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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1006.)
© 2000 American Heart Association, Inc.

Vascular Biology

Lysophosphatidylcholine Inhibits Endothelial Cell Migration and Proliferation via Inhibition of the Extracellular Signal–Regulated Kinase Pathway

Yoshiyuki Rikitake; Seinosuke Kawashima; Tomoya Yamashita; Tomomi Ueyama; Satoshi Ishido; Hak Hotta; Ken-ichi Hirata; Mitsuhiro Yokoyama

From the First Department of Internal Medicine (Y.R., S.K., T.Y., T.U., K.H., M.Y.), and the Department of Microbiology (S.I., H.H.), Kobe University School of Medicine, Kobe, Japan.

Correspondence to Seinosuke Kawashima, MD, First Department of Internal Medicine, Kobe University School of Medicine, 7-5-2, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. E-mail kawashim{at}med.kobe-u.ac.jp

	Abstract

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Abstract—Lysophosphatidylcholine (lysoPC), a major lipid component of oxidized low density lipoprotein, inhibits endothelial cell (EC) migration and proliferation, which are critical processes during angiogenesis and the repair of injured vessels. However, the mechanism(s) of lysoPC-induced inhibition of EC migration and proliferation has not been clarified. In this report, we demonstrate the critical role of extracellular signal–regulated kinase (ERK) in growth factor–stimulated EC migration and proliferation as well as their inhibition by lysoPC. EC migration and proliferation stimulated by basic fibroblast growth factor (FGF-2) were blocked by inhibition of ERK activity by both the specific mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 and the overexpression of a dominant-negative mutant of MEK1. Conversely, overexpression of a constitutively active mutant of MEK1 increased EC migration and proliferation, which were comparable to those of ECs stimulated with FGF-2. LysoPC inhibited FGF-2–induced ERK activation via prevention of Ras activation without inhibiting tyrosine phosphorylation of phospholipase C-. Taken together, our data demonstrate that ERK activity is required for FGF-2–induced EC migration and proliferation and suggest that inhibition of the Ras/ERK pathway by lysoPC contributes to the reduced EC migration and proliferation.

Key Words: angiogenesis • lysophosphatidylcholine • signal transduction • basic fibroblast growth factor

	Introduction

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Migration and proliferation of vascular endothelial cells (ECs) play a critical role in diverse normal and abnormal biological processes, including angiogenesis, embryonic development, wound healing, and tumor metastasis (reviewed in References 1 and 2^{1 2} ). Growth factors such as basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor are well known as initiators of EC migration and proliferation through their ability to activate intracellular signaling pathways. FGF-2 causes the activation of Ras with subsequent activation of downstream effector molecules, including extracellular signal–regulated kinase (ERK).³ Ras is activated through conversion of the GDP-bound inactive form to the GTP-bound active form. Activated Ras interacts with Raf-1 and translocates it to the plasma membrane.⁴ Activated Raf-1 then phosphorylates and activates mitogen-activated protein kinase kinase (MEK), leading to the phosphorylation and activation of ERK.⁵ Although the critical role of the ERK pathway in signaling events that lead to cell proliferation is established,^{6 7} the role of the ERK pathway in EC migration is poorly understood.

Lysophosphatidylcholine (lysoPC), a component of oxidized LDL, has been implicated in the pathological states of vascular ECs that are related to atherosclerosis. LysoPC induces the expression of adhesion molecules^{8 9} and genes for growth factors, such as platelet-derived growth factor and heparin-binding epidermal growth factor–like protein¹⁰ in cultured ECs. We^{11 12 13} and others^{14 15 16} have demonstrated that lysoPC suppresses production and release of endothelium-derived nitric oxide through the blockade of receptor-mediated intracellular signaling that activates endothelial nitric oxide synthase, resulting in the impairment of endothelium-dependent relaxation in isolated arterial strips. It was reported that lysoPC inhibits injury-induced migration¹⁷ and growth factor–stimulated DNA synthesis¹⁸ in cultured ECs. However, the molecular mechanism(s) of the lysoPC-induced inhibition of EC migration and proliferation remains unknown.

Here we examined the role of the ERK pathway in EC migration and proliferation. We show that ERK activity plays an important role in these processes, since EC migration and proliferation stimulated by FGF-2 were blocked by inhibition of ERK activity by a specific MEK1 inhibitor and overexpression of a dominant-negative mutant of MEK1. Conversely, overexpression of a constitutively active mutant of MEK1 increased EC migration and proliferation, which were comparable to those in ECs stimulated with FGF-2. We also show that lysoPC suppresses FGF-2–induced ERK activation by inhibiting Ras activation. Our results suggest that inhibition of the Ras/ERK pathway by lysoPC plays a critical role in decreased EC migration and proliferation.

