Sphingosine 1-Phosphate May be a Major Component of Plasma

Sphingosine 1-Phosphate May be a Major Component ofPlasma Lipoproteins Responsible for the CytoprotectiveActions in Human Umbilical Vein Endothelial Cells*

Takao Kimura , Koichi Sato , Atsushi Kuwabara , Hideaki Tomura ,

Mitsuteru Ishiwara , Isao Kobayashi , Michio Ui§and Fumikazu Okajima

From the†Laboratory of Signal Transduction, Institute for Molecular and Cellular

Regulation, Gunma University, Maebashi 371-8512, ‡Department of Laboratory

Medicine, School of Medicine, Gunma University, Maebashi 371-8511, and § the

Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Tokyo,

JAPAN

Short title: Cytoprotection by Plasma Lipoproteins

¶To whom correspondence should be addressed:

Fumikazu Okajima

Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation,

Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, JAPAN

Tel. +81-27-220-8850

Fax. +81-27-220-8895

E-mail. [email protected]

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(Abstract)

Sphingosine 1-phosphate (S1P), a novel lipid mediator, is concentrated in

the fraction of lipoproteins including high-density lipoprotein (HDL) and low-

density lipoprotein (LDL) in human plasma. Here, we showed that oxidation

of LDL resulted in a marked reduction in the S1P level in association with a

marked accumulation of lysophosphatidylcholine (LPC). We therefore

investigated the role of the lipoprotein-associated lipids especially S1P in the

lipoprotein-induced cytoprotective or cytotoxic actions in human umbilical

vein endothelial cells. The viability of the cells gradually decreased in the

absence of serum or growth factors in the culture medium. The addition of

oxidized LDL (ox-LDL) accelerated the decrease in the cell viability. LPC and

7-ketocholesterol mimicked ox-LDL actions. On the other hand, HDL and

LDL almost completely reversed the serum deprivation- or ox-LDL-induced

cytotoxicity. Exogenous S1P mimicked cytoprotective actions. Moreover, the

S1P-rich fraction and chromatographically purified S1P from HDL exerted

cytoprotective actions, but the rest of the fraction did not. The cytoprotective

actions of HDL and S1P were associated with extracellular signal-regulated

kinase (ERK) activation and almost completely inhibited by pertussis toxin

and PD98059, an ERK kinase inhibitor. The HDL-induced action was

specifically desensitized in the S1P-pretreated cells. Taken together, these

results indicate that the lipoprotein-associated S1P and the lipid receptor-

mediated signal pathways may be responsible for the lipoprotein-induced

cytoprotective actions. Furthermore, the decrease in the S1P content, in

addition to the accumulation of the cytotoxic substances such as LPC, may be

important for the acquisition of the cytotoxic property to ox-LDL.


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(Introduction)

Plasma lipoproteins are responsible for lipid transport to cells and control of

cholesterol synthesis. Low-density lipoprotein (LDL)1 provides cholesterol to cells

through LDL receptors, and this lipoprotein is thought to play an important role in

atherosclerosis after undergoing oxidative modifications (1-4). Thus, ox-LDL is

present in atherosclerotic lesions and exerts a variety of biological actions,

including cytotoxicity on the cells of the artery wall, potentially involved in

atherogenesis (1-4). Recent studies show that LPC mimics part of ox-LDL-induced

actions (5-8). On the other hand, HDL levels have been shown to be inversely

correlated with the risk of cardiovascular disease (1-4). Several mechanisms have

been proposed for the anti-atherogenic functions of HDL. These include the

promotion of the efflux of cholesterol from atherosclerotic plaques, inhibition of

the oxidative modification of LDL, and inhibition of the expression of adhesion

molecules such as vascular cell adhesion molecule-1 (VCAM-1) (1-4). HDL has

also been shown to protect endothelial cells from serum deprivation- and ox-LDL-

induced cytotoxicity (1-4, 9, 10), but the mechanisms by which HDL exerts the

cytoprotective action are not fully understood.

S1P, one of the sphingolipid metabolites, has been shown to participate in a

variety of cellular responses including proliferation, differentiation, adhesion,

motility and apoptosis (11-16). These cellular responses elicited by S1P were first

thought to be mediated through an intracellular target(s), but extracellular

mechanisms through G-protein-coupled S1P receptors have also been suggested.

Supporting the latter extracellular mechanisms, several isoforms of S1P receptors

have been identified (11-16). These S1P receptor subtypes are expressed and

functioning in a variety of cells including endothelial cells. In vascular endothelial

cells, S1P has been shown to regulate a wide range of cellular activities involved in

angiogenesis, wound healing, apoptosis and atherosclerosis (17-20). Thus, S1P

induces cell migration, expression of several cell adhesion molecules, DNA

synthesis, and cell survival (17-20).


