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(Hypertension. 1998;31:248.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Lysophosphatidylcholine Stimulates MAP Kinase Activity in Rat Vascular Smooth Muscle Cells

Tadashi Yamakawa; Satoru Eguchi; Yuko Yamakawa; Evangeline D. Motley; Kotaro Numaguchi; Hirotoshi Utsunomiya; Tadashi Inagami

From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee (T.Y., S.E., Y.Y., K.N., H.U., T.I.); and the Department of Anatomy and Physiology, Meharry Medical College, Nashville, Tennessee (E.D.M.)

Correspondence to Tadashi Inagami, PhD, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232

	Abstract

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Lysophosphatidylcholine (lyso-PC) has been implicated in atherogenesis and the inflammatory process. Although lyso-PC has been reported to contribute to the mitogenic effect of oxidized LDL on rat cultured vascular smooth muscle cells (VSMCs), the signaling mechanisms by which lyso-PC promotes its proliferation are poorly characterized. Mitogen-activated protein (MAP) kinases are important mediators involved in the intracellular network of interacting proteins that transduces extracellular cues to intracellular responses. We therefore examined the effect of lyso-PC on MAP kinase activation, proto-oncogene expression, and AP-1 binding activity using cultured rat VSMC. Marked activation of MAP kinase occurred within 10 minutes of lyso-PC treatment, whereupon rapid inactivation ensued. MAP kinase activation by lyso-PC was concentration-dependent (6.25 to 25 µmol/L). Pertussis toxin treatment did not affect lyso-PC-induced MAP kinase phosphorylation. Lyso-PC (25 µmol/L) also increased the mRNA expression of c-fos and c-jun genes. An electrophoretic mobility shift assay showed that AP-1 binding activity was enhanced by lyso-PC. To examine the upstream signaling of MAP kinase, we used several inhibitors on MAP kinase activation induced by lyso-PC. Although lyso-PC induced sustained increase in intracellular Ca²⁺ concentration, EGTA had no effect on MAP kinase activation induced by lyso-PC. However, protein kinase C inhibitor GF109203X and downregulation of protein kinase C activity by prolonged treatment with phorbol ester inhibited lyso-PC-induced MAP kinase activation. These data suggest that lyso-PC transmits its mitogenic activity through a MAP kinase-AP-1 pathway, which exists downstream of its protein kinase C activation in VSMCs.

Key Words: lysophosphatidylcholine • mitogen-activated protein kinase • VSMC • AP-1 • PKC

	Introduction

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Lyso-PC is a natural phospholipid that can be generated intracellularly by the action of phospholipase. A₂ on membrane phosphatidylcholine, the most abundant cellular phospholipid.¹ It is also known that the oxidation of low-density lipoprotein (LDL) ultimately leads to the conversion of phosphatidylcholine to lysophosphatidylcholine. Indeed, during the oxidation of LDL, as much as 40% of its phosphatidylcholine can be converted to lyso-PC,² suggesting that it may play an important pathophysiological role in vascular remodeling associated with hypertension and atherosclerosis. In addition, lyso-PC plays an important role in inflammation and wound healing. Lyso-PC also selectively induces vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in arterial endothelial cells.³

Lyso-PC also induces heparin-binding EGF-like protein and platelet-derived growth factor gene expression in cultured endothelial cells and thus may contribute to the migration and proliferation of VSMCs.⁴ In addition, an increase in the lyso-PC level of oxidized LDL was shown to be directly related to the ability of oxidized LDL to induce endothelial dysfunction.^5–7 Recently, lyso-PC has been shown to stimulate proliferation of VSMC.^8,9

MAP kinases, also known as extracellular signal-regulated kinases, are a family of protein-serine/threonine kinases that are believed to function as integrators for mitogenic signals.^10,11 Although MAP kinases were initially characterized by their rapid activation in response to mitogens that bind to receptors with intrinsic tyrosine kinase activity, they are now recognized to respond to several distinct classes of receptors agonists and physiological forces such as fluid shear stress and stretch.¹² Despite the growth-promoting activity of lyso-PC on VSMCs, no definitive evidence exists for the activation of MAP kinase by lyso-PC in VSMCs. In the present study, we found that lyso-PC induces activation of the MAP kinase and subsequent c-fos and c-jun mRNA expression leading to the activation of AP-1 DNA binding in cultured rat VSMC.