	Methods

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Materials
Synthetic palmitoyl-lysoPC was obtained from Sigma Chemical Co. Human recombinant FGF-2 was from R&D Systems. Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were from Gibco BRL. PD98059 was from Calbiochem. Phosphospecific ERK antibody was from New England Bio Labs. Monoclonal anti-ERK1 antibody, monoclonal anti-MEK1 antibody, and anti-phosphotyrosine antibody (PY-20) were from Transduction Laboratories.

Cell Culture
Cells were isolated from bovine aortas as previously described¹² and cultured in DMEM supplemented with 15% FCS. Bovine aortic ECs (BAECs) were cultured at 37°C in a humidified atmosphere of 95% air, 5% CO₂ and used between passages 5 and 12.

Adenoviral Transfection
Transient transfection was carried out by using replication-defective recombinant adenoviral vectors prepared as described previously.¹⁹ In brief, a constitutively activated mutant of MEK1 (both Ser²¹⁸ and Ser²²² to Glu; MEK1 EE) and a dominant-negative mutant of MEK1 (Asp²⁰⁸ to Ala; MEK1 DN), which were generously provided by Dr K. Okazaki (Biomolecular Engineering Research Institute, Osaka, Japan),^{20 21} were placed into pAdex1CAwt, a cassette cosmid vector (kindly provided by Dr I. Saito, University of Tokyo, Tokyo, Japan) under a CA promoter comprising a cytomegalovirus enhancer and a chicken ß-actin promoter²² (pAdex MEK1 EE and pAdex MEK1 DN, respectively). A recombinant adenovirus was constructed by in vitro homologous recombination in 293 cells²³ with the use of pAdex MEK1 EE, pAdex MEK1 DN, or pAdex LacZ and the adenovirus DNA–terminal protein complex. Cells were grown on 60-mm culture dishes. After reaching confluence, cells were infected with recombinant adenovirus expressing MEK1 EE, MEK1 DN, or LacZ (Ad.MEK1 EE, Ad.MEK1 DN, or Ad.LacZ, respectively) for 1 hour at 37°C in a 95% air, 5% CO₂ atmosphere. The viral suspension was removed and cells were cultured for 48 hours.

Migration Assay
Migration assay was performed in modified Boyden chambers. Cell migration was examined in a 48-well microchemotaxis chamber with a gelatin-coated polycarbonate membrane with 5-µm pores. Cells were suspended in DMEM–1% FCS at a concentration of 5.0x10⁵ cells/mL. After treatment with lysoPC, PD98059, or vehicle (0.1% dimethyl sulfoxide) at room temperature before being seeded, the cell suspension was added to each upper well. DMEM–1% FCS containing FGF-2 (10 ng/mL) was placed in the lower compartment, and cells were then incubated at 37°C (95% air, 5% CO₂). After 6 hours, unmigrated cells were removed from the upper side of the filters, which were then fixed with methanol, followed by counterstaining with hematoxylin. The number of cells was counted microscopically at x400 magnification. In each experiment, migration for each concentration of a given test substance was assessed in 8 separate wells under a specified condition.

Proliferation Assay
Cell proliferation was quantified by total cell number as previously described.²⁴ Cells (5x10³ per well) were seeded in 96-well microtiter plates in 0.1 mL of DMEM–15% FCS. After adherence (3 hours), the medium was replaced by DMEM–0.5% to 1% FCS containing FGF-2 (10 ng/mL) with or without PD98059 or lysoPC at the indicated concentrations. After 48 to 72 hours, cells were fixed by addition of 10 µL of glutaraldehyde and shaken for 15 minutes. After being washed 3 times with deionized water, plates were air dried and stained for 20 minutes with 0.1% crystal violet solution in 200 mmol/L MES, pH 6.0. After being washed 3 times with deionized water to remove excess dye, plates were air dried before solubilization of bound dye in 10% acetic acid. The optical density of dye extracts was measured at 595 nm by using a microplate reader (Bio-Rad). In each experiment, proliferation for each concentration of a given test substance was assessed in 8 separate wells under a specified condition.

