°C for 30 minutes. Transcribed RNA was isolated by a RNA isolation reagent (Isogen-LS, Wako Pure Chemical), followed by denaturation with sodium hydroxide and ethanol precipitation. Linearized target cDNAs (5 µg in plasmid form) were alkali-denatured and immobilized onto Hybond-N+ membranes by use of a slot-blot apparatus (Schleicher & Schuell Inc). The membranes were hybridized with transcribed RNAs containing an equal amount of radioactivity in a solution containing 50% (vol/vol) formamide, 5x SSPE (1x SSPE consists of 0.15 mol/L NaCl, 10 mmol/L NaH₂PO₃, and 1 mmol/L EDTA, pH 7.4), 0.1% (wt/vol) SDS, 10% (vol/vol) Denhardt’s solution, and denatured salmon sperm DNA at 42°C for 36 hours. Filters were washed in 1x SSC with 0.1% (wt/vol) SDS for 15 minutes at room temperature, washed with 0.2x SSC supplemented with 0.1% (wt/vol) SDS for 10 minutes at 42°C, and then autoradiographed with Fujix Bioimage Analyzer BAS2000 (Fuji Photo Film).

Plasmid Constructs
Oligonucleotides were synthesized by CyberSyn and purified by using reverse-phase C18 cartridges. The nucleotide sequence of oligo A, which consists of the nucleotide sequence corresponding to the PDGF-A chain core promoter located between -76 and -47 bp upstream from the transcription start site, is 5'-GGGGGGGGCGGGGGCGGGG-GCGGGGGAGGG-3' (sense strand), and the complementary strand was annealed. The nucleotide sequence of mutant oligo A is 5'-GGGGGGGGCGGGGGGCGGGGGGCGGGGGGAGGG-3' (G insertion sites are underlined). Oligo A and the mutant oligo A were subcloned into the Xho/HindIII site of the pGL2 vector (Promega) and were designated A.pGL2-Luc and Am.PGL2-Luc, respectively.

DNA Transfection and Luciferase Assay
Transfections were performed with 2 µg of each construct in combination with 50 ng of pRL-SV40 vector (Promega), which contains the simian virus 40 (SV40) early enhancer/promoter region and thereby provides strong and constitutive expression of Renilla luciferase as an internal control. BAECs cultured in a 12-well plate were transfected with each plasmid construct by the lipofection method with the use of LipofectAMINE (Life Technologies, Inc; GIBCO-BRL). Eight hours after transfection, the cells were washed with PBS, replaced with DMEM with 1% FCS, and grown to confluence and near quiescence (3 or 4 days after transfection). After reaching confluence, BAECs were incubated for 1 hour with lyso-PC in serum-free DMEM and subsequently incubated with DMEM in the absence of lyso-PC for an additional 6 hours. Firefly and Renilla luciferase activities in the BAEC lysates were measured by using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activities were normalized for the protein concentrations of cell lysates.

Nuclear Protein Extraction
BAECs were lysed in buffer A (10 mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl₂, 20 mmol/L KCl, 0.5 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, and 0.5% Nonidet P-40) by incubation for 10 minutes at 4°C. The cell lysate was recentrifuged at 16 000 rpm, and the pelleted nuclei were lysed in buffer C (20 mmol/L HEPES, pH 7.9, 25% glycerol, 420 mmol/L NaCl, 0.2 mmol/L EDTA, 1.5 mmol/L MgCl₂, 0.5 mmol/L dithiothreitol, and 0.2 mmol/L phenylmethylsulfonyl fluoride) by gentle shaking for 20 minutes at 4°C. The nuclear extract was clarified by centrifugation, and the supernatant was stored at -80°C until use.

Western Blot Analysis
Nuclear protein extracts were prepared as described above. Protein concentrations were determined by Bradford protein assay reagent (Bio-Rad Laboratories). Nuclear fractions of proteins were resuspended in 20 µL of SDS sample buffer, boiled with 15% 2-mercaptoethanol, and then subjected to SDS–polyacrylamide (8%) gel electrophoresis. Electrophoresed proteins were electroblotted at 100 V for 1 hour onto nitrocellulose membranes (ECL, Amersham) in a buffer containing 25 mmol/L Tris-Cl, 192 mmol/L glycine, and 5% methanol. The membranes were incubated with a rabbit polyclonal antibody directed to Egr-1 (Santa Cruz) for 1 hour as a primary antibody (1:10 000 dilution), washed vigorously, and then incubated with peroxidase-conjugated anti-rabbit IgG (Amersham) for 1 hour. After a wash with PBS containing 0.05% Tween 20, the bands were visualized by ECL reagents (Amersham). Blots were exposed to x-ray films for 1 to 10 minutes.

