sphingosine 1-phosphate receptors mediate stimulatory and inhibitory signalings for expression of...

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Cellular Signalling 18

Sphingosine 1-phosphate receptors mediate stimulatory and inhibitory

signalings for expression of adhesion molecules in endothelial cells

Takao Kimura a,e, Hideaki Tomura a, Chihiro Mogi a, Atsushi Kuwabara a,e, Mitsuteru Ishiwara a,

Kunihiko Shibasawa a, Koichi Sato a, Susumu Ohwada b, Doon-Soon Im c, Hitoshi Kurose d,

Tamotsu Ishizuka a, Masami Murakami e, Fumikazu Okajima a,*

a Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japanb Second Department of Surgery, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan

c Laboratory of Pharmacology, College of Pharmacy, Pusan National University, Busan, 609-735, Republic of Koread Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University,

3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japane Department of Clinical Laboratory Medicine, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan

Received 5 May 2005; received in revised form 14 July 2005; accepted 18 July 2005

Available online 18 August 2005

Abstract

Sphingosine 1-phosphate (S1P) stimulates expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in

human umbilical vein endothelial cells. S1P-induced actions were associated with nuclear factor kappa-B activation and inhibited by

pertussis toxin as well as by antisense oligonucleotides specific to S1P receptors, especially, S1P3. S1P also stimulated endothelial

nitric oxide synthase (eNOS) and its activation was markedly inhibited by the antisense oligonucleotide for the S1P1 receptor rather

than that for the S1P3 receptor. The dose–response curve of S1P to stimulate adhesion molecule expression was shifted to the left in

the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin and the NOS inhibitor NN-nitro-l-arginine methyl ester. NO

donor S-nitroso-N-acetylpenicillamine inhibited S1P-induced adhesion molecule expression. Moreover, tumor necrosis factor-a-induced

adhesion molecule expression was markedly inhibited by S1P in a manner sensitive to inhibitors for PI3-K and NOS. These results

suggest that S1P receptors are coupled to both stimulatory and inhibitory pathways for adhesion molecule expression. The stimulatory

pathway involves nuclear factor kappa-B and inhibitory one does phosphatidylinositol 3-kinase and NOS.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Sphingosine 1-phosphate; TNF-a; Vascular cell adhesion molecule-1; Intercellular adhesion molecule-1; Adhesion molecule; eNOS; Nuclear factor

kappa-B; Endothelial cells

0898-6568/$ - s

doi:10.1016/j.ce

Abbreviation

umbilical vein e

Inhibitor of nBhydrochloride;

Phenyl-5-trifluo

carbamoyl)-ethy

3-kinase; GAPD

serum albumin;

* Correspondi

E-mail addr

(2006) 841 – 850

ee front matter D 2005 Elsevier Inc. All rights reserved.

llsig.2005.07.011

s: S1P, sphingosine 1-phosphate; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; HUVECs, human

ndothelial cells; NO, nitric oxide; eNOS, endothelial NO synthase; TNF-a, tumor necrosis factor-a; NF-nB, nuclear factor kappa-B; InB,; HDL, high-density lipoprotein; l-NAME, l-NG-nitroarginine methyl ester hydrochloride; d-NAME, d-NG-nitroarginine methyl ester

BAY 11-7085, (E)3-[(4-t-Butylphenyl)sulfonyl]-2-propenenitrile; CAY10444, 2-undecyl-thiazolidine-4-carboxylic acid; SEW2871, 5-(4-

romethylthiophen-2-yl)-3-(3-trifluoromethylphenyl)-1,2,4-oxadiazole; VPC23019, (R)-Phosphoric acid mono-[2-amino-2-(3-octyl-phenyl-

l] ester; PTX, pertussis toxin; G-protein, GTP-binding regulatory protein; SNAP, S-nitroso-N-acetylpenicillamine; PI3-K, phosphatidylinositol

H, glyceraldehydes 3-phosphate dehydrogenase; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; BSA, bovine

FBS, fetal bovine serum; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; RT, reverse transcription.

ng author. Tel.: +81 27 220 8850; fax: +81 27 220 8895.

ess: [email protected] (F. Okajima).