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Sachinidis et al. were the first to show that S1P-like lipids are associated with

plasma lipoproteins (21). Recently, we specified one of the S1P-like lipids as S1P

(22). We also succeeded in quantifying the S1P content in plasma components: this

lipid was concentrated, per unit amount of protein, in lipoprotein fractions with the

rank order of HDL>LDL=VLDL>lipoprotein-deficient plasma (albumin fraction)

(22). These results raise the possibility that S1P mediates some of the lipoprotein-

induced actions in endothelial cells. In the present paper, we show that S1P may

mediate the lipoprotein-induced cytoprotective actions through S1P receptors and

their intracellular signaling pathways. We also found that oxidation of LDL

markedly reduced its S1P content in association with a marked increase in

cytotoxic LPC content. Thus, plasma lipoprotein-associated S1P may be an

important factor to determine whether they are cytoprotective or cytotoxic.

MATERIALS AND METHODS

Materials---- S1P was purchased from Cayman Chemical Co., and 1-oleoyl-

sn-glycero-3-phosphate (lysophosphatidic acid; LPA), 7-ketocholesterol, 25-

hydroxycholesterol, 1-palmitoyl (C16:0) lysophosphatidylcholine (LPC) and other

lipids were purchased from Sigma unless otherwise noted. A p44/p42 MAP kinase

(ERK 1/2) enzyme assay kit was purchased from Amersham Corp. and an ERK

specific antibody (K-23, amino acids 305-327 of rat ERK 1 which recognizes both

ERK 1 and ERK 2) was from Santa Cruz Biotechnology. Plasma lipoproteins were

prepared by density gradient centrifugation; LDL (1.019-1.063 g/ml) and HDL

(1.063-1.21 g/ml) were separated from freshly isolated human plasma by sequential

ultracentrifugation as described previously (22). Human plasma was collected from

normal healthy volunteers. Ox-LDL was prepared by oxidizing for 20 h with 10

µΜ CuSO4 after extensive dialysis against 150 µΜ NaCl/PBS (9:1) (5, 6). For

preparation of charcoal-treated lipoproteins, the lipoproteins (2.5 mg proteins in 1


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ml) were treated with 250 mg of BSA-pretreated charcoal, which was prepared by

mixing with 1% BSA and the subsequent washing with PBS and then filtered (0.45

µm). The sources of all other reagents were the same as described previously (15,

19, 22-24).

Cell Culture---HUVECs with passages of 3 were purchased from Whittaker

Bioproducts (Walkersville, MD). The cells (passage number between 5 and 12)

were cultured in RPMI1640 medium supplemented with 15% (v/v) FBS (Sigma)

and several growth factors as previously described (19). Where indicated, PTX

(100 ng/ml) or its vehicle (final 2 mM urea) was added to the culture medium 24 h

before experiments, unless otherwise stated. CHO cells, which were expressing

Edg-1 or Edg-3, were cultured as previously described (15, 22, 23).

Cell Survival Assay-----HUVECs were cultured for 24 h with test agents in

fresh RPMI 1640 medium containing 0.1% BSA unless otherwise specified. In the

experiments with PD98059 (10 µΜ) or SB203580 (1 µΜ), the cells were pretreated

with these inhibitors for 1 h and then cultured for another 24 h with test agents in

the presence of these inhibitors. The cells were then washed twice with PBS and

harvested with trypsin. The viable cells were determined by trypan blue (0.2%)

exclusion assay. The results were expressed as percentages of the value obtained

with 15% FBS in the control cells.

Measurement of ERK1/2 Activity------HUVECs were incubated for 4 h in

fresh RPMI 1640 medium containing 0.1% BSA unless otherwise noted. Where

indicated, the cells were treated without or with PD98059 (10 µΜ) or SB203580 (1

µΜ) for the last 1 h during this incubation period. The cells were then washed once

and preincubated for 20 min with or without these inhibitors at 37°C in a HEPES-

buffered medium (15) and finally incubated for 5 min with test agents in the same

medium. In the case for the desensitization experiments with S1P (Fig. 5), the cells

were incubated for another 5 h with or without S1P (1 µΜ) after the 4 h-culture

with RPMI 1640 medium containing 0.1% BSA. After the S1P pretreatment

procedure, the cells were preincubated for 20 min in Hepes-buffered medium and


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incubated for 5 min with the test agents as described above. The incubation was

terminated by washing twice with ice-cold PBS and adding 0.5 ml of a lysis buffer

as previously described (24). The kinase activity was determined with an assay kit

(Amersham Corp.) that measures the incorporation of [γ-32P] ATP into a synthetic

peptide (KRELVEPLTPAGEAPNQALLR) as a specific substrate. The enzyme

activity was expressed as percentages of the basal activity without test agents in

control cells. The same lysate was also analyzed by Western blotting with an ERK

specific antibody to detect the change in gel mobility reflecting phosphorylation of

the enzyme as described previously (24).