In-Gel Kinase Assay
ERK activity was measured by the in-gel kinase assay.²⁵ Nearly confluent cells were starved in serum-free DMEM for at least 24 hours and then stimulated with FGF-2 for the number of indicated minutes after pretreatment with PD98059, lysoPC, or vehicle for 60 minutes. Cells were washed twice with ice-cold PBS and lysed in a buffer containing 10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 2 mmol/L EGTA, 2 mmol/L DTT, 1 mmol/L Na₃VO₄, 1 mmol/L PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin. Cells were scraped off the dish, sonicated, and centrifuged at 15 000 rpm at 4°C for 20 minutes. Supernatants were boiled with 5x Laemmli sample buffer for 5 minutes. Protein concentrations were determined by the Bradford protein assay (Bio-Rad). Equal amounts of proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) through a gel containing 0.5 mg/mL myelin basic protein. The gel was incubated 3 times in 50 mmol/L Tris-HCl, pH 8.0, and 20% 2-propanol for 20 minutes; once in buffer A (50 mmol/L Tris-HCl, pH 8.0, 5 mmol/L 2-mercaptoethanol) for 1 hour; 3 times in 6 mol/L guanidine-HCl for 20 minutes; and in buffer A containing 0.04% Tween 20 at 4°C for at least 16 hours. The gel was incubated in 40 mmol/L HEPES, pH 7.4, 100 µmol/L EGTA, 2 mmol/L DTT, 5 mmol/L MgCl₂, 25 µmol/L ATP, and 2.5 µCi/mL [-³²P]ATP at 22°C for 1 hour. Then the gel was washed 5 times with 1% sodium pyrophosphate and 5% trichloroacetic acid for 20 minutes. After being dried, the gel was autoradiographed with a BAS-2000 system (Fuji). Mitogen-activated protein kinase activity was obtained as the radioactivity levels of 44- and 42-kDa proteins.

Evaluation of Ras Activation
The active GTP-bound form of Ras was detected by using glutathione S-transferase (GST) fusion protein corresponding to the Ras-binding domain (RBD) of Raf-1 bound to glutathione-agarose (Upstate Biotechnology Inc) according to the manufacturer’s protocol. After each stimulation, cells were lysed in a magnesium-containing lysis buffer (25 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 25 mmol/L NaF, 10 mmol/L MgCl₂, 1 mmol/L EDTA, 1 mmol/L Na₃VO₄, 10 µg/mL leupeptin, and 10 µg/mL aprotinin). Lysates were incubated with 5 µL of Raf-1 (RBD)-GST-agarose beads at 4°C for 30 minutes. Beads then were washed 3 times with the lysis buffer and resuspended in 2x Laemmli buffer. After being boiled for 5 minutes, the supernatant was collected by centrifugation, and the protein samples were resolved by SDS-PAGE and transferred onto membranes. Ras was detected by immunoblot analysis with anti-Ras antibody (Transduction Laboratories).

Immunoprecipitation
After each stimulation, BAECs were washed twice with ice-cold PBS and lysed in a buffer (10 mmol/L Tris-HCl, pH 7.4, 50 mmol/L NaCl, 1% Triton X-100, 1 mmol/L Na₃VO₄, 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 1 mmol/L PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin). Precleared lysates were incubated with antisera against phospholipase C (PLC)- (Santa Cruz Biotechnology) for 2 hours, followed by incubation with protein A–Sepharose beads (Pharmacia Biotech Inc) at 4°C for 1 hour. After being washed 3 times with lysis buffer, the immnoprecipitates were dissolved in 2x Laemmli buffer.

Immunoblotting
Samples were separated by SDS-PAGE and transferred to membranes (Immobilon, Millipore). Membranes were immunoblotted with appropriate antibodies, followed by incubation with the secondary antibody, and developed by using the ECL detection assay (Amersham).

Statistical Analysis
Data are expressed as mean±SE. The significance of the difference between group means was analyzed by 1-way ANOVA followed by the Bonferroni test for samples. Values of P<0.05 were considered statistically significant.

	Results

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Effect of the MEK Inhibitor PD98059 on FGF-2–Stimulated EC Migration and Proliferation
To determine the role of ERK activity in FGF-2–stimulated migration and proliferation of BAECs, the effect of the specific MEK inhibitor PD98059²⁶ was examined. Pretreatment of the cells with PD98059 (10 to 100 µmol/L) blocked the FGF-2–induced ERK activation in a concentration-dependent manner, as measured by the in-gel kinase assay (Figure 1A). As shown in Figures 1B and 1C, PD98059 inhibited the FGF-2–stimulated migration and proliferation of BAECs in a concentration-dependent manner, suggesting that ERK activity is involved in the migration and proliferation of BAECs in response to FGF-2.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of the MEK inhibitor PD98059 on FGF-2–stimulated ERK activation and EC migration and proliferation. A, Serum-starved BAECs were pretreated with PD98059 at the indicated concentrations or vehicle (0.1% dimethyl sulfoxide, DMSO) for 60 minutes followed by stimulation with FGF-2 (10 ng/mL) for 10 minutes. Cell lysates were subjected to the in-gel kinase assay. ERK activity was determined by autoradiography. Values are mean±SE of 6 independent experiments. *P<0.01 vs FGF-2. B, BAECs (2.5x104/well) were incubated in a 48-well chemotaxis chamber in the absence or presence of FGF-2 (10 ng/mL) with either PD98059 at the indicated concentrations or vehicle (0.1% DMSO) for 6 hours. Values are mean±SE, a representative of 3 separate experiments. *P<0.01 vs FGF-2. C, BAECs (5x103/well) were cultured in the absence or presence of FGF-2 (10 ng/mL) with either PD98059 at the indicated concentrations or vehicle (0.1% DMSO) for 48 hours. Values are mean±SE of 3 independent experiments. *P<0.05 vs FGF-2. HPF indicates high-power field.

