This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morimoto, M. Articles by Kita, T. Search for Related Content PubMed PubMed Citation Articles by Morimoto, M. Articles by Kita, T. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Related Collections Lipid and lipoprotein metabolism Mechanism of atherosclerosis/growth factors Pathophysiology Gene regulation

(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

Top Abstract Introduction Methods Results Discussion References

 
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

Top Abstract Introduction Methods Results Discussion References

 
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

Top Abstract Introduction Methods Results Discussion References

 
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

Top Abstract Introduction Methods Results Discussion References

 
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

Top Abstract Introduction Methods Results Discussion References

 
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.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Gimbrone MA Jr, Cybulsky MI, Kume N, Collins T, Resnick N. Vascular endothelium: an integrator of pathophysiological stimuli in atherogenesis. Ann N Y Acad Sci. 1995;748:122–132.[Medline] [Order article via Infotrieve]
2. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity [see comments]. N Engl J Med. 1989;320:915–924.[Medline] [Order article via Infotrieve]
3. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991;88:1785–1792.
4. Vadas P, Pruzanski W. Role of secretory phospholipases A2 in the pathobiology of disease. Lab Invest. 1986;55:391–404.[Medline] [Order article via Infotrieve]
5. Kume N, Gimbrone MA Jr. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest. 1994;93:907–911.
6. Nakano T, Raines EW, Abraham JA, Klagsbrun M, Ross R. Lysophosphatidylcholine upregulates the level of heparin-binding epidermal growth factor-like growth factor mRNA in human monocytes. Proc Natl Acad Sci U S A. 1994;91:1069–1073.[Abstract/Free Full Text]
7. Nishi E, Kume N, Ochi H, Moriwaki H, Wakatsuki Y, Higashiyama S, Taniguchi N, Kita T. Lysophosphatidylcholine increases expression of heparin-binding epidermal growth factor–like growth factor in human T lymphocytes. Circ Res. 1997;80:638–644.[Abstract/Free Full Text]
8. Kume N, Cybulsky MI, Gimbrone MA Jr. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest. 1992;90:1138–1144.
9. Zembowicz A, Tang JL, Wu KK. Transcriptional induction of endothelial nitric oxide synthase type III by lysophosphatidylcholine. J Biol Chem. 1995;270:17006–17010.[Abstract/Free Full Text]
10. Hirata K, Miki N, Kuroda Y, Sakoda T, Kawashima S, Yokoyama M. Low concentration of oxidized low-density lipoprotein and lysophosphatidylcholine upregulate constitutive nitric oxide synthase mRNA expression in bovine aortic endothelial cells. Circ Res. 1995;76:958–962.[Abstract/Free Full Text]
11. Zembowicz A, Jones SL, Wu KK. Induction of cyclooxygenase-2 in human umbilical vein endothelial cells by lysophosphatidylcholine. J Clin Invest. 1995;96:1688–1692.
12. Sata M, Walsh K. Oxidized LDL activates fas-mediated endothelial cell apoptosis. J Clin Invest. 1998;102:1682–1689.[Medline] [Order article via Infotrieve]
13. Kugiyama K, Kerns SA, Morrisett JD, Roberts R, Henry PD. Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature. 1990;344:160–162.[Medline] [Order article via Infotrieve]
14. Murugesan G, Fox PL. Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein. J Clin Invest. 1996;97:2736–2744.[Medline] [Order article via Infotrieve]
15. Inoue N, Hirata K, Yamada M, Hamamori Y, Matsuda Y, Akita H, Yokoyama M. Lysophosphatidylcholine inhibits bradykinin-induced phosphoinositide hydrolysis and calcium transients in cultured bovine aortic endothelial cells. Circ Res. 1992;71:1410–1421.[Abstract/Free Full Text]
16. Ochi H, Kume N, Nishi E, Moriwaki H, Masuda M, Fujiwara K, Kita T. Tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 induced by lysophosphatidylcholine in cultured endothelial cells. Biochem Biophys Res Commun. 1998;243:862–868.[Medline] [Order article via Infotrieve]
17. Flavahan NA. Lysophosphatidylcholine modifies G protein-dependent signaling in porcine endothelial cells. Am J Physiol. 1993;264:H722–H727.[Abstract/Free Full Text]