Extraction of Active Components of HDL---HDL (about 4 mg in 2 ml) was

extensively mixed with chloroform (3 ml), methanol (2 ml), water (0.5 ml) and 10

N NaOH (0.1 ml), and phases were separated. The upper alkaline phase was

collected. To the lower phase, 4 ml of synthetic upper-phase mixture was added,

and phases were separated again. The lower phase containing the majority of

phospholipids and neutral lipids evaporated to dryness (Fraction a). The pooled

upper alkaline phase containing S1P (about 8 ml), chloroform (4 ml) and HCl (0.2

ml) were mixed extensively and phases were separated. The lower chloroform

phase was collected. This extraction procedure was repeated another four times

more by adding chloroform (4 ml) to the upper aqueous solution and phase

separation. The pooled chloroform phase (about 20 ml) containing S1P was

evaporated to dryness (Fraction b). S1P recovery was about 90% as determined by

including [3H]S1P as an internal standard in the lipid purification procedure. The

upper aqueous phase containing water-soluble substances was also dried by

evaporation (Fraction c). Fraction b was further processed by a silica gel high-

performance thin layer chromatography (HPTLC) (Merck) using a solvent system

consisting of 1-butanol : acetic acid : water (3:1:1). The silica gel with the resolved

lipids (about 1-cm length each) was scraped off to obtain lipids covering the entire

area of migration. The lipids were then eluted with chloroform : methanol : HCl

(100:100:1) and dried by evaporation. All fractions thus separated were dissolved


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in PBS containing 0.4% BSA (2 ml) and were used at the final concentration of

10% of this solution.

Detection of LPC in Lipoproteins----The total lipids were extracted from

lipoproteins as described for Fraction a in the previous section except that 1 N HCl

was used instead of 10 N NaOH. LPC was then separated by an HPTLC using a

solvent system consisting of chloroform : methanol : 20% NH4OH (60:35:8). The

bands were staining with primulin and visualized under UV light. The LPC fraction

was scraped and the lipid content was quantified by malachite green method (25)

Lipoprotein Electrophoresis ---Agarose gel film, TITAN GEL LIPO KIT

J3045 (Helena Laboratories, Japan), was used. After electrophoresis, the film was

dried and thereafter stained with Fat Red 7B. Other experimental conditions are

described in the previous paper (26).

Quantitative Measurement of S1P----S1P in plasma lipoproteins was

selectively extracted and its content was measured by a radioreceptor binding assay

using Edg-1expressing CHO cells as described previously (22, 23).

Measurement of Inositol phosphate production in S1P Receptor-Expressing

CHO cells--- This was performed as described previously (22). In brief, vector- or

Edg-3-transfected CHO cells, which had been labeled with [3H]inositol, were

harvested from the 10-cm dishes with trypsin, washed by sedimentation (250 x g

for 5 min) and resuspended in the Hepes-buffered medium. The cells were then

incubated to measure the production of [3H]-labeled IP2 and IP3. In order to

normalize the effects of lipoproteins in vector-transfected or Edg-3-transfected

cells, data were first normalized to 105 dpm of the total radioactivity incorporated

into the cellular inositol lipids in each experiment, and then the results were

expressed as percentages of the maximal activity obtained at 1 µΜ S1P in Edg-3-

transfected cells.

Data presentation----All experiments were performed in duplicate or triplicate.

The results of multiple observations were presented as means + S.E.M. of at least

three separate experiments unless otherwise stated. Statistical significance was


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assessed by Student t test.

RESULTS

Oxidation of LDL Resulted in a Decrease in the Lipoprotein-associated S1P

Content----In Fig. 1A, we measured the S1P content in the lipoprotein particles of

human plasma by a radioreceptor binding assay, which was recently established by

us (23). For this quantitative measurement, S1P was extracted from lipoproteins.

Consistent with the previous result (22), S1P contents in LDL and HDL reached

approximately 100 to 200 pmol/mg protein, respectively, which are 20 to 40 times

higher than the S1P content in the lipoprotein-deficient plasma (22). Since

oxidation of LDL is thought to be a major risk factor for the development of

atherosclerosis, we examined the effect of oxidation on the S1P content in LDL.

The CuSO4 treatment of LDL induced degradation of apolipoprotein B (Fig.1B).

The copper treatment also induced a marked accumulation of LPC at an expense of

reduction of phosphatidylcholine (Fig. 1C) (5, 6). Under these conditions, the S1P

content was reduced to about 25% of the initial value (Fig. 1A).

In order to examine whether the change in the S1P content reflects in the

functional activity, we measured S1P receptor-mediated phospholipase C

stimulating activity by the intact lipoprotein samples without the extraction

procedure of S1P. In the vector-transfected CHO cells, inositol phosphate

production in response to lipoproteins regardless of the lipoprotein species was

very small (Fig. 1D, upper panel). On the other hand, in the S1P receptor Edg-3-

overexpressing CHO cells, HDL and LDL markedly stimulated inositol phosphate

production reflecting activation of phospholipase C, whereas ox-LDL exerted only

a small effect on the activity (Fig. 1D, lower panel). It is reasonable to assume that

the increase in the activity induced by the receptor transfection may be mediated by

the S1P receptor. Thus, the change in the S1P content in lipoprotein particles seems


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to reflect in their ability to stimulate the S1P receptor.