Oligonucleotide Synthesis and Radiolabeling
Oligo A (5'-GGGGGGGGCGGGGGCGGGGGCGGGGGAGGG-3') was annealed with the antisense strand, and the double-stranded oligonucleotides were end-labeled with [-³²P]dATP (Amersham) by use of T4 polynucleotide kinase (New England Biolabs, Inc). The unbound nucleotides were removed by Chromaspin-10 columns (Clontech Laboratories).

Electrophoretic Mobility Shift Assay
Binding reactions were carried out in a total volume of 10 µL containing 5 to 10 µg nuclear extract, 1 µg poly(dIdC)-poly(dIdC) (Sigma), and ³²P-labeled oligonucleotide probe (100 000 cpm) in 10 mmol/L Tris HCl, pH 7.5, 10 mmol/L NaCl, 1 mmol/L EDTA, 5% glycerol, and 1 mmol/L dithiothreitol at 25°C for 30 minutes. Nuclear extract was preincubated with 1 µL of an affinity-purified anti-peptide antibody (Santa Cruz Biotechnology) for 10 minutes before the addition of the oligonucleotide probe. Nuclear extract–oligonucleotide mixtures were subjected to electrophoresis through 5% (wt/vol) polyacrylamide gels containing 10% glycerol. After they were dried, the gels were autoradiographed and analyzed by Fuji Bioimage Analyzer BAS2000 (Fuji Photo Film Co Ltd).

	Results

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Transient Expression of Egr-1 mRNA by Lyso-PC Precedes PDGF-A Chain mRNA Expression
To determine the effects of lyso-PC on Egr-1 mRNA levels, Northern blot analyses were carried out. Lyso-PC concentrations as low as 10 µmol/L were able to induce Egr-1 mRNA, and a maximal increase in the amount of Egr-1 mRNA was observed at 15 µmol/L lyso-PC (data not shown) after 1 hour of the treatment. Time-course experiments showed that Egr-1 mRNA levels were increased within 30 minutes, peaked after 1 hour, and then declined (Figure 1). Egr-1 mRNA was not induced by treatment with the cell culture medium alone (data not shown). Transient expression of Egr-1 mRNA by lyso-PC preceded the induced expression of PDGF-A chain mRNA by lyso-PC (Figure 1).

View larger version (58K): [in this window] [in a new window]   Figure 1. Time-dependent expression of Egr-1 mRNA induced by lyso-PC. Total cellular RNA isolated from BAECs exposed to 15 µmol/L lyso-PC for the indicated time periods was subjected to Northern blot analyses (15 µg RNA per lane) with 32P-labeled cDNA probes. Bands for 28S and 18S ribosomal RNA visualized by ethidium bromide staining to control the amounts of RNA loaded are also shown.

Lyso-PC–Induced Expression of Egr-1 mRNA Depends on Gene Transcription
To examine whether lyso-PC–induced Egr-1 mRNA expression depends on new RNA synthesis, we pretreated BAECs with 5 µg/mL actinomycin D for 30 minutes before exposure to lyso-PC. Actinomycin D pretreatment abolished lyso-PC–induced expression of Egr-1 mRNA as well as that induced by phorbol 12-myristate 13-acetate (PMA, data not shown). To obtain direct evidence that lyso-PC activates transcription of the Egr-1 gene, a nuclear runoff assay was carried out. As shown in Figure 2, 15 µmol/L lyso-PC treatment for 1 hour significantly activated transcription of the Egr-1 but not the GAPDH gene. These results indicate that increased levels of Egr-1 mRNA induced by lyso-PC result primarily from gene transcription.

View larger version (30K): [in this window] [in a new window]   Figure 2. Lyso-PC–induced Egr-1 mRNA expression depends on gene transcription. After BAECs were treated with or without lyso-PC (15 µmol/L) for 1 hour, nuclear extracts were isolated and subjected to nuclear runoff assay to measure transcriptional activities for Egr-1 and GAPDH genes.

Induced Expression of Egr-1, but Not Sp-1, in Nuclei of Lyso-PC–Treated BAECs
Nuclear extracts of BAECs exposed to lyso-PC for 1 hour were subjected to Western blot analysis to measure the amount of Egr-1 protein in nuclei. Lyso-PC significantly increased nuclear Egr-1 protein levels in a dose-dependent fashion; a maximal increase in Egr-1 was observed at 15 µmol/L lyso-PC (Figure 3A). Egr-1 protein levels in nuclei were induced within 30 minutes and remained elevated for 1 hour (Figure 3B). In contrast, nuclear levels of Sp-1 were not significantly induced by lyso-PC (Figure 3B). We performed the same experiments in human umbilical vein endothelial cells, and the results were almost the same as those observed in BAECs (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 3. Lyso-PC–induced expression of Egr-1 protein in nuclei. BAECs were treated with different concentrations of lyso-PC for 1 hour (A) or with 15 µmol/L lyso-PC for the indicated periods of time (B), and immunoblotting was performed with antibodies directed to Egr-1 and Sp-1.