18. Yuan Y, Schoenwaelder SM, Salem HH, Jackson SP. The bioactive phospholipid, lysophosphatidylcholine, induces cellular effects via G-protein-dependent activation of adenylyl cyclase. J Biol Chem. 1996;271:27090–27098.[Abstract/Free Full Text]
19. Ozaki H, Ishii K, Arai H, Kume N, Kita T. Lysophosphatidylcholine activates mitogen-activated protein kinases by a tyrosine kinase-dependent pathway in bovine aortic endothelial cells. Atherosclerosis. 1999;143:261–266.[Medline] [Order article via Infotrieve]
20. Fang X, Gibson S, Flowers M, Furui T, Bast RC Jr, Mills GB. Lysophosphatidylcholine stimulates activator protein 1 and the c-Jun N-terminal kinase activity. J Biol Chem. 1997;272:13683–13689.[Abstract/Free Full Text]
21. Sugiyama S, Kugiyama K, Ohgushi M, Fujimoto K, Yasue H. Lysophosphatidylcholine in oxidized low-density lipoprotein increases endothelial susceptibility to polymorphonuclear leukocyte-induced endothelial dysfunction in porcine coronary arteries: role of protein kinase C. Circ Res. 1994;74:565–575.[Abstract/Free Full Text]
22. Ares MP, Kallin B, Eriksson P, Nilsson J. Oxidized LDL induces transcription factor activator protein-1 but inhibits activation of nuclear factor-kappa B in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1995;15:1584–1590.[Abstract/Free Full Text]
23. Sugiyama S, Kugiyama K, Ogata N, Doi H, Ota Y, Ohgushi M, Matsumura T, Oka H, Yasue H. Biphasic regulation of transcription factor nuclear factor-kappaB activity in human endothelial cells by lysophosphatidylcholine through protein kinase C-mediated pathway. Arterioscler Thromb Vasc Biol. 1998;18:568–576.[Abstract/Free Full Text]
24. Ochi H, Kume N, Nishi E, Kita T. Elevated levels of cAMP inhibit protein kinase C–independent mechanisms of endothelial platelet-derived growth factor-B chain and intercellular adhesion molecule-1 gene induction by lysophosphatidylcholine. Circ Res. 1995;77:530–535.[Abstract/Free Full Text]
25. Cieslik K, Lee CM, Tang JL, Wu KK. Transcriptional regulation of endothelial nitric-oxide synthase by an interaction between casein kinase 2 and protein phosphatase 2A. J Biol Chem. 1999;274:34669–34675.[Abstract/Free Full Text]
26. Cieslik K, Zembowicz A, Tang JL, Wu KK. Transcriptional regulation of endothelial nitric-oxide synthase by lysophosphatidylcholine. J Biol Chem. 1998;273:14885–14890.[Abstract/Free Full Text]
27. Bonthron DT, Morton CC, Orkin SH, Collins T. Platelet-derived growth factor A chain: gene structure, chromosomal location, and basis for alternative mRNA splicing. Proc Natl Acad Sci U S A. 1988;85:1492–1496.[Abstract/Free Full Text]
28. Rorsman F, Bywater M, Knott TJ, Scott J, Betsholtz C. Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: alternative exon usage predicts two different precursor proteins. Mol Cell Biol. 1988;8:571–577.[Abstract/Free Full Text]
29. Takimoto Y, Wang ZY, Kobler K, Deuel TF. Promoter region of the human platelet-derived growth factor A-chain gene. Proc Natl Acad Sci U S A. 1991;88:1686–1690.[Abstract/Free Full Text]
30. Dynan WS, Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983;35:79–87.[Medline] [Order article via Infotrieve]
31. Kadonaga JT, Carner KR, Masiarz FR, Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987;51:1079–1090.[Medline] [Order article via Infotrieve]
32. Sukhatme VP, Cao XM, Chang LC, Tsai-Morris CH, Stamenkovich D, Ferreira PC, Cohen DR, Edwards SA, Shows TB, Curran T, et al. A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell. 1988;53:37–43.[Medline] [Order article via Infotrieve]
33. McCaffrey TA, Fu C, Du B, Eksinar S, Kent KC, Bush H Jr, Kreiger K, Rosengart T, Cybulsky MI, Silverman ES, et al. High-level expression of Egr-1 and Egr-1-inducible genes in mouse and human atherosclerosis [see comments]. J Clin Invest. 2000;105:653–662.[Medline] [Order article via Infotrieve]
34. Khachigian LM, Williams AJ, Collins T. Interplay of Sp1 and Egr-1 in the proximal platelet-derived growth factor A-chain promoter in cultured vascular endothelial cells. J Biol Chem. 1995;270:27679–27686.[Abstract/Free Full Text]
35. Silverman ES, Khachigian LM, Lindner V, Williams AJ, Collins T. Inducible PDGF A-chain transcription in smooth muscle cells is mediated by Egr-1 displacement of Sp1 and Sp3. Am J Physiol. 1997;273:H1415–H1426.[Abstract/Free Full Text]