	Methods

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Reagents
Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), penicillin, and streptomycin were obtained from Life. Technologies Inc. Polyclonal antibodies for Tyr204-phosphorylated p44/42 MAP kinase was purchased from New England Biolabs. Rabbit polyclonal antibodies against p42 MAP kinase, c-Fos, and c-Jun subunits of AP-1 were from Santa Cruz Biotechnology, Inc. NF-B and AP-1 consensus oligonucleotides were from Promega. GF109203X was purchased from Calbiochem. Palmitoyl-lyso-PC (lyso-PC), which was 100% pure by HPLC and mass spectrum, 1-palmitoyl-2-oleyl-phosphatidylcholine (PC), and palmitoyllyso-PA (LPA) were purchased from Avanti Polar Lipids. Phorbol 12-myristate 13-acetate (PMA), EGTA, and pertussis toxin (PTX) were obtained from Sigma.

Cell Culture
VSMCs were prepared from the thoracic aorta of 12-week-old Sprague-Dawley rats (Charles River Breeding Laboratories) by the explant method and cultured in DMEM containing 10% FCS, penicillin, and streptomycin as described previously.¹³ Subcultured VSMC from passages 3 through 15, used in the experiments, showed more than 99% positive immunostaining of smooth muscle -actin antibody (Sigma) and were negative for mycoplasma infection by the polymerase chain reaction kit (Stratagene). For the experiments, cells at about 80% confluence in culture wells were used after 2 days of serum depletion.

MAP Kinase Assay
VSMCs grown on 24-well plates were stimulated with agonists for the indicated periods. The reaction was terminated by the replacement of medium with ice-cold lysis buffer (10 mmol/L Tris, pH 7.4, 20 mmol/L NaCl, 2 mmol/L EGTA, 2 mmol/L dithiotreitol, and 1 mmol/L orthovanadate with 10 µmol/L leupeptin, 10 µmol/L aprotinin). After brief sonication, the samples were centrifuged for 5 minutes at 14 000g, and the supernatant was assayed for MAP kinase activity with a BIOTRAKT MAP kinase enzyme assay kit (Amersham) that measured the incorporation of [-³³P] ATP into a synthetic peptide (KRELVERPLTPAGEAPNQALLR) as a specific MAP kinase substrate. The reaction was carried out with the cell lysate (1 µg of protein) in 75 mmol/L HEPES buffer, pH 7.4, containing 1.2 mmol/L MgCl₂, 2 mmol/L substrate peptide, and 1.2 mmol/L ATP, 1 µCi of [-³³P] ATP for 30 minutes at 4°C. The resultant solution was applied to a phosphocellulose membrane and extensively washed in 1% acetic acid and then in H₂O. The radioactivity trapped on the membrane was measured by liquid scintillation counting.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from VSMCs by the method of Dignam et al¹⁴ with minor modifications. After washing with PBS, 5 x 10⁶ cells were harvested and centrifuged at 2000g for 5 minutes. The pellet was resuspended in 400 µL of buffer A (10 mmol/L HEPES/KOH, pH 7.9, 1.5 mmol/L MgCl₂, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol [DTT], 0.1% Nonidet P-40), supplemented with the following protease inhibitors: 0.2 mmol/L phenylmethylsulfonyl fluoride, 2 µmol/L aprotinin, and 2 µmol/L antipain, pepstatin, and leupeptin. The pellet was mixed well and centrifuged at 5000 rpm for 1 minute. The nuclear pellet was then resuspended in 100 µL of buffer C (20 mmol/L HEPES/KOH, pH 7.9, 420 mmol/L KCl, 5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 0.5 mmol/LDTT, 20% glycerol, and the mixture of protease inhibitors described above). After 30 minutes at 4°C under constant agitation, nuclear debris were centrifuged at 15 000g for 15 minutes. The supernatant was distributed into 15 µL aliquots and stored at -80°C. Electrophoretic mobility shift assays (EMSA) were performed with a commercial kit following the instructions of the manufacturer (Promega). Briefly, NF-B (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3') consensus oligonucleotides were [³²P] endlabeled in a mixture containing 10 µCi of [-³²P] ATP (3000 Ci/mmol) (NEN). Ten µg of nuclear proteins were equilibrated for 10 minutes in a binding buffer containing 4% glycerol, 1 mmol/L MgCl₂, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 50 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 7.5, and 50 µg/mL of poly (dI-dC). A labeled probe (0.35 pmol) was added to the reaction and incubated for 20 minutes at room temperature. The samples were run on a nondenaturing, 4% polyacrylamide gel at 100 V for 120 minutes. The gel was dried and exposed to x-ray film.