T. Kimura et al. / Cellular Signalling 18 (2006) 841–850842

1. Introduction

Sphingosine 1-phosphate (S1P), a bioactive lipid

mediator, exerts a variety of actions in many types of

cells, including vascular endothelial cells [1–5]. For

example, S1P stimulates proliferation [6], survival [7–9],

migration [6,9–14], NO synthesis [8,11,15], and angio-

genesis [16,17] in endothelial cells. These actions are

thought to be anti-atherogenic [4,5]. On the other hand,

S1P has also been shown to stimulate the expression of

adhesion molecules, including vascular cell adhesion

molecule-1 (VCAM-1) and intercellular adhesion mole-

cule-1 (ICAM-1) [18–22]. An increase in the expression

of adhesion molecules stimulates monocyte and lympho-

cyte interaction with endothelial cells and cell penetration

into subendothelial space or the intima of arterial walls,

thereby resulting in inflammatory responses and foam cell

formation in the intima [23,24]. Thus, the expression of

adhesion molecules is thought to be an early step in the

onset of atherosclerosis. These studies suggest that S1P has

dual aspects on atherosclerosis, i.e., anti-atherogenic and

pro-atherogenic factors [4,5].

In the earlier study [18], intracellular S1P action was

claimed to play a role in adhesion molecule expression;

the increase in intracellular S1P, through sphingosine

kinase activation, was postulated to mediate tumor

necrosis factor (TNF)-a-induced adhesion molecule

expression. In that study, the involvement of sphingosine

kinase was evaluated from the finding that non-selective

sphingosine kinase inhibitor dimethylsphingosine [25]

inhibited the TNF-a-induced adhesion molecule expres-

sion. Recent studies, however, have reported that S1P-

induced actions are sensitive to PTX, suggesting the

involvement of G-protein-coupled receptors (GPCRs)

[19–21]. However, in one paper, an argument was

presented against the physiological role of S1P in

adhesion molecule expression because of its low activity

compared with its expression by TNF-a [19]. Thus, the

role and its target of S1P in the expression of adhesion

molecules are controversial. This situation prompted us to

investigate the role and action mechanism of S1P in the

expression of adhesion molecules, including VCAM-1 and

ICAM-1, in human umbilical vein endothelial cells

(HUVECs). We found that S1P receptors are coupled to

dual signaling pathways for the expression of adhesion

molecules: a stimulatory pathway involves NF-nB, and an

inhibitory pathway, PI3-K and NOS. The balance of the

activities of the NF-nB pathway and PI3-K/NOS pathway

may be important for determining the activity of S1P on

adhesion molecule expression, which might explain, in

part, differences among the previous studies with regard

to the activity elicited by S1P [18–22]. Such an inhibitory

pathway may be important under physiological and

pathological circumstances, when endothelial cells are

stimulated with a potent stimulator of adhesion molecule

expression, such as TNF-a.

2. Materials and methods

2.1. Materials

S1P, CAY10444, an S1P3-selective antagonist [26], and S-

nitroso-N-acetylpenicillamine (SNAP) were purchased from

Cayman Chemical Co.; wortmannin was from Calbiochem-

Novabiochem; BAY 11-7085, l-NG-nitroarginine methyl

ester hydrochloride (l-NAME), and d-NG-nitroarginine

methyl ester hydrochloride (d-NAME) were from BIOMOL

Research Laboratories Inc.; anti-endothelial nitric oxide

synthase (eNOS) antibody, anti-phospho-Ser-1179 eNOS

antibody, anti-InBa antibody, and anti-h-actin antibody werefrom Cell Signaling Technology Inc.; primary mouse anti-

bodies for VCAM-1 and ICAM-1 were from Chemicon

International; and SEW2871, an S1P1-selective agonist, [27]

was from Sigma-Aldrich. VPC23019, an S1P1-selective

antagonist, was generously provided by Prof. Kevin R.