Effect of Overexpression of the Dominant-Negative and Constitutive Active Mutants of MEK1 on EC Migration and Proliferation
To obtain more insight into the role of ERK in EC migration and proliferation, we overexpressed kinase-inactive and constitutive active mutants of MEK1 in BAECs by using recombinant adenovirus vectors (Ad. MEK1 DN and Ad.MEK1 EE). A recombinant adenovirus vector encoding ß-galactosidase (Ad.LacZ) was used as a negative control. The transfection efficiency was 100% (when measured 48 hours after transfection) by ß- galactosidase staining at a multiplicity of infection of 30 for Ad.LacZ (data not shown). As shown in Figure 2A, FGF-2 activated ERK in Ad.LacZ-infected cells, whereas infection with Ad.MEK1 DN effectively blocked FGF-2–induced ERK activation. In Ad.MEK1 EE–infected cells, ERK was activated under unstimulated conditions, a finding that was comparable to the FGF-2–induced ERK activation. Infection with Ad.MEK1 DN and Ad.MEK1 EE, but not with Ad.LacZ, increased MEK1 expression as measured by immunoblotting with an anti-MEK1 antibody (Figure 2B). Ad.MEK1 DN–infected cells showed the reduced migratory and proliferative responses compared with the Ad.LacZ-infected control cells (Figures 2C and 2D). Conversely, infection with Ad.MEK1 EE significantly stimulated EC migration and proliferation. Treatment with FGF-2 showed only insignificant effects on cell migration and proliferation in Ad.MEK1 EE–infected BAECs (data not shown). Thus, although signaling pathways other than the ERK pathway may play a role in cell migration and proliferation, it is likely that FGF-2–induced migration and proliferation are mainly mediated by the ERK pathway.

View larger version (32K): [in this window] [in a new window]   Figure 2. Effect of overexpression of the dominant-negative and constitutive active mutants of MEK1 on EC migration and proliferation. A and B, BAECs were infected with Ad.MEK1 EE, Ad.MEK1 DN, or Ad.LacZ at a multiplicity of infection of 30. Forty-eight hours after infection, cells were stimulated with FGF-2 (10 ng/mL) for 10 minutes. Phosphorylation of ERK was determined by immunoblot analysis with phosphospecific ERK antibody (A, upper). Membrane was reprobed with anti-ERK1 antibody (A, lower). To confirm overexpression of MEK1 EE and MEK1 DN, cells lysates from the same samples were used for immunoblot analysis with anti-MEK1 antibody (B). C, BAECs (2.5x104/well), which were infected with Ad.MEK1 EE, Ad.MEK1 DN, or Ad.LacZ at a multiplicity of infection of 50, were incubated in a 48-well chemotaxis chamber in the absence or presence of FGF-2 (10 ng/mL) for 6 hours. Values are mean±SE, for a representative of 3 separate experiments. *P<0.01 vs LacZ-FGF-2, P<0.01 vs LacZ+FGF-2. D, BAECs (5x103/well), which were infected with Ad.MEK1 EE, Ad.MEK1 DN, or Ad.LacZ at a multiplicity of infection of 50, were cultured in the absence or presence of FGF-2 (10 ng/mL) for 48 hours. Values are mean±SE of 3 independent experiments. *P<0.05, P<0.01 vs LacZ-FGF-2, P<0.01 vs LacZ+FGF-2. HPF indicates high-power field.