HDL and LDL Protect HUVECs from Cytotoxicity Induced by Serum-

deprivation and Ox-LDL---When HUVECs were cultured without serum or growth

factors, the cells gradually lost their viability and were detached from the dishes. At

24 h after serum deprivation, only 50% of the cells had survived (Fig. 2A). Under

these conditions, both HDL and LDL at 100 µg/ml almost completely reversed the

serum deprivation-induced cytotoxicity (Fig. 2A). On the contrary, ox-LDL

accelerated the cytotoxicity (Fig. 2A). As shown in Fig. 2B, the oxidative lipids,

including 7-ketocholesterol, 25-hydroxycholesterol and LPC, which were

accumulated during LDL oxidation (5, 6, 27, 28) mimicked the ox-LDL-induced

action. The cytotoxicity induced by these agents including ox-LDL was reversed

by HDL (Fig. 2B). LDL was also effective for inhibiting the ox-LDL-induced

action (Fig. 2C).

S1P has been shown to protect HUVECs from cytotoxicity or apoptosis

induced by serum deprivation (18-20). We confirmed this observation (Fig. 2D).

Furthermore, we found that S1P also inhibited the ox-LDL-induced cytotoxicity

(Fig. 2D). Thus, S1P mimicked cytoprotective action of HDL or LDL. LPA has

also been shown to regulate the variety of functions of HUVECs (29), but this lipid

was ineffective for cytoprotection of HUVECs at concentrations less than 10 µΜ and exerted a rather cytotoxic effect at higher concentrations (data not shown). We

also examined the effects of other lipids including platelet-activating factor,

phosphatidic acid, phosphatidylserine, phosphatidylinositol and

phosphatidylethanolamine, but we could not detect any significant cytoprotective

effect at concentrations less than 10 µΜ (data not shown).

HDL and S1P-induced Cytoprotective Actions May be Mediated by Gi/Go

Protein-Regulated ERK Pathways----- We next examined the signaling pathways

involved in the S1P and HDL-induced cytoprotective actions. For this, we used

PTX, an inhibitor for Gi/Go protein functions, PD98059, an inhibitor for ERK

kinase (MEK), and SB203580, an inhibitor for p38 MAP kinase. Any drug


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treatment hardly affected the viability of the cells in the presence of serum (Fig.

3A). Among these agents, a prior treatment of the cells with PTX or PD98059, but

not SB203580, almost completely inhibited S1P- or HDL-induced cytoprotective

actions against the cytotoxicity induced by serum deprivation and ox-LDL (Fig.

3B). When LDL was used instead of HDL, we observed a similar cytoprotective

action that was sensitive to both PTX and PD98059 (data not shown).

These results suggest involvement of Gi/Go proteins and ERK in the S1P- and

HDL-induced actions. Actually, S1P and HDL induced the phosphorylation of the

ERK 1/2 as evidenced by the gel mobility-shift (Fig. 4A) and activated the enzyme

as evidenced by the phosphorylation of the ERK-specific substrate peptide (Fig. 4,

B and C). As expected, the activation of ERK was completely suppressed by the

treatment of the cells with PTX and PD98059 (Fig. 4, A and D). These results

indicate that HDL and S1P-induced cytoprotective actions may be mediated by

ERK signaling pathways that are regulated by Gi/Go protein-coupled receptors.

S1P May be a Major Component Mediating the HDL-Induced Cytoprotective

Actions---- Thus, we could not discriminate the action mode of HDL from that of

S1P. This suggests that HDL-induced cytoprotective actions may be mediated by

S1P. To demonstrate this possibility, we performed desensitization experiments as

shown in Fig. 5. When the cells were treated with S1P, the ERK activity peaked at

around 5 min and then gradually decreased to the initial level at around 5 h (data

not shown). After the S1P pretreatment, the cells no longer responded to the

secondly applied S1P, but ATP-induced ERK activation was hardly affected by the

S1P pretreatment (Fig. 5). Thus, the cells were undergoing homologous

desensitization when the cells were pretreated with S1P. Under these conditions,

HDL-induced ERK activation was also completely lost (Fig. 5). Thus, S1P seems

to mediate HDL-induced ERK activation and hence the cytoprotective action of the

lipoprotein.

The participation of S1P in the HDL action was further confirmed in Fig. 6. In

this experiment, components of HDL were separated into three fractions: Fraction


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a, lipid fractions containing the majority of lipids including fatty acids, neutral

lipids and phospholipids; Fraction b, lipids soluble under an alkaline aqueous

solution such as S1P and LPA; Fraction c, substances soluble in an aqueous

solution. The cytoprotective activity (Fig. 6A) and ERK-activating activity (Fig.