Lyso-PC Activates PDGF-A Chain Core Promoter–Dependent Gene Transcription
To determine whether lyso-PC can activate the PDGF-A chain core promoter–dependent gene transcription, oligonucleotides corresponding to the PDGF-A chain core promoter (oligo A) were linked to the luciferase reporter gene and transiently transfected into BAECs. After treatment with lyso-PC, luciferase activities were measured. As shown in Figure 4, BAECs transfected with oligo A construct showed an 5-fold increase in the luciferase activity after exposure to lyso-PC. In contrast, the luciferase activity was not significantly induced by lyso-PC in BAECs transfected with the mutant (3 guanine insertions) oligo A (oligo Am) construct (Figure 4).

View larger version (17K): [in this window] [in a new window]   Figure 4. Lyso-PC stimulates transcription of PDGF-A core promoter–luciferase fusion genes. BAECs were transiently transfected with luciferase reporter gene linked to either wild-type (A.pGL2-Luc) or mutant (Am.pGL2-Luc) oligo A and subsequently treated with or without lyso-PC (15 µmol/L) for 2 hours. After additional incubation without lyso-PC for 6 hours, activities of luciferase were measured. Each value (arbitrary units) is indicated as mean±SD from 6 independent experiments.

Lyso-PC–Induced Egr-1 Binds to the Core Promoter of PDGF-A Chain Gene
To determine whether lyso-PC–induced Egr-1 in nuclei can bind to the core promoter of the PDGF-A chain, an electrophoretic mobility shift assay (EMSA) was carried out with the use of radiolabeled oligo A. As shown in Figure 5A, a nucleoprotein complex was induced by treatment with lyso-PC for 1 hour, and this complex appeared identical to that induced by PMA, a potent inducer of Egr-1 and PDGF-A chain gene transcription. This complex declined after 2 hours (Figure 5A). Addition of a 100-fold excess amount of unlabeled oligo A completely abolished the lyso-PC–induced shift band, indicating that this binding is specific to oligo A (Figure 5B). The same molar excess of oligo Am slightly reduced the appearance of the lyso-PC–induced shift band; however, the effect was much less prominent compared with that of oligo A(Figure 5B). Inclusion of a polyclonal antibody directed to Egr-1, but not c-Jun or c-Fos, eliminated the lyso-PC–induced shift band without affecting the other bands (Figure 5B), indicating that Egr-1 binds to this site of the promoter in lyso-PC–treated BAECs. An anti–Sp-1 antibody did not affect the constitutive or lyso-PC–inducible shift bands with radiolabeled oligo A (data not shown). These results provide evidence that Egr-1 induced by lyso-PC can specifically bind to the core promoter of the PDGF-A chain gene.

View larger version (29K): [in this window] [in a new window]   Figure 5. Nuclear proteins isolated from lyso-PC–treated BAECs bind to core promoter of PDGF-A chain. A, Nuclear extracts from BAECs exposed to 15 µmol/L lyso-PC for 0, 1, 2, and 4 hours or 100 ng/mL PMA for 1 hour were subjected to EMSA with [32P]oligo A. B, Nuclear extracts from BAECs exposed to 15 µmol/L lyso-PC for 1 hour were subjected to EMSA with [32P]oligo A. A 100-fold molar excess of unlabeled oligo A, oligo Am, and polyclonal antibodies directed to Egr-1, c-Jun, or c-Fos were included in the nuclear extracts 15 minutes before the addition of [32P]oligo A.

Lyso-PC Induces Egr-1 in BAECs via Activation of the MEK-ERK Pathway
To explore whether the MAP kinase kinase (MEK)-ERK pathway is involved in lyso-PC–induced Egr-1 expression, the effects of PD98059, a specific inhibitor for MEK1, were examined. As shown in Figure 6, PD98059, but not the p38 MAP kinase inhibitor SB202190, dose-dependently inhibited lyso-PC–induced Egr-1 expression. PMA-induced expression of Egr-1 was also blocked by PD98059, but not by SB202190, as previously shown.³⁹ These results thus indicate that the MEK-ERK pathway, but not p38 MAP kinase, is involved in lyso-PC–induced expression of Egr-1.

View larger version (32K): [in this window] [in a new window]   Figure 6. Lyso-PC–induced Egr-1 expression depends on MEK1-ERK pathway. After pretreatment for 30 minutes with 5 or 50 µmol/L PD98059 (PD, A) or 1 or 10 µmol/L SB202190 (SB, B), BAECs were incubated with lyso-PC (15 µmol/L) for 1 hour, and their nuclear extracts were subjected to immunoblotting with an anti–Egr-1 antibody.