Effect of LysoPC on FGF-2–Stimulated EC Proliferation and Migration
It was shown that lysoPC inhibits wound injury–promoted BAEC movement¹⁷ and DNA synthesis in human coronary ECs.¹⁸ We tested the effects of lysoPC on FGF-2–stimulated BAEC migration and proliferation. As shown in Figures 3A and 3B, lysoPC (1 to 10 µg/mL) inhibited the FGF-2–stimulated BAEC migration and proliferation in a concentration-dependent manner. On the other hand, in the absence of stimulation with FGF-2, lysoPC itself exhibited no significant inhibition of BAEC migration and proliferation. The concentrations of lysoPC used in the current study were below the critical micellar concentration of 20 to 25 µg/mL and not cytotoxic to BAECs.^{12 17}

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of lysoPC on FGF-2–stimulated EC migration and proliferation. A, BAECs (2.5x104/well) were incubated in a 48-well chemotaxis chamber in the absence or presence of FGF-2 (10 ng/mL) with or without lysoPC at the indicated concentrations for 6 hours. Values are mean±SE, for a representative of 4 separate experiments. *P<0.01 vs FGF-2. B, BAECs (5x103/well) were cultured in the absence or presence of FGF-2 (10 ng/mL) with or without lysoPC at the indicated concentrations for 72 hours. Values are mean±SE of 4 independent experiments. *P<0.05, P<0.01 vs FGF-2. HPF indicates high-power field.

LysoPC Inhibits the FGF-2–Induced Activation of ERK
The effect of lysoPC on FGF-2–induced ERK activation was examined. We found that treatment of BAECs with lysoPC (1 to 10 µg/mL) showed a concentration-dependent inhibition of FGF-2–induced ERK activation (Figures 4A and 4B). However, lysoPC (10 µg/mL) did not inhibit the phorbol 12-myristate 13-acetate ([PMA] 200 nmol/L)–induced ERK activation (135.2±11.5% of values without lysoPC, n=4; Figure 4C and data not shown). As shown in Figure 5, treatment with lysoPC (10 µg/mL) strongly prevented ERK activation at any time period after stimulation with FGF-2. These results suggest that prevention of FGF-2–induced ERK activation plays a role in the lysoPC-induced inhibition of BAEC migration and proliferation.

View larger version (28K): [in this window] [in a new window]   Figure 4. Concentration-dependent inhibition of FGF-2–induced ERK activation by lysoPC. A and B, Serum-starved BAECs were pretreated with lysoPC (1, 5, or 10 µg/mL) for 60 minutes followed by stimulation with FGF-2 (10 ng/mL) for 10 minutes. Cell lysates were subjected to the in-gel kinase assay. A representative result of 3 independent experiments is shown in A. B, Quantification of ERK activity determined by autoradiography. Values are mean±SE of 3 experiments, each of which was performed in duplicate. *P<0.05, P<0.01 vs FGF-2. C, Serum-starved BAECs were pretreated with lysoPC (10 µg/mL) for 60 minutes followed by stimulation with FGF-2 (10 ng/mL) or PMA (200 nmol/L) for 10 minutes. Cell lysates were subjected to the in-gel kinase assay. A representative result of 4 independent experiments is shown.

View larger version (34K): [in this window] [in a new window]   Figure 5. Time course of the inhibitory effect of lysoPC on FGF-2–induced ERK activation. Serum-starved BAECs were pretreated with or without lysoPC (10 µg/mL) for 60 minutes followed by stimulation with FGF-2 (10 ng/mL) for the indicated periods. Cell lysates were subjected to the in-gel kinase assay. The upper panel shows a representative result of 4 independent experiments. The lower panel shows quantification of ERK activity as determined by autoradiography. Values are mean±SE of 4 independent experiments. *P<0.01 vs -lysoPC.

LysoPC Inhibits FGF-2–Induced Activation of Ras, but not Tyrosine Phosphorylation of PLC-
To elucidate the mechanism by which lysoPC inhibited the FGF-2–induced activation of ERK, we examined the effect of lysoPC on the activity of Ras, a critical upstream regulator of the ERK pathway. Ras activity was measured as the amount of Ras precipitated with Raf-1 (RBD)-GST as described previously.^{27 28} As shown in Figure 6A, stimulation with FGF-2 resulted in rapid activation of Ras, and treatment with lysoPC (10 µg/mL) significantly blocked FGF-2–induced Ras activation.

View larger version (32K): [in this window] [in a new window]   Figure 6. LysoPC inhibits FGF-2–induced activation of Ras, but not tyrosine phosphorylation of PLC-. A, Serum-starved BAECs were pretreated with or without lysoPC (10 µg/mL) for 60 minutes followed by stimulation with FGF-2 (10 ng/mL) for the indicated periods. Ras activity was determined by immunoblot analysis of proteins precipitated with GST-RBD. A representative result of 3 independent experiments is shown. B, Serum-starved BAECs were pretreated with or without lysoPC (10 µg/mL) for 60 minutes followed by stimulation with FGF-2 (10 ng/mL) for the indicated periods. PLC- was immunoprecipitated with anti–PLC- antibody. Immunoprecipitates were blotted with anti-phosphotyrosine antibody (upper) and reprobed with anti–PLC- antibody (bottom). A representative result of 3 independent experiments is shown.