Lynch of University of Virginia School of Medicine. This

chemical is about 50-fold less potent in antagonizing S1P3compared with S1P1 and inactive for S1P2 [28]. The sources

of all other reagents were the same as described previously

[6,7,9].

2.2. Cell culture and transfection of oligonucleotides

HUVECs (passage number 3) were purchased from

Whittaker Bioproducts. The cells were cultured in RPMI

1640 medium supplemented with 15% (v /v) fetal bovine

serum (FBS) and several growth factors as previously

described [6]. We usually used 5–8 passage of the cells and

checked the cobble-stone like cell shape before experi-

ments. Where indicated, pertussis toxin (PTX, 100 ng/ml)

or its vehicle (PBS) was added to the culture medium 24 h

before experiments, unless otherwise stated. Transfection of

antisense oligonucleotides to block the expression of S1P1and S1P3 receptors was performed using NovaFECTORTM

reagent (VennNova) according to the method of Paik et al.

[12] as described previously [9]. 18-mer phosphothioate

oligonucleotides used are as follows: antisense EDG-1/

S1P1, 5V-GAC GCT GGT GGG CC C CAT-3V and

antisense EDG-3/S1P3, 5V-CGG GAG GGC AGT TGC

CAT-3V. We also transfected sense oligonucleotides for

S1P1 and S1P3 as described previously [9]. The expression

of these S1P receptor mRNAs was measured at 12 h and

experiments were started at 16 h after the transfection.

THP-1 monocytic cells were cultured in RPMI 1640

medium containing 10% FBS.

2.3. Adenovirus infection

The cDNA for InBa was generated by RT-PCR from

mouse brain. The phosphorylation-deficient S32A, S36A

mutant was generated by PCR [29]. Eighty percent

confluent HUVECs were infected with recombinant adeno-

viruses coding for green fluorescent protein or dominant-

T. Kimura et al. / Cellular Signalling 18 (2006) 841–850 843

negative mutant InBa at a multiplicity of infection of 30 for

2 h at 37 -C in RPMI1640 containing 5% FBS. Cells were

then cultured for an additional 48 h with RPMI1640

containing 15% FBS and other supplements before treat-

ment. Under these conditions, infection with adenoviruses

coding for green fluorescent protein resulted in almost 100%

cells positive to green fluorescent protein.

2.4. NF-jB transcription assays

HUVECs were transfected with the lipofectin/nucleic

acid mixture including 1 Ag of lipofectin reagent (Invi-

trogen), 1 Ag of pNFnB-Luc (Stratagene), and 240 ng of

pRL (Renilla luciferase)-SV40. Luciferase reporter assay

was performed 48 h after transfection using a Dual-

Luciferase Reporter Assay System (Promega) according

to the manufacturer’s instructions. Renilla luciferase

activity was used to normalize transfection efficiencies

among experiments.

2.5. Determination of cell surface expression of adhesion

molecules by enzyme immunoassay

HUVECs were plated on 96-well-plates and transfected

with antisense oligonucleotides or infected with adenovirus

as described above. Cells were then washed twice and

incubated in RPMI 1640 containing 0.1% bovine serum

albumin (BSA) with test agents for 8 h. Thereafter cells

were washed with PBS twice and fixed with PBS

containing 3% formamide under 4-C. The plates were

blocked at 4-C overnight with 5% skim milk powder in

PBS. Cell surface expression of adhesion molecules was

determined by primary binding with specific mouse

antibody for VCAM-1 or ICAM-1, followed by secondary

binding with a horseradish peroxidase-conjugated goat

anti-mouse IgG antibody [30]. Quantification was per-

formed by determination of colorimetric conversion at

optical density at 450 nm of 3,3’,5,5’-tetramethylbenzidine

using TMB peroxidase EIA substrate kit (Bio-Rad

Laboratories).