Immunoblotting
VSMCs grown on 6-well plates were stimulated with an agonist at 37°C in serum-free DMEM for specified doses and durations. The reaction was terminated by the replacement of medium with 100 µL of SDS-polyacrylamide gel electrophoresis buffer, pH 6.8, containing 62.5 mmol/L Tris-HCl, 2% SDS, 10% glycerol, 50 mmol/L dithiothreitol, and 0.1% bromophenol blue. After brief sonication (5 seconds), samples were boiled for 5 minutes at 95°C and centrifuged (14 000g, 5 minutes) at 4°C, and the supernatant (25 µL) was subjected to SDS-polyacrylamide gel electrophoresis. Proteins in the gel were electrophoretically transferred to a nitrocellulose membrane (Hybond-C extra, Amersham). The membrane was treated with indicated primary antibodies. After incubation with secondary anti-rabbit or anti-mouse antibodies, immunoreactive proteins were detected by the ECL Western blotting detection system (Amersham).

Isolation of Total RNA and Quantitation of c-fos and c-jun mRNA by Northern Blot Hybridization
VSMCs grown on 100-mm-diameter culture dishes were stimulated with agonist in serum-free DMEM. After the indicated duration, total RNA was isolated by a one-step preparation.¹⁵ Total RNA (20 µg) was size-separated by electrophoresis on 1% agarose/formaldehyde gels and then transferred to Hybond-N membranes (Amersham). The RNA was immobilized on nylon filters by UV transillumination for 5 minutes. The membranes were prehybridized for 2 hours at 65°C in 1 mol/L NaCl, 10% dextran, 1% SDS, and 0.1 mg/mL denatured salmon sperm DNA. Hybridization was carried out at 65°C overnight with the same solution and [³²P] labeled denatured c-fos or c-jun probe. The membranes were washed with 2 x SSC (1 x, 150 mmol/L NaCl and 15 mmol/L sodium citrate) for 5 minutes at room temperature and then with 0.2 x SSC, 0.1% SDS at 65°C for 30 minutes. The membranes were exposed to x-ray film.

Measurement of Intracellular Calcium
Fura-2 was used to monitor changes in intracellular calcium concentration ([Ca²⁺]i) using a slight modification of a previously described procedure.¹⁶ After incubation in serum-free DMEM for 48 hours, cells were trypsinized, incubated with 4 µmol/L fura-2 acetoxymethylester at 37°C for 30 minutes in Krebs Ringer HEPES solution (20 mmol/L HEPES, pH 7.4, 130 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl₂, 1.5 mmol/L CaCl₂, 10 mmol/L glucose, 0.1% bovine serum albumin), and resuspended to 2 x 10⁶ cells/mL. Measurement of fluorescence was made at 37°C using a SPEX dual-wavelength spectrofluorometer (two excitations at 340 and 380 nm; emission at 510 nm).

	Results

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MAP Kinase Activity
To assess the potential effect of lyso-PC on MAP kinase activity, VSMCs were incubated with lyso-PC, and the MAP kinase activity was examined by Western blotting using phosphospecific anti-MAP kinase antibody and MAP kinase assay. As shown in Fig 1A, stimulation of p44/42 MAP kinase phosphorylation by 25 µmol/L lyso-PC occurred within 5 minutes of treatment and was maximal by 10 minutes after lyso-PC exposure. Rapid inactivation of p44/42 MAP kinase ensued, with a return to basal p44/42 MAP kinase levels occurring within 1 hour of exposure to lyso-PC. However, the abundance of MAP kinase protein expressed remained unchanged during exposure to lyso-PC. MAP kinase activity assay showed that it was also stimulated by lyso-PC with a similar time course (Fig 1B). Concentration-dependent increases in the phosphorylation of p44/42 MAP kinase were observed from 6.25 to 25 µmol/L of lyso-PC (Fig. 1C). Compared with lyso-PC, PC had no effect on MAP kinase phosphorylation in VSMCs (Fig 1D).