6B) of HDL were recovered in the S1P-rich Fraction b but not in Fraction a or

Fraction c. The lipid components of Fraction b were further separated by an

HPTLC (Fig. 6E), in which S1P was mostly recovered in the fraction 4. The S1P-

containing fraction 4 clearly induced the cytoprotective action (Fig. 6C) and ERK

activation (Fig. 6D).

Charcoal Treatment Attenuated Not Only Cytoprotective Actions of HDL and

LDL But Also Cytotoxic Action of ox-LDL---Finally, we examined the effects of

charcoal treatment, which would remove low-molecular weight substances such as

S1P and LPC, on the lipoprotein actions. Charcoal treatment reduced the S1P

content to 10-20% of initial value in either LDL or ox-LDL (Fig. 7A) without any

significant change in the apolipoprotein composition (Fig. 7B). This treatment also

markedly removed LPC from the lipoprotein particles (Fig. 7C). Under these

conditions, not only cytoprotective action of LDL but also cytotoxic action of ox-

LDL were reversed (Fig. 7D). In the case of HDL, however, charcoal treatment

only partially (50%) removed S1P from the lipoprotein particles (Fig. 7A) probably

due to its tight binding to the lipoprotein (22). Thus, the charcoal treatment exerted

a small but significant inhibitory effect on the cytoprotective action of HDL (Fig.

7D).

DISCUSSION

HDL has been shown to exhibit a wide range of anti-atherogenic functions,

some of which may be cytoprotective actions against cytotoxicity or apoptosis

induced by several cytokines, Fas, growth factors-deprivation, and ox-LDL (1-4, 9,


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10). Consistent with the previous studies (9, 10), HDL protected HUVECs from the

serum deprivation- and ox-LDL-induced cytotoxic actions. We also found that

LDL exerted the cytoprotective action to the extent comparable to HDL.

Considering the characteristics of LDL as a risk factor for atherogenesis, one might

wonder if this observation were peculiar. In the previous studies, ox-LDL has been

repeatedly shown to be cytotoxic, but, to our knowledge, there is no report showing

the cytotoxic action of the native LDL. Thus, we postulate that native LDL itself

possesses potentially cytoprotective functions, but this lipoprotein might acquire

the cytotoxic character during its oxidation.

The present studies indicate that S1P and its receptor-mediated signaling

pathways are important for the HDL and LDL-induced cytoprotective actions. First,

the S1P-rich fraction and HPTLC-purified S1P from HDL exerted the

cytoprotective action, but the rest of the fraction did not (Fig. 6). Second, the

removal of S1P by charcoal treatment of HDL and LDL inhibited the

cytoprotective action of these lipoproteins, although the effect was small in the case

of HDL because of the insufficient removal of S1P (Fig. 7). Third, S1P- and HDL-

induced cytoprotective actions were associated with the activation of ERK, and

these responses were suppressed by PTX, an inhibitor of Gi/Go-protein function, or

PD98059, an inhibitor of ERK kinase (Figs. 3 and 4). These results suggest that

Gi/Go-protein-regulated ERK activation may play an important role in the

cytoprotective actions of S1P and HDL. The role of Ca2+ signaling and/or ERK

pathway in the S1P-induced cell survival has recently been proposed by other

groups besides ours (18, 20). Forth, S1P or HDL-induced, but not ATP-induced,

ERK activation was specifically desensitized by a prior stimulation of the cells

with S1P, suggesting an involvement of S1P receptors in the HDL action (Fig. 5).

In relation to this, it has been reported that TNF-α increases the intracellular S1P

level by activation of sphingosine kinase and thereby induces anti-apoptotic action

in HUVECs (17). This suggests that an accumulation of intracellular S1P may also

exhibit cytoprotective action. However, the same authors also reported that HDL


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decreased rather than increased the intracellular level of S1P by inhibiting

sphingosine kinase (30). Thus, it would be a minor mechanism, if not negligible,

that HDL-associated S1P would be incorporated into the cells and thereby induce

cytoprotective action. Although we did not specify the subtype of the S1P receptor

involved in the HDL actions in the present study, both Edg-1 and Edg-3 may be

responsible for the cytoprotective action (18-20, 31). The present results are quite

consistent with the recent study by Sachinidis et al (21), in which it was suggested

that S1P-like lipids in lipoproteins may mediate the activation of ERK and

stimulation of DNA synthesis in vascular SMCs.

In the previous study (9), Apo A as well as HDL exhibited cytoprotective

action against ox-LDL-induced cytotoxicity in endothelial cell lines, although HDL

was more effective than Apo A. This suggests that not only the lipid component,

probably S1P as shown here, but also Apo A may possess the potential

cytoprotective activity against cytotoxicity of ox-LDL. However, in that study, the

endothelial cell lines seem to be stable for serum deprivation and ox-LDL: the cells

survived for at least 48 h even without serum and more than 24 h was required for

the induction of significant cytotoxic effect by ox-LDL. This was somehow

different from our system using HUVECs: about 50% of the cells lost their

viability during 24 h-culture without serum or growth factors even in the absence

of ox-LDL. Similar susceptibility to serum deprivation of HUVECs has been

observed by other groups (10, 18, 20). Thus, Apo A might participate in the

cytoprotective action of HDL against predominantly late or chronic phase of

cytotoxicity. Alternatively, the cytoprotective mechanisms might differ between the

different sources of endothelial cells.