2.6. Quantitative RT-PCR analysis

Total RNA was isolated using TRI REAGENT (Sigma-

Aldrich) according to the instructions from the manufac-

turer. After DNase I (Promega, Madison, UI) treatment to

remove possible traces of genomic DNA contaminating in

the RNA preparations, 5 Ag of the total RNA was reverse-

transcribed using High Capacity cDNA Archive kit

according to the instructions from the manufacturer

(Applied Biosystems). To evaluate the expression level of

the VCAM-1 and ICAM-1 mRNA, quantitative RT-PCR

was performed using real-time TaqMan technology with a

Sequence Detection System model 7700 (Applied Bio-

systems). The human VCAM-1 and ICAM-1-specific probe

was obtained from TaqMan Gene Expression Assays

(Applied Biosystems). The ID number of the products is

Hs00365486 for VCAM-1, Hs00164932 for ICAM-1, and

Hs99999905 for GAPDH. The expression level of the

target mRNA was normalized to the relative ratio of the

expression of GAPDH mRNA [31]. Each RT-PCR assay

was performed at least three times, and the results are

expressed as meanTSE.

2.7. Western blotting

HUVECs were cultured and pretreated with several

reagents as described above and then incubated for

indicated times with test agents. For detection of eNOS

phosphorylation and InBa, the reaction was terminated by

washing twice with ice-cold PBS and adding 0.1 ml of lysis

buffer containing 1% Triton X-100, 50 mM Tris–HCl pH

7.5, 150 mM NaCl, 2 mM EDTA, 8 mM EGTA, 25 mM

NaF, 10 mM Na4P2O7, 1 mM Na3VO4, 5 Ag/ml leupeptin,

5 Ag/ml pepstatin, 5 Ag/ml aprotinin, and 0.5 mM PMSF.

The lysate was analyzed by Western blotting [6,9];

separation with 6% SDS-polyacrylamide gel electrophore-

sis and detection with eNOS-specific and phospho-eNOS-

Ser473 antibodies for eNOS phosphorylation, and 10%

SDS-polyacrylamide gel electrophoresis and detection with

InBa specific antibody for InBa detection.

2.8. NOS enzymatic activity in cell lysate

NOS enzymatic activity was measured according to the

method previously described [9]. Briefly, HUVECs were

incubated with test agents for 10 min, and then rinsed twice

in ice-cold PBS, harvested from the dishes, and resus-

pended in ice-cold lysis buffer [32]. The cells were

disrupted by sonication (Branson Ultrasonics, Chicago,

IL) three times for 10 s each. NOS enzymatic activity in the

resulting cell lysates was determined by measuring the

conversion of l-[3H]arginine to l-[3H]citrulline. Fifty

microliters of cell lysate were added to 50 Al of reaction

mixtures containing 2 mM cold l-arginine and 2 ACi/ml of

l-[3H]arginine. After incubation at 37 -C for 1 h, the assay

was terminated by the addition of 400 Al of 40 mM HEPES

buffer, pH 5.5, with 2 mM EDTA and 2 mM EGTA. The

terminated reactions were applied to 1-ml columns of

Dowex AG50WX-8 (Tris form) and eluted with 1 ml of 40

mM HEPES buffer. The l-[3H]citrulline generated was

collected into scintillation vials and quantified by liquid

scintillation spectroscopy.

2.9. THP-1 cell adhesion assay

THP-1 monocytic cells were washed twice and resus-

pended in RPMI 1640 containing 0.1% BSA. The cell

suspensions were overlaied (1.5�106/ml, 500 Al/well) onthe confluent monolayers of HUVECs that had been grown

in 12-well-plates and treated with various reagents. After

incubation for 15 min at 37 -C, non-adherent THP-1


monocytic cells were removed by washing four times with

prewarmed RPMI 1640 medium containing 0.1% BSA.

The number of THP-1 monocytic cells adhered to

HUVECs was counted in four places under microscopy

at �400 magnification (4HPF) as adhering cells.

2.10. Data presentation

All experiments were performed in duplicate or

triplicate. The results of multiple observations are pre-

sented as the meanTSE or as representative results from

more than three different batches of cells, unless other-

wise stated. Statistical significance was assessed by the

Student’s t-test; values were considered significant at

p <0.05.