Binding of FGF-2 to FGF receptors stimulates receptor tyrosine kinase activities, leading to the interaction with FGF receptors and PLC-.²⁹ Stimulation of FGF receptor tyrosine kinase activities promotes tyrosine phosphorylation of PLC-. To examine whether lysoPC may modulate FGF receptor tyrosine kinase activities, we determined the effect of lysoPC on the FGF-2–dependent tyrosine phosphorylation of PLC-. As shown in Figure 6B, treatment with lysoPC (10 µg/mL) did not affect the FGF-2–induced increase in tyrosine phosphorylation of PLC-, suggesting that lysoPC does not interfere with FGF-2 binding to FGF receptors or inhibit their tyrosine kinase activities.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The results of the current study demonstrate that ERK activity plays a critical role in EC migration and proliferation stimulated by FGF-2. Both migration and proliferation of ECs were blocked by the MEK1 inhibitor in a concentration-dependent fashion. In comparison with Ad.LacZ-infected cells, Ad.MEK1 DN–infected cells showed reduced migratory and proliferative activities with or without stimulation with FGF-2. In contrast to the effect of Ad.MEK1 DN, constitutive activation of ERK by overexpression of MEK1 EE enhanced EC migration and proliferation. Taken together, ERK activity is necessary and sufficient for EC migration and proliferation.

In contrast to the considerable knowledge on the role of ERK activity in cell proliferation,^{6 7} the mechanism(s) by which ERK activity regulates EC migration is poorly understood. The rapid activation of ERK and the immediate induction of cell migration in response to growth factors suggest that ERK leads to direct activation of the intracellular motility machinery, independent of de novo gene transcription. Klemke et al³⁰ have demonstrated that ERK activity controls cell motility by regulating the myosin light-chain kinase/myosin light-chain pathway. Phosphorylated ERK directly phosphorylates and activates myosin light-chain kinase, leading to the phosphorylation of myosin light chain. Active MEK-induced cell migration was blocked by the myosin light-chain kinase inhibitor or transfection of an inactive mutant of myosin light-chain kinase.

Another possible mechanism is that ERK regulates cell migration by increasing PLA₂ activity. Sa et al³¹ showed that the FGF-2–induced BAEC movement is dependent on PLA₂ activity via an ERK-dependent mechanism. Activated ERK phosphorylates and activates cytosolic PLA₂,³² leading to translocation of PLA₂ from the cytosol to the plasma membrane and induction of arachidonic acid release. It was suggested that arachidonic acid and its metabolites could be implicated in the migration of a variety of cells.^{33 34 35} Thus, myosin light-chain kinase– and PLA₂-dependent pathways may play a role in ERK-regulated BAEC migration.

The current study showed that lysoPC inhibited FGF-2–stimulated EC migration and proliferation, which is consistent with previous studies showing the antimigratory and antiproliferative activities of lysoPC.^{17 18} Conversely, as for vascular smooth muscle cells, lysoPC showed a stimulatory effect.^{36 37} This phenomenon was observed not only in the action of lysoPC, because similar opposite effects on cell migration and proliferation between ECs and vascular smooth muscle cells have been reported with respect to the effect of nitric oxide.^{38 39} It is likely that migratory and proliferative cell responses differ, depending on cell type and/or experimental conditions.

We determined the molecular mechanism(s) of lysoPC-induced inhibition of EC migration and proliferation. We found that lysoPC inhibited the FGF-2–induced activation of the Ras/ERK pathway. Our data demonstrating that lysoPC did not inhibit PMA-induced ERK activation suggest that lysoPC-induced inhibition of ERK activity is not due to direct prevention of either Raf-1–induced MEK activation or MEK-induced ERK activation, because PMA activates ERK through Raf-1 activation. Therefore, it is likely that the lysoPC-induced inhibition of ERK activity is mainly due to its prevention of FGF-2–induced Ras activation, although inhibition of the Ras-dependent activation of Raf-1 may also be involved.

The mechanism(s) involved in the inhibition of Ras activity by lysoPC is unclear. The result showing that lysoPC inhibited the Ras/ERK signaling pathway without affecting tyrosine phosphorylation of PLC- suggests that inhibition of the Ras/ERK pathway by lysoPC is not due to either interference with FGF-2 binding to FGF receptors or inhibition of FGF receptor tyrosine kinase activity. LysoPC inhibition of Ras activity could be mediated via interference with the receptor-mediated signaling cascades for activation of Ras and/or activation of Ras-GTPase activating protein.