View larger version (45K): [in this window] [in a new window]   Figure 1. Effect of lyso-PC on MAP kinase activation. Cells were stimulated with lyso-PC for indicated doses (µmol/L) and durations (minutes). (A) Time course of MAP kinase phosphorylation. (B) Time course of kinase activity. (C) Dose response of MAP kinase phosphorylation. (D) Effect of PC on MAP kinase phosphorylation. Data are expressed as fold p44/42 MAP kinase phosphorylation, in which the p44/42 phosphorylation in unstimulated cells was defined as 1.0. Values shown represent mean ± SE from three separate experiments, each performed in duplicate. Immunoblots represent phospho-p44/42 MAP kinase (top) and total p42 MAP (bottom) from single representative experiments. * and **, greater than time 0. (*, P<.01; **, P<.001; Student’s t test.)

The Effect of PTX on MAP Kinase Activation
It was reported that LPA was formed from lyso-PC by lysophospholipase D in rat plasma.¹⁷ In addition, it is well known that LPA activates MAP kinase via a PTX-sensitive pathway.^18–20 Thus, it is possible that LPA, but not lyso-PC, may activate MAP kinase in VSMCs. To elucidate whether lyso-PC itself or LPA activates MAP kinase, we studied the effect of PTX on lyso-PC-induced MAP kinase activation in VSMCs. Pretreatment of 50 to 1000 ng/mL PTX dose dependently inhibited MAP kinase phosphorylation induced by LPA but not by lyso-PC (Fig 2).

View larger version (50K): [in this window] [in a new window]   Figure 2. Effect of pertussis toxin on lyso-PC- and LPA-induced MAP kinase phosphorylation. VSMCs were pretreated with or without 50 to 1000 ng/mL PTX for 24 hours and stimulated with either lyso-PC (25 µmol/L) or LPA (25 µmol/L) for 10 minutes. Data are expressed as fold p44/42 MAP kinase phosphorylation, in which the p44/42 phosphorylation in unstimulated cells was defined as 1.0. Values shown represent mean ± SE from three separate experiments, each performed in duplicate. Immunoblots represent phospho-p44/42 MAP kinase (top) and total p42 MAP (bottom) from single representative experiments. * and **, lower than LPA-stimulated control. (*, P<.01; **, P<.001; Student’s t test.)

c-fos, c-jun mRNA Expression
To determine whether proto-oncogenes, c-fos and c-jun mRNA expression is stimulated by lyso-PC in cultured rat VSMCs, we performed Northern blot analysis. Lyso-PC induced a marked c-fos and c-jun mRNA expression. Both were detected as early as 30 minutes and sustained at least 2 hours after lyso-PC stimulation (Fig 3).

View larger version (31K): [in this window] [in a new window]   Figure 3. Effect of lyso-PC on c-fos and c-jun mRNA expression. (A) and (B) VSMCs were stimulated by lyso-PC (25 µmol/L) for indicated durations. Total RNA was extracted and Northern blot analysis was performed using c-fos and c-jun as a probe. The filters were stained by methylene blue to check relative loading of total RNA. Results were representative of three individual experiments.

AP-1 Binding Activity
To examine whether the MAP kinase activation and subsequent c-Fos and c-Jun expression by lyso-PC leads to an enhanced AP-1 binding activity, electrophoretic mobility shift assays were performed on nuclear extracts from VSMCs treated with lyso-PC. The AP-1 binding activity of the nuclear extracts was markedly enhanced after 60 minutes of the lyso-PC stimulation, which was sustained up to 4 hours (Fig 4). The reaction was proven to be specific because addition of an excess amount of cold AP-1 but not cold NF-B oligonucleotide eliminated the signals of the retarted bands. AP-1 are composed of several distinct DNA binding subunits, referred to as c-Jun families and c-Fos families. To determine the identity of proteins in the lyso-PC-induced AP-1 binding complex, we performed a supershift assay using selective antibodies directed to the AP-1 proteins. Incubation with antibodies to c-Fos elicited a supershift complex, but not antibodies to c-Jun, suggesting that lyso-PC induced a binding complex containing the heterodimer of c-Fos and other members of the c-Jun family.