Fig. 1. S1P induced adhesion molecule expression through Gi/o-proteins and NF-nB24 h and incubated for 8 h with or without S1P (1 AM). THP-1 monocytic cells

monocytic cells were removed. A representative photograph is shown in (A) and

similarly treated with PTX or its vehicle and then incubated for 8 h with the indi

ICAM-1 (right panel) protein expression. (D) HUVECs were similarly treated wit

methods, and then incubated with or without S1P (1 AM) or TNF-a (TNF; 60 pM

expression. Infection of adenovirus coding for green fluorescent protein hardly aff

BAY 11-7085 effects, the cells were incubated with test agents in the presence

adenovirus coding for green fluorescent protein (GFP) or coding for dominant

HUVECs were transfected with pNFnB-Luc and/or dnInBa and 24 h after the tran

cells were treated with S1P (1 AM) or TNF-a (TNF; 60 pM) for last 8 h and lucife

green fluorescent protein was ineffective for the NF-nB activity. (G) HUVECs wer

expression of VCAM-1 (left panel) and ICAM-1 (right panel) by a quantitative RT

ratio to the expression of GAPDH mRNA.

3. Results

3.1. S1P stimulates VCAM-1 and ICAM-1 expression

through Gi/o-proteins and NF-jB

As shown in Fig. 1A and B, S1P promoted THP-1

monocytic cell adhesion to HUVECs and its adhesion

was almost completely inhibited by PTX. The change in

cell adhesion was associated with the change in the

expression of VCAM-1and ICAM-1 (Fig. 1C). Thus, S1P

promoted the cell surface expression of VCAM-1 and

ICAM-1 in HUVECs in a dose-dependent manner, and

PTX completely inhibited the S1P-induced actions with-

out any significant effect on the TNF-a-induced action

(Fig. 1C,D). Although S1P stimulated adhesion molecule

in HUVECs. (A and B) HUVECs were treated with PTX or its vehicle for

were then overlaid on the cells. Fifteen minutes later, non-adherent THP-1

adhered THP-1 monocytic cells were counted in (B). (C) HUVECs were

cated concentrations of S1P for measurement of VCAM-1 (left panel) and

h PTX or dominant negative InBa (dnInBa) as described in Materials and

) for 8 h to measure VCAM-1 (left panel) and ICAM-1 (right panel) protein

ected the adhesion molecule expression induced by these test agents. To see

or absence of BAY 11-7085 (10 AM). (E) HUVECs were infected with

negative InBa (dnInBa) to measure the expression of IkBa protein. (F)

sfection the cells were treated for another 24 h with PTX or its vehicle. The

rase reporter assay was performed as NF-nB activity. Adenovirus coding for

e treated in a similar way with the indicated agents, and analyzed for mRNA

-PCR assay. The expression level of mRNAwas normalized to the relative


expression, it should be noted that the S1P action

required higher concentrations of S1P (more than 100

nM) than other S1P-induced actions, such as cytoprotec-

tion [7,9], migration [6,9], or NOS activation (see Fig.

3C), in which the response was almost submaximal or

maximal at the same concentration of S1P [7,9]. S1P-or

TNF-a-induced cell surface expression of adhesion

molecules seems to involve NF-nB activation, as evi-

denced by the findings that the expression of VCAM-1

and ICAM-1 (Fig. 1D) was completely or markedly

inhibited by an NF-nB-specific inhibitor, BAY 11-7085,

and an overexpression (Fig. 1E) of dominant-negative

mutant or phosphorylation-deficient InBa. Actually, S1P

and TNF-a stimulated NF-nB-dependent transcription

activity in a manner dependent on the dominant negative

InBa (Fig. 1F). As expected, S1P-induced but not TNF-

a-induced action was inhibited by PTX (Fig. 1F).