In conclusion, our data suggest that ERK activity is sufficient to induce EC migration as well as proliferation. LysoPC inhibits activation of the Ras/ERK pathway and accordingly, both migration and proliferation of BAECs in response to FGF-2. From these experimental results, we speculate that antimigratory and antiproliferative activities of lysoPC may contribute to the limited reendothelialization observed in injured blood vessels and in arterial grafts in vivo.⁴⁰

	Acknowledgments

 
This study was supported by grants-in-aid for scientific research (No. 09281222) from the Ministry of Education, Science, and Culture, a research grant for cardiovascular diseases (A8–1) from the Ministry of Health and Welfare of Japan, and a grant from Japan Cardiovascular Research Foundation (to M.Y.). We are very thankful to Dr K. Okazaki (Biomolecular Engineering Research Institute, Osaka, Japan) and Dr I. Saito (University of Tokyo, Tokyo, Japan) for providing expression vectors containing MEK1 EE and MEK1 DN constructs and pAdex1CAwt, respectively. We are grateful to Dr T. Takahashi for helpful discussion and S. Tsutsui and K. Matsui for excellent technical assistance.

Received August 17, 1999; accepted December 7, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Folkman J, Klagsbrun M. Angiogenic factors. Science. 1987;235:442–447.[Abstract/Free Full Text]

2. Folkman J, Shing Y. Angiogenesis. J Biol Chem.. 1992;267:10931–10934.[Free Full Text]

3. Huang J, Mohammadi M, Rodrigues GA, Schlessinger J. Reduced activation of RAF-1 and MAP kinase by a fibroblast growth factor receptor mutant deficient in stimulation of phosphatidylinositol hydrolysis. J Biol Chem. 1995;270:5065–5072.[Abstract/Free Full Text]

4. Barnard D, Diaz B, Hettich L, Chuang E, Zhang XF, Avruch J, Marshall M. Identification of the sites of interaction between c-Raf-1 and Ras-GTP. Oncogene. 1995;10:1283–1290.[Medline] [Order article via Infotrieve]

5. Alessi DR, Saito Y, Campbell DG, Cohen P, Sithanandam G, Rapp U, Ashworth A, Marshall CJ, Cowley S. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO J. 1994;13:1610–1619.[Medline] [Order article via Infotrieve]

6. Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995;80:179–185.[Medline] [Order article via Infotrieve]

7. Hill CS, Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell. 1995;80:199–211.[Medline] [Order article via Infotrieve]

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. 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]

10. Kume N, Gimbrone MA Jr. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest. 1994;93:907–911.

11. Yokoyama M, Hirata K, Miyake R, Akita H, Ishikawa Y, Fukuzaki H. Lysophosphatidylcholine: essential role in the inhibition of endothelium-dependent vasorelaxation by oxidized low density lipoprotein. Biochem Biophys Res Commun. 1990;168:301–308.[Medline] [Order article via Infotrieve]

12. Inoue N, Hirata K, Yamada M, Hamamori Y, Matsuda Y, Akita H, Yokoyama M. Lysophosphatidylcholine inhibits bradykinin-induced phosphatidylinositide hydrolysis and calcium transients in cultured bovine aortic endothelial cells. Circ Res. 1992;71:1410–1421.[Abstract/Free Full Text]

13. Miwa Y, Hirata K, Kawashima S, Akita H, Yokoyama M. Lysophosphatidylcholine inhibits receptor-mediated Ca²⁺ mobilization in intact endothelial cells of rabbit aorta. Arterioscler Thromb Vasc Biol. 1997;17:1561–1567.[Abstract/Free Full Text]

14. Kugiyama M, 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]

15. Kugiyama K, Ohgushi M, Sugiyama S, Murohara T, Fukunaga K, Miyamoto E, Yasue H. Lysophosphatidylcholine inhibits surface receptor–mediated intracellular signals in endothelial cells by a pathway involving protein kinase C activation. Circ Res. 1992;71:1422–1428.[Abstract/Free Full Text]

16. Flavahan NA. Lysophosphatidylcholine modifies G protein-dependent signaling in porcine endothelial cells. Am J Physiol. 1993;264:H722–H727.[Abstract/Free Full Text]

17. 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]

18. Chen C-H, Nguyen HH, Weilbaecher D, Luo S, Gotto AM, Henry PD. Basic fibroblast growth factor reverses atherosclerotic impairment of human coronary angiogenesis-like responses in vitro. Atherosclerosis. 1995;116:261–268.[Medline] [Order article via Infotrieve]