View larger version (59K): [in this window] [in a new window]   Figure 4. Effect of lyso-PC on AP-1 DNA binding activity. (A) VSMCs were stimulated by lyso-PC (25 µmol/L) for indicated durations. Nuclear protein (10 µg) was used for EMSA. The specificity of AP-1 binding was verified by excess (50x) of unlabeled competitor (AP-1 or NF B). (B) Supershift analysis of AP-1 family members. Antibodies against AP-1 subunits (c-Fos and c-Jun) was added to binding mixture for 45 minutes at room temperature before EMSA was performed on lyso-PC-treated extracts. Results are representative of three individual experiments.

The Effect of Extracellular Ca²⁺ and PKC on MAP Kinase Activation
We next assessed the upstream signal of MAP kinase. It has been reported that lyso-PC from 10^-7 to 10^-5 mol/L dose dependently induced a sustained increase in [Ca²⁺]i.⁸ We measured intracellular Ca²⁺ concentrations stimulated by lyso-PC using Fura-2. Like previous reports, lyso-PC dose-dependently induced sustained increased in [Ca²⁺]i, which was totally abolished when extracellular Ca²⁺ was removed (data not shown). To elucidate the association of Ca²⁺ with MAP kinase activation by lyso-PC, we examined the effect of the extracellular calcium chelator, EGTA on MAP kinase activation. The activation induced by lyso-PC was slightly inhibited by EGTA, but not significantly (Fig 5).

View larger version (72K): [in this window] [in a new window]   Figure 5. Involvement of PKC in lyso-PC-induced MAP kinase phosphorylation in VSMC. Before stimulation, VSMCs were treated for 24 hours with PMA (1 µmol/L), 30 minutes with GF109293X (2 µmol/L), or 5 minutes with EGTA (5 mmol/L). Cells were stimulated with 25 µmol/L lyso-PC for 10 minutes and p44/42 MAP kinase phosphorylation was determined. Values shown represent mean ± SE from three separate experiments, each performed in duplicate. N.S., not stimulated. * and **, lower than lyso-PC stimulated control. (*, P<.01; **, P<.001; Student’s t test.)

Since it has been reported that lyso-PC significantly potentiated protein kinase C (PKC)-mediated cellular responses,^21–23 we examined whether PKC is essential for lyso-PC-induced MAP kinase activation in VSMC. Downregulation of PKC was achieved by a 24-hour exposure of cells to PMA, after which cells were treated with lyso-PC. Depletion of PKC inhibited MAP kinase activation induced by lyso-PC. We further examined the effect of the specific PKC inhibitor GF109203X on MAP kinase phosphorylation. Pretreatment with 2 µmol/L GF109203X significantly suppressed the phosphorylation of MAP kinase by lyso-PC (Fig 5).

	Discussion

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Previous studies showed that the lyso-PC caused the proliferation of cultured VSMCs.^8,9 In the present study, we have investigated the mechanisms and have shown that it induced p44/42 MAP kinase activation, c-fos and c-jun mRNA expression, and subsequent enhancement of AP-1 binding activity. Our results also suggest that MAP kinase activation is mediated by activation of PKC, but not by intracellular Ca²⁺ elevation.

LPA is an intercellular lipid mediator with growth-factorlike activities.^24–26 LPA induces proliferation and DNA synthesis in VSMC.²⁷ LPA also stimulates MAP kinase in VSMC.^27,28 LPA-induced MAP kinase activation is via the Gi-Ras/Raf pathway in fibroblast cell lines.^18–20 Although LPA is an abundant constituent of serum, LPA was reported to be formed from lyso-PC by lysophospholipase D in rat plasma.¹⁷ Thus, it is important to determine whether MAP kinase activation stimulated by lyso-PC might be mediated by LPA generated from phosphatidylcholine or lyso-PC via the phospholipase D activation. To solve this question, we examined the effect of PTX on lyso-PC- and LPA-induced MAP kinase activation in VSMC. As shown in the results, lyso-PC-induced MAP kinase phosphorylation was not inhibited by PTX although LPA-induced MAP kinase phosphorylation was completely abolished by PTX. The lack of PTX sensitivity of lyso-PC activation of MAP kinase clearly differentiates it from the effects of LPA reported in fibroblasts.¹⁸ In addition, LPA also mobilizes intracellular Ca²⁺, but it was different from the case of lyso-PC. LPA increases [Ca²⁺]i transiently; on the other hand, lyso-PC induces sustained increase in [Ca²⁺]i.²⁸ Taken together, lyso-PC-induced MAP kinase activation is not mediated by LPA but by lyso-PC itself.