Furthermore, S1P weakly induced InBa phosphorylation,

which was completely blocked by BAY 11-7085 (data

not shown). S1P also stimulated the mRNA expression of

VCAM-1 and ICAM-1 in a PTX-or BAY 11-7085-

dependent manner (Fig. 1G). These results suggest that

S1P stimulates VCAM-1 and ICAM-1 expression at

transcriptional levels through PTX-sensitive Gi/o-proteins

and NF-nB.

Fig. 2. Effects of antisense oligonucleotide transfection on VCAM-1 and ICAM

with (C) or without (A and B) pNFnB-Luc for 24 h and then treated with

receptors for another 16 h as described in Materials and methods. The cells we

or TNF-a (60 pM) to measure VCAM-1 or ICAM-1 expression in (A and

experiments.

3.2. S1P stimulates adhesion molecule expression through

S1P-specific receptors

In order to clarify the involvement of S1P receptors,

we examined the effects of an antisense oligonucleotide

specific to the S1P receptor subtypes. In HUVECs, S1P1and S1P3 are predominant S1P receptors [6,16]. We have

not succeeded to detect a significant S1P receptor protein

band by Western blotting. However, transfection of

antisense oligonucleotides specific to S1P1 and S1P3receptors caused almost complete inhibition of mRNA

expression of the respective receptor (data not shown), as

previously described [9]. Under the conditions used, S1P-

induced cell surface expression of VCAM-1 and ICAM-1

was partially but significantly inhibited by an antisense

oligonucleotide against either S1P1 or S1P3, although the

S1P3antisense oligonucleotide was more effective than the

S1P1 antisense oligonucleotide (Fig. 2A). Neither S1P1nor S1P3 sense oligonucleotide was effective in S1P-or

TNF-a-induced adhesion molecule expression (data not

shown). Furthermore, TNF-a-induced VCAM-1 expres-

sion (Fig. 2B) and ICAM-1 expression (data not shown)

were hardly affected even by a combination of antisense

oligonucleotides. Thus, the antisense oligonucleotide

method was specific. The change in adhesion molecule

-1 expression, and NF-nB activation in HUVECs. The cells were treated

antisense oligonucleotide against S1P1 receptor, S1P3 receptor or both

re incubated for 8 h with or without the indicated concentrations of S1P

B) and NF-nB activity in (C). Data are meansTSE of three separate


expression was associated with a parallel change in the

NF-nB activity (Fig. 2C). The predominant role of S1P3receptors rather than S1P1 receptors was also supported

by the experiments using S1P receptor subtype-selective

antagonists as shown later (see Fig. 5A). These results

suggest that S1P-stimulated VCAM-1 and ICAM-1

expression is mediated through S1P-specific receptors

especially S1P3 receptors.

3.3. S1P receptors are also coupled to inhibitory signaling

pathways on adhesion molecule expression

It has been shown that S1P activates eNOS and produces

NO in HUVECs [8]. In fact, S1P induced eNOS phosphor-

ylation (Fig. 3A), reflecting the activation of the enzyme

(Fig. 3B). The contribution of the respective S1P receptor

was examined in Fig. 3C. In contrast to the adhesion

molecule expression, S1P-induced NOS activation was

Fig. 3. S1P exerts both stimulatory and inhibitory action on VCAM-1 expression. (

min with or without S1P (1 AM) in the presence or absence of wortmannin (100

experiments is shown. (B) HUVECs were first treated with PTX or wortmannin

enzymatic activity of NOS in control cells was 55T8 pmol/mg protein/min and re

with antisense oligonucleotides as described in Fig. 2 and NOS activity was then m

inhibitor l-NAME and PI3-K inhibitor wortmannin on S1P-induced VCAM-1 exp

in the presence or absence of l-NAME (1 AM), d-NAME (1 AM), or wortmannin (

in the presence of the indicated concentrations of NO donor SNAP for 8 h to measu

h and incubated with or without S1P (1 AM) or TNF-a (60 pM) in the presence or

meansTSE of three separate experiments in (B–G).

markedly inhibited by the antisense oligonucleotide for the

S1P1 receptor and moderately by the antisense oligonucleo-

tide for S1P3 receptor. It is noteworthy that the potency of

S1P to activate NOS (Fig. 3C) was roughly 100 times higher

than that of S1P to stimulate adhesion molecule expression

(Fig. 2A) and NF-nB transcriptional activity (Fig. 2C).