The present study indicates that S1P mediates the HDL-induced

cytoprotective actions through ERK-involving pathways, but it should be noted

that there was a considerably large difference in their potency between ERK

activation (about 3 nM, see Fig. 4B) and cytoprotective action (30-100 nM, see Fig.

2D), when exogenous S1P effects were compared. On the other hand, in the case of


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HDL, the difference was small: 10 µg/ml for ERK activation (Fig. 4C) vs. 30

µg/ml for cytoprotective action (Fig. 2A). This peculiar observation may be

explained by the notion that S1P is metabolized very fast especially in the absence

of lipoproteins. Under the present assay conditions using HUVECs, we observed

that a half-life of HDL-associated S1P was about 2 h at 100 µg/ml HDL, which

corresponds to approximately 20 nM S1P, whereas the half-life of exogenous S1P

was about 30 min at the same concentration in the absence of HDL but presence of

0.1% BSA (data not shown). For the ERK assay, the activity was measured at 5

min after the addition of test agents, while it was measured at 24 h after for the

cytoprotective activity. Thus, it is reasonable to speculate that a higher

concentration of S1P is necessary to observe the long-term cytoprotective action

compared to the short-term ERK activation especially in the absence of HDL.

The mechanism by which ox-LDL induces a variety of responses involved in

the development of atherosclerosis was recently extensively investigated, but it is

still not completely defined (1-4). During oxidation of LDL, several products such

as lipid hydroperoxides, oxysterols, and LPC are produced (5, 6, 27, 28). In

addition, the production of lipid mediators such as LPA and platelet-activating

factor has also been reported (29, 32). Among these oxidative lipid products, LPC

has been shown to duplicate a variety of ox-LDL-induced actions including

monocyte migration and expression of adhesion molecules on endothelial cells (5-

8). As for cytotoxicity, LPC and oxysterols such as 7-ketocholesterol have been

shown to mimic the ox-LDL-induced action in vascular endothelial cells (8, 28).

Thus, these lipids may be components of ox-LDL responsible for the induction of

cytotoxicity, although their molecular targets and their mechanisms causing

cytotoxicity remain unknown. This conclusion is further supported by the

observation that charcoal treatment of ox-LDL reversed its cytotoxic activity in an

association with a marked decrease in LPC content without any apparent change in

apolipoprotein components (Fig. 7).

In vascular smooth muscle cells, LDL- and HDL-associated S1P-like lipids


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stimulated DNA synthesis (21). Based on these results, the authors postulated that

the S1P-like lipids might behave as atherogenic mediators and might be increased

by oxidation of lipoproteins (21). However, in the present study, we demonstrated

that oxidation of LDL markedly reduced, but not increased, its S1P content. The

reduction of S1P content by copper treatment was blocked by an antioxidant

butylated hydroxytoluene, indicating an oxidation-dependent reaction (data not

shown). At present, however, the metabolic pathway of S1P degradation and its

mechanism remains uncharacterized. This is an important future subject. In any

event, during LDL oxidation, the contents of cytotoxic LPC and cytoprotective S1P

changed reciprocally. The decrease in the S1P content may also be involved in the

acquisition of cytotoxicity to ox-LDL. Thus, we propose that the balance between

the contents of cytotoxic lipids including LPC and cytoprotective S1P may be an

important factor that determines whether plasma lipoproteins are cytotoxic or

cytoprotective. This balance might also be an important determinant for

lipoproteins to be atherogenic or anti-atherogenic. In this proposal, S1P is

postulated to be an anti-atherogenic mediator. In the endothelial cells, S1P has been

shown to stimulate nitric oxide production, cell migration, and cell proliferation

(18-20, 33). Furthermore, in vascular smooth muscle cells, S1P is a potent inhibitor

of cell migration (34). These responses in addition to cytoprotective action seem to

favor anti-atherogenic properties. On the other hand, S1P has been shown to induce

expression of adhesion molecules such as VCAM-1 and E-selectin in endothelial

cells (16). These actions suggest rather atherogenic properties of S1P. Thus, further

experiments are necessary to conclude whether S1P is atherogenic or anti-

atherogenic. However, these findings together with the present study suggest that

control of the S1P content in plasma lipoproteins and the S1P receptor function in

vascular cells may provide potentially useful means for the therapy of

cardiovascular disease.

In conclusion, HDL-associated S1P is a major component for the lipoprotein-

induced cytoprotective action in HUVECs. This action is probably mediated by


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ERK pathways that are regulated by S1P receptors such as Edg-1 and Edg-3.