19. Miyake S, Makimura M, Kanegae Y, Harada S, Sato Y, Takamori K, Tokuda C, Saito I. Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci U S A. 1996;93:1320–1324.[Abstract/Free Full Text]

20. Okazaki K, Sagata N. MAP kinase activation is essential for oncogenic transformation of NIH3T3 cells by Mos. Oncogene. 1995;10:1149–1157.[Medline] [Order article via Infotrieve]

21. Okazaki K, Sagata N. The Mos/MAP kinase pathway stabilizes c-Fos by phosphorylation and augments its transforming activity in NIH 3T3 cells. EMBO J. 1995;14:5048–5059.[Medline] [Order article via Infotrieve]

22. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991;108:193–199.[Medline] [Order article via Infotrieve]

23. Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977;36:59–74.[Abstract/Free Full Text]

24. Kueng W, Silber E, Eppenberger U. Quantification of cells cultured on 96-well plates. Anal Biochem. 1989;182:16–19.[Medline] [Order article via Infotrieve]

25. Takahashi T, Kawahara Y, Okuda M, Ueno H, Takeshita A, Yokoyama M. Angiotensin II stimulates mitogen-activated protein kinases and protein synthesis by a Ras-independent pathway in vascular smooth muscle cells. J Biol Chem. 1997;272:16018–16022.[Abstract/Free Full Text]

26. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995;92:7686–7689.[Abstract/Free Full Text]

27. de Rooij J, Bos JL. Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras. Oncogene. 1997;14:623–625.[Medline] [Order article via Infotrieve]

28. Foschi M, Chari S, Dunn MJ, Sorokin A. Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase. EMBO J. 1997;16:6439–6451.[Medline] [Order article via Infotrieve]

29. Mohammadi M, Dikic I, Sorokin A, Burgess WH, Jaye M, Schlessinger J. Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction. Mol Cell Biol. 1996;16:977–989.[Abstract]

30. Klemke RL, Cai S, Giannini AL, Gallagher PJ, Lanerolle PD, Cheresh DA. Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol. 1997;137:481–492.[Abstract/Free Full Text]

31. Sa G, Murugesan G, Jaye M, Ivashchenko Y, Fox PL. Activation of cytosolic phospholipase A₂ by basic fibroblast growth factor via p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells. J Biol Chem. 1995;270:2360–2366.[Abstract/Free Full Text]

32. Lin L-L, Wartmann M, Lin AY, Knopf JL, Seth A, Davis RJ. cPLA₂ is phosphorylated and activated by MAP kinase. Cell. 1993;72:269–278.[Medline] [Order article via Infotrieve]

33. Sa G, Fox PL. Basic fibroblast growth factor-stimulated endothelial cell movement is mediated by a pertussis toxin-sensitive pathway regulating phospholipase A₂ activity. J Biol Chem. 1994;269:3219–3225.[Abstract/Free Full Text]

34. Peppelenbosch MP, Qiu RG, de Vries-Smits AM, Tertoolen LG, de Laat SW, McCormick F, Hall A, Symons MH, Bos JL. Rac mediates growth factor-induced arachidonic acid release. Cell. 1995;81:849–856.[Medline] [Order article via Infotrieve]

35. Locati M, Lamorte G, Luini W, Introna M, Bernasconi S, Mantovani A, Sozzani S. Inhibition of monocyte chemotaxis to C-C chemokines by antisense oligonucleotide for cytosolic phospholipase A2. J Biol Chem. 1996;271:6010–6016.[Abstract/Free Full Text]

36. Chai Y-C, Howe PH, Di Corleto PE, Chisolm GM. Oxidized low density lipoprotein and lysophosphatidylcholine stimulate cell cycle entry in vascular smooth muscle cells. J Biol Chem. 1996;271:17791–17797.[Abstract/Free Full Text]

37. Stiko A, Regnstrom J, Shar PK, Cercek B, Nilsson J. Active oxygen species and lysophosphatidylcholine are involved in oxidized low density lipoprotein activation of smooth muscle cell DNA synthesis. Arterioscler Thromb Vasc Biol. 1996;16:600–605.[Abstract/Free Full Text]

38. Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774–1777.

39. Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, Ledda F. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest. 1994;94:2036–2044.

40. Reidy MA, Clowes AW, Schwartz SM. Endothelial regeneration, V: inhibition of endothelial regrowth in arteries of rat and rabbit. Lab Invest. 1983;49:569–575.[Medline] [Order article via Infotrieve]

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