Fig. 3D shows the effects of the NOS inhibitor l-NAME

and the inactive form d-NAME on S1P-induced VCAM-1

expression. l-NAME, but not d-NAME, shifted the S1P

dose–response curve to the left. These results suggest that

S1P-induced NO production plays an inhibitory role in

adhesion molecule expression. Thus, S1P exerts both

stimulatory and inhibitory actions on adhesion molecule

expression; the stimulatory action dominates the inhibitory

action at concentrations higher than 1 AM. The S1P-induced

dose–response curve was also shifted to the left by PI3-K

inhibitor wortmannin (Fig. 3E), which was associated with

the inhibition of eNOS phosphorylation (Fig. 3A) and NOS

A) HUVECs were treated with PTX or its vehicle, and then incubated for 10

nM) to measure eNOS phosphorylation. A representative of three separate

and then incubated with or without S1P to measure NOS activity. Basal

sults are expressed as percentages of this value. (C) HUVECs were treated

easured with the indicated concentrations of S1P. (D and E) effects of NOS

ression. HUVECs were incubated with the indicated concentrations of S1P

100 nM). (F) HUVECs were incubated with S1P (1 AM) or TNF-a (60 pM)

re VCAM-1 expression. (G) HUVECs were treated with pNFnB-Luc for 48absence of SNAP (1 AM) for last 8 h to measure NF-nB activity. Results are


activation (Fig. 3B). We observed similar inhibition profiles

by l-NAME and wortmannin on ICAM-1 expression (data

not shown). PTX also inhibited eNOS phosphorylation (Fig.

3A) and NOS activation (Fig. 3B). These results suggest

that the inhibitory action of S1P on adhesion molecule

expression may be mediated by eNOS activation through

Gi/o-proteins and PI3-K. Supporting this speculation, NO

donor S-nitroso-N-acetylpenicillamine (SNAP) inhibited

S1P-and TNF-a-induced VCAM-1 expression (Fig. 3F),

ICAM-1 expression (data not shown), and NF-nB activation

(Fig. 3G).

Since TNF-a-induced adhesion molecule expression was

inhibited by the NO donor, we next examined whether S1P

is able to inhibit TNF-a-induced adhesion molecule

expression. The maximal response of S1P on adhesion

molecule expression was about 30~50% of that of TNF-a

(see Figs. 1D 2A,B and 3F). Consistent with this observa-

tion, TNF-a promoted THP-1 monocytic cell adhesion to

HUVECs more effectively than S1P did (Fig. 4A,B). More

importantly, the TNF-a-induced action in the presence of

S1P was reduced to the level elicited by S1P alone (Fig.

4A,B). The change in the cell adhesion activity was

associated with the changes in the expression of adhesion

molecule (Fig. 4C) and the activation of NF-nB-dependent

Fig. 4. S1P inhibits TNF-a-induced adhesion molecule expression and NF-nB ac

monolayer of HUVECs, which had been stimulated without (None), with S1P (1 Alater, non-adherent THP-1 monocytic cells were removed. A representative photo

monocytic cells were counted in (B). (C) HUVECs were incubated for 8 h with th

pM) to measure VCAM-1 expression (left panel) and ICAM-1 expression (right pa

shown in Fig. 1F, then treated with or without PTX, and finally incubated with S1P

wortmannin (Wort; 100 nM) for 8 h.

transcriptional activity (Fig. 4D). Thus, S1P inhibited TNF-

a-induced adhesion molecule expression and NF-nB acti-

vation, while the lysolipid alone induced small effects,

compared with TNF-a, on these cellular activities. The S1P-

induced inhibition of adhesion molecule expression was

reversed by PTX, l-NAME, and wortmannin (