	Discussion

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Endothelial activation by oxidized LDL and its lipid constituents, including lyso-PC, has been implicated in atherogenesis.^{1 2 3} The present study demonstrates, for the first time, that lyso-PC can rapidly induce the expression of Egr-1 and activate the core promoter of PDGF-A chain gene, suggesting that lyso-PC–induced expression of Egr-1 appears to mediate lyso-PC–induced transcription of the PDGF-A chain gene in vascular endothelial cells. Because binding sites for Egr-1 are present in the promoters of transforming growth factor-ß,⁴⁰ basic fibroblast growth factor,⁴¹ tissue factor,^{42 43 44} ICAM-1,⁴⁵ and CD44,⁴⁶ the induced expression of Egr-1 by lyso-PC may be a common molecular mechanism involved in lyso-PC–induced gene expression. In fact, our previous studies have demonstrated that lyso-PC can induce ICAM-1 gene expression.^{8 24}

The data in the present study indicate that Egr-1 appears to be involved in lyso-PC–induced PDGF-A chain expression; however, we do not know, at present, whether nuclear expression of Egr-1 alone is sufficient for lyso-PC–induced gene expression. In fact, previous reports have shown that lyso-PC can activate NF-B²³ and AP-1.^{20 22} Furthermore, our recent studies have demonstrated that lyso-PC can also activate Jun2 12-O-tetradecanoylphorbol 13-acetate response element.⁴⁷ Therefore, other transcriptional regulatory mechanisms may also be involved in lyso-PC–induced gene transcription. In addition, because NF-B can act in cooperation with other transcription factors, such as high-mobility group protein I(Y),⁴⁸ Sp-1,⁴⁹ activating transcription factor-2,⁵⁰ and AP-1,⁵¹ Egr-1 might also interact with other transcription factors. These points remain to be clarified.

In the present study, we were unable to demonstrate constitutive bindings of Sp-1 to the core promoter of the PDGF-A chain, although we used the anti–Sp-1 antibody from the same commercial source as the previous studies.^{36 37} We do not know the exact reason; however, this might result from the difference in the experimental conditions in EMSA, in the quality of the antibody, or in the basal culture conditions. In addition, previous studies with the ecNOS promoter have shown that protein phosphatase 2A–dependent enhancement of Sp-1 binding is involved in lyso-PC–induced ecNOS transcription.²⁵ In contrast to lyso-PC–induced ecNOS transcription,²⁶ our preliminary results have shown that okadaic acid, which inhibits the activities of phosphatases, does not affect lyso-PC–induced PDGF-A chain expression (data not shown). Therefore, lyso-PC may stimulate multiple transcriptional regulatory mechanisms, some of which remain to be elucidated.

Lyso-PC appears to activate the transcription of Egr-1, because inhibition of de novo RNA synthesis blocked lyso-PC–induced Egr-1 mRNA expression (data not shown). Direct evidence that lyso-PC stimulates Egr-1 gene transcription was demonstrated by nuclear runoff assay (Figure 2). A recent report has indicated that induced expression of Egr-1 by cyclic strain depends on the Ras-ERK pathway but not on Rac-JNK.⁵² In cultured vascular smooth muscle cells, Egr-1 expression by phorbol ester also depends on ERK1 but not p38 MAP kinase.³⁹ Our previous studies have shown that lyso-PC can activate ERK¹⁹ as well as JNK^{19 20} ; therefore, lyso-PC–induced transcription of Egr-1 may involve the Ras-ERK pathway. In fact, a specific inhibitor of MEK1 suppressed lyso-PC–induced Egr-1 expression in the present study (Figure 6).

In summary, the present study demonstrates that lyso-PC can induce the expression of Egr-1 in nuclei and activate the core promoter of the PDGF-A chain. Transcriptional regulation of Egr-1 by this atherogenic lipid may be one of the common molecular mechanisms involved in atherogenesis, because Egr-1 expression is also implicated in the transcriptional regulation of basic transcription element binding protein-2,³⁹ which plays a key role in phenotypic modulation of vascular smooth muscle cells in atherogenesis.⁵³ Further studies related to the roles of Egr-1 and its regulation by atherogenic lipids in atherogenesis may provide new insight into the pathogenesis of this complex disease.

	Acknowledgments

 
This work was supported by the Center of Excellence Grant (12CE2006) and Grants-in-Aid (Nos. 11307018, 11838008, 09281103, and 09281104 from the Ministry of Education, Science and Culture of Japan). We thank Kumiko Kanai and Akemi Saito for excellent technical assistance.

Received April 12, 2000; accepted January 16, 2001.

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