It was reported that lyso-PC significantly activates PKC and potentiates PKC-mediated cellular responses such as primary T-lymphocyte activation and HL-60 cell differentiation into macrophages.^21–23,29 PKC is known to activate MAP kinase presumably by directly phosphorylating Raf-1.³⁰ In the present study, PKC depletion by prolonged PMA treatment significantly inhibited MAP kinase activation by lyso-PC. We also showed that the selective PKC inhibitor GF109203X, which has been shown to inhibit PKC-, -ßI, -ßII, and -,³¹ suppressed MAP kinase activation by lyso-PC. These data indicate that a class of PKC that is sensitive to GF109203X and PMA are involved in MAP kinase activation pathway induced by lyso-PC. However, inhibitory effect of GF109203X or downregulation of PKC by PMA on MAP kinase activation by lyso-PC was not complete. Thus, we need to perform further investigation about a PKC independent pathway.

It has been reported recently that lyso-PC from 10^-7 to 10^-5 mol/L dose-dependently induced a sustained increase in [Ca²⁺]i that was totally abolished when extracellular Ca²⁺ was chelated by EGTA in VSMCs.⁸ Because MAP kinase activation-induced angiotensin II is Ca²⁺-dependent in our VSMCs,³² we have examined the role of Ca²⁺ on MAP kinase activation by lyso-PC. In the present study, MAP kinase activation by lyso-PC was not significantly affected by EGTA, suggesting that the major pathway of MAP kinase activation by lyso-PC might be Ca²⁺-independent.

AP-1 is a sequence-specific transcriptional activator composed of jun and fos subunits that is involved in mitogenesis, differentiation, transformation, and inflammation. A key role in stimulation of AP-1 activity is played by various MAP kinases. Upon activation in response to lyso-PC, MAP kinase translocates to the nucleus where they phosphorylate transcription factors, such as TCF/ElK-1, which is bound to the c-fos promoter. Increased c-Fos synthesis results in elevated AP-1 activity. c-Jun N-terminal kinase (JNK) have also been known to activate the AP-1 binding activity through c-jun. It has recently been reported that lyso-PC stimulated JNK-1 and AP-1 in Rat-1 cells, bovine aortic endothelial cells, and Hela cells. Whether lyso-PC stimulates JNK in VSMCs is under investigation.

Phosphatidylcholine is the major phospholipid in LDL. During oxidation of LDL, up to approximately 50% of this phospholipid can be converted to lyso-PC.³³ It has been reported that MAP kinase was stimulated by oxidized-LDL in U937 macrophage-like cells.³⁴ Very recently, oxidized-LDL has been shown to stimulate MAP kinase in VSMCs³⁵ and this stimulation seemed to be PKC-dependent. As shown in the results, MAP kinase activation induced by lyso-PC was also in part PKC-dependent. Therefore, it is reasonable to speculate that oxidized LDL contributes to the proliferation of VSMCs by the MAP kinase activation via lyso-PC.

In conclusion, the present results show that lyso-PC stimulates MAP kinase, c-fos, c-jun, and AP-1 in VSMCs, providing a new insight into the mechanisms of vascular remodeling associated with abnormal lipid metabolism.

	Acknowledgments

 
This work was supported in part by National Institutes of Health Grants HL-35323, HL-58205, HL-03320, and DK-20593. We thank Trinita Fizgerald and Edward Price for excellent technical assistance and Tina Stack for secretarial assistance.

Received September 17, 1997; first decision October 10, 1997; accepted October 27, 1997.

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