Oxidation of LDL resulted in a marked decrease in S1P content in association with

a marked increase in LPC content. Such a reciprocal change in the

lysophospholipid composition may be important for the acquisition of cytotoxic

property to ox-LDL.


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(Footnote)

*This work was supported in part by a research grant grants-in-aid for scientific

research from the Japan Society for the Promotion of Science. The costs of

publication of this article were defrayed in part by the payment of page charges.

This article must therefore be hereby marked “advertisement” in accordance with

18 U.S.C. Section 1734 solely to indicate this fact.

¶ To whom correspondence should be addressed: Laboratory of Signal

Transduction, Institute for Molecular and Cellular Regulation, Gunma University,

3-39-15 Showa-machi, Maebashi 371-8512, JAPAN.

Tel.: +81-27-220-8850; Fax.: +81-27-220-8895;

E-mail.: [email protected]

1The abbreviations used are: LDL, low-density lipoprotein; ox-LDL, oxidized low-

density lipoprotein; HDL, high-density lipoprotein; VLDL, very low-density

lipoprotein; S1P, sphingosine 1-phosphate; LPC, lysophosphatidylcholine; LPA, 1-

oleoyl-sn-glycero-3-phosphate or lysophosphatidic acid; HPTLC, high-

performance thin layer chromatography; ERK, extracellular signal-regulated

kinase; Edg, endothelial differentiation gene; HUVECs, human umbilical vein

endothelial cells; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline;

BSA, bovine serum albumin; FBS, fetal bovine serum; Apo, apolipoprotein; Hepes,

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IP2, inositol bisphosphate; IP3,

inositol trisphosphate.


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(Figure Legends)

FIG. 1. Characterization of lipoproteins. Respective lipoprotein was processed

for measurement of S1P content (pmol/mg protein) in A; electrophoresis analysis

of apolipoprotein (Apo) composition in B, where the major band in ox-LDL at a

size similar to Apo A is a degradation product of Apo B; HPTLC analysis of LPC,

phosphatidylcholine (PC), sphingomyelin (SM), and other lipid composition in C,

where O and F stand for “origin” and “front”, respectively; and inositol phosphate

production depending on the S1P receptor Edg-3 stimulation at the indicated

concentrations of ox-LDL (●), LDL (○) and HDL (△) in D. In C, authentic SM

from Sigma was also doublet possibly reflecting a difference in a fatty acid

composition. The LPC fraction in the HPTLC was scraped off and LPC content

(nmol/mg protein) was quantified as 245 + 29, 34 + 2 and 19 + 2 for ox-LDL, LDL

and HDL, respectively (number of observation was four). For other experimental

procedures and expression of results, see Materials and Methods. In A and D, data

are means + S.E.M. of four separate experiments. In B and C, a representative

result from four separate experiments is shown.

FIG. 2. Effects of plasma lipoproteins, ox-LDL and several components of

lipoproteins on cell survival. HUVECs were cultured with the indicated

concentrations of HDL (○,●), LDL (□,■), or ox-LDL (△,▲) in the presence of

0.1% BSA (closed symbol) or 15% FBS (open symbol) in A. HUVECs were

cultured with the indicated concentrations of HDL (B), LDL (C), or S1P (D) in the

presence of 0.1% BSA with or without the indicated agents; ox-LDL (100 µg/ml),

7-ketocholesterol (7keto; 30 µΜ), LPC (30 µΜ), 25-hydroxycholesterol (25h; 30

µΜ) or FBS (Serum; 15%). Data are means + S.E.M. of four separate experiments.

FIG. 3. Effects of PTX, PD98059 and SB203580 on the cytoprotective action

of S1P or HDL. HUVECs, which had been treated or untreated with PTX,

PD98059 (PD) or SB203580 (SB), were cultured in the presence of 15% FBS (A)


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or 0.1% BSA (B) with the indicated agents; ox-LDL (100 µg/ml), S1P (1 µΜ),

HDL (100 µg/ml), or their combination. Data are means + S.E.M. of four

separate experiments.

FIG. 4. Effects of PTX and PD98059 on the S1P- or HDL-induced ERK

activation. HUVECs, which had been untreated (Control) or treated with PTX or

PD98059, were incubated for 5 min without (None) or with S1P (1 µΜ) or HDL

(100 µg/ml) to detect the change in the phosphorylation of ERK (A) or the activity

of the enzyme (D). The control cells were also assayed for ERK activity with the

indicated concentrations of S1P (B) or HDL (C). In A, a representative result from

three separate experiments is shown. In B-D, data are means + S.D. of three values

from a representative experiment. Other two experiments gave similar results.

FIG. 5. Effects of S1P pretreatment on the ERK activation induced by S1P,

HDL, or ATP, a P2-purinergic agonist. The cells were preincubated for 5 h

without (Control) or with S1P (1 µΜ) and then incubated for 5 min with S1P (1

µΜ), HDL (100 µg/ml) or ATP (100 µΜ) to detect the change in the activity of the

enzyme. Data are means + S.E.M. of three separate experiments.