36. Delbridge GJ, Khachigian LM. FGF-1-induced platelet-derived growth factor-A chain gene expression in endothelial cells involves transcriptional activation by early growth response factor-1. Circ Res. 1997;81:282–288.[Abstract/Free Full Text]
37. Khachigian LM, Anderson KR, Halnon NJ, Gimbrone MA Jr, Resnick N, Collins T. Egr-1 is activated in endothelial cells exposed to fluid shear stress and interacts with a novel shear-stress-response element in the PDGF A-chain promoter. Arterioscler Thromb Vasc Biol. 1997;17:2280–2286.[Abstract/Free Full Text]
38. Khachigian LM, Lindner V, Williams AJ, Collins T. Egr-1-induced endothelial gene expression: a common theme in vascular injury. Science. 1996;271:1427–1431.[Abstract]
39. Kawai-Kowase K, Kurabayashi M, Hoshino Y, Ohyama Y, Nagai R. Transcriptional activation of the zinc finger transcription factor BTEB2 gene by Egr-1 through mitogen-activated protein kinase pathways in vascular smooth muscle cells. Circ Res. 1999;85:787–795.[Abstract/Free Full Text]
40. Kim SJ, Glick A, Sporn MB, Roberts AB. Characterization of the promoter region of the human transforming growth factor-beta 1 gene. J Biol Chem. 1989;264:402–408.[Abstract/Free Full Text]
41. Biesiada E, Razandi M, Levin ER. Egr-1 activates basic fibroblast growth factor transcription: mechanistic implications for astrocyte proliferation. J Biol Chem. 1996;271:18576–18581.[Abstract/Free Full Text]
42. Mackman N, Morrissey JH, Fowler B, Edgington TS. Complete sequence of the human tissue factor gene, a highly regulated cellular receptor that initiates the coagulation protease cascade. Biochemistry. 1989;28:1755–1762.[Medline] [Order article via Infotrieve]
43. Cui MZ, Parry GC, Oeth P, Larson H, Smith M, Huang RP, Adamson ED, Mackman N. Transcriptional regulation of the tissue factor gene in human epithelial cells is mediated by Sp1 and EGR-1. J Biol Chem. 1996;271:2731–2739.[Abstract/Free Full Text]
44. Cui MZ, Penn MS, Chisolm GM. Native and oxidized low density lipoprotein induction of tissue factor gene expression in smooth muscle cells is mediated by both Egr-1 and Sp1. J Biol Chem. 1999;274:32795–32802.[Abstract/Free Full Text]
45. Maltzman JS, Carmen JA, Monroe JG. Transcriptional regulation of the ICAM-1 gene in antigen receptor- and phorbol ester-stimulated B lymphocytes: role for transcription factor EGR1. J Exp Med. 1996;183:1747–1759.[Abstract/Free Full Text]
46. Maltzman JS, Carman JA, Monroe JG. Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol Cell Biol. 1996;16:2283–2294.[Abstract]
47. Ueno Y, Kume N, Miyamoto S, Ochi H, Nishi E, Moriwaki H, Hashimoto N, Kita T. Lysophosphatidylcholine phosphorylates CREB and transactivates jun2TRE site of c-jun promoter in vascular endothelial cells. FEBS Lett. 1999;457:241–245.[Medline] [Order article via Infotrieve]
48. Thanos D, Maniatis T. The high mobility group protein HMG I(Y) is required for NF-kappa B-dependent virus induction of the human IFN-beta gene. Cell. 1992;71:777–789.[Medline] [Order article via Infotrieve]
49. Perkins ND, Edwards NL, Duckett CS, Agranoff AB, Schmid RM, Nabel GJ. A cooperative interaction between NF-kappa B and Sp1 is required for HIV-1 enhancer activation. EMBO J. 1993;12:3551–3558.[Medline] [Order article via Infotrieve]
50. Du W, Thanos D, Maniatis T. Mechanisms of transcriptional synergism between distinct virus-inducible enhancer elements. Cell. 1993;74:887–898.[Medline] [Order article via Infotrieve]
51. Stein B, Baldwin AS Jr. Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B. Mol Cell Biol. 1993;13:7191–7198.[Abstract/Free Full Text]
52. Wung BS, Cheng JJ, Chao YJ, Hsieh HJ, Wang DL. Modulation of Ras/Raf/extracellular signal-regulated kinase pathway by reactive oxygen species is involved in cyclic strain-induced early growth response-1 gene expression in endothelial cells. Circ Res. 1999;84:804–812.[Abstract/Free Full Text]
53. Watanabe N, Kurabayashi M, Shimomura Y, Kawai-Kowase K, Hoshino Y, Manabe I, Watanabe M, Aikawa M, Kuro-o M, Suzuki T, et al. BTEB2, a Kruppel-like transcription factor, regulates expression of the SMemb/nonmuscle myosin heavy chain B (SMemb/NMHC-B) gene. Circ Res. 1999;85:182–191. [Abstract/Free Full Text]

This article has been cited by other articles:

				M. Takahashi, Y. Hiyama, M. Yokoyama, S. Yu, Y. Hu, K. Melford, A. Bensadoun, and I. J. Goldberg In Vivo Arterial Lipoprotein Lipase Expression Augments Inflammatory Responses and Impairs Vascular Dilatation Arterioscler. Thromb. Vasc. Biol., March 1, 2008; 28(3): 455 - 462. [Abstract] [Full Text] [PDF]

				J. L. Ingram, A. Antao-Menezes, J. B. Mangum, O. Lyght, P. J. Lee, J. A. Elias, and J. C. Bonner Opposing Actions of Stat1 and Stat6 on IL-13-Induced Up-Regulation of Early Growth Response-1 and Platelet-Derived Growth Factor Ligands in Pulmonary Fibroblasts J. Immunol., September 15, 2006; 177(6): 4141 - 4148. [Abstract] [Full Text] [PDF]

				L. M. Khachigian Early Growth Response-1 in Cardiovascular Pathobiology Circ. Res., February 3, 2006; 98(2): 186 - 191. [Abstract] [Full Text] [PDF]

				A. Md Sheikh, H. Ochi, A. Manabe, and J. Masuda Lysophosphatidylcholine posttranscriptionally inhibits interferon-{gamma}-induced IP-10, Mig and I-Tac expression in endothelial cells Cardiovasc Res, January 1, 2005; 65(1): 263 - 271. [Abstract] [Full Text] [PDF]

				H. Lum, J. Qiao, R. J. Walter, F. Huang, P. V. Subbaiah, K. S. Kim, and O. Holian Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1786 - H1789. [Abstract] [Full Text] [PDF]

				E. Hurt-Camejo, G. Camejo, H. Peilot, K. Oorni, and P. Kovanen Phospholipase A2 in Vascular Disease Circ. Res., August 17, 2001; 89(4): 298 - 304. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morimoto, M. Articles by Kita, T. Search for Related Content PubMed PubMed Citation Articles by Morimoto, M. Articles by Kita, T. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Related Collections Lipid and lipoprotein metabolism Mechanism of atherosclerosis/growth factors Pathophysiology Gene regulation