FIG. 6. Cytoprotective property of HDL is recovered in the S1P-rich fraction.

HUVECs were incubated without (None) or with HDL (100 µg/ml) or each

fraction (a-c) of HDL corresponding to 200 µg/ml HDL as described in Materials

and Methods for measurement of cell survival activity (A) or ERK activity (B). The

fraction b was further processed for HPTLC in which authentic S1P migrated at the

position marked (Rf = 0.44) (E). The cell survival activity (C) or ERK activity (D)

of each fraction corresponding to 200 µg/ml HDL was measured. Data are means +

S.D. of three values from a representative experiment. Other two experiments gave

similar results.


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FIG. 7. Inhibition by charcoal treatment of HDL- or LDL-induced

cytoprotective actions and ox-LDL-induced cytotoxic action. HDL, LDL or ox-

LDL was treated with charcoal, and then the respective lipoprotein was processed

for measurement of S1P content (pmol/mg protein) in A; electrophoresis analysis

of Apo A and Apo B in B; HPTLC analysis of LPC, phosphatidylcholine (PC),

sphingomyelin (SM), and other lipid composition in C, where O and F stand for

“origin” and “front”, respectively; and cell survival activity by 15% FBS (serum)

or 100 µg/ml each lipoprotein in the presence of 0.1% BSA in D. Data are means

+ S.E.M. of four separate experiments in A and D. In B and C, a representative

result from four separate experiments is shown. *P<0.05, **P<0.01; charcoal

treatment is significantly different from the respective control sample (Control).


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Figure 1Kimura T. et al

0

100

200

S1P

co

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nt

(pm

ol/m

g p

rote

in)

LDLox-LDL HDL

←Apo A

←Apo B

→F

→PC

→SM

→LPC

→Oox-LDL LDL HDL

AB

C

Lipoproteins (µg/ml)

Vector/CHO

10000

10

20

30

40

50

10010

0

10

Edg-3/CHO

IP2

+ IP

3 (%

)

D

ox-LDL LDL HDL by guest on April 10, 2018

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Cel

l su

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%)

100101020

40

60

80

100

120

Lipoproteins (µg/ml)

A

10.10.010

S1P alone+ox-LDL+Serum

S1P (µM)

D

LDL (µg/ml)

C

1001010

LDL alone

+ox-LDL

20

40

60

80

100

120

HDL (µg/ml)

Cel

l su

rviv

al (

%)

BHDL alone

+7keto+LPC+25h

+ox-LDL

1001010



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Con PTX PD SB0

20

40

60

80

100

120

Cel

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%)

A

None

S1P

HDL

ox-LDL

ox-LDL+ S1P

ox-LDL + HDL

Cel

l su

rviv

al (

%)

Con PTX PD SB0

20

40

60

80

100

120B



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Control PTX PD98059A

p42

p44p42pp44p

None S1P HDL None S1P HDL None S1P HDL

B

ER

K a

ctiv

ity

(%)

Control PTX0

100

200

300

400

500

C

PD98059

S1P (µM) 10.10.010.0010

0

100

200

300

400

500

ER

K a

ctiv

ity

(%)

10001001010

HDL (µg/ml)

D

None

S1P

HDL

p42p44


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Control Pretreatment with S1P

0

100

200

300

400

500

600

ER

K a

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None

S1P

HDL

ATP



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0

20

40

60

80

100

None HDL a b c

A

None HDL a b c

ER

K a

ctiv

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(%)

0

100

200

300

400B

Cel

l su

rviv

al (

%)

020406080

100C

ER

K a

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(%)

0

100

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300 D

Origin S1P Front

E 1 2 3 4 5 6 7

Cel

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%)


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B

D

No

ne

Ser

um

0

20

40

60

80

100

120

Cel

l su

rviv

al (

%)

LDLox-LDL

ControlCharcoal

＊＊

＊

＊＊

HDL

0

100

200

300

LDLox-LDLS1P

co

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nt

(pm

ol/m

g p

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in)

Control

Charcoal

A

HDL

→F

→PC

→SM

→LPC

→O

ox-LDL LDL

- + - + - + Charcoal

HDL

Charcoal- +

ox-LDL

- +

LDL

←Apo B

- +

HDL

←Apo A

C

＊＊＊＊

＊＊


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Kobayashi, Michio Ui and Fumikazu OkajimaTakao Kimura, Koichi Sato, Atsushi Kuwabara, Hideaki Tomura, Mitsuteru Ishiwara, Isaoresponsible for the cytoprotective actions in human umbilical vein endothelial cells

Sphingosine 1-phosphate may be a major component of plasma lipoproteins

published online June 26, 2001J. Biol. Chem.

10.1074/jbc.M104353200Access the most updated version of this article at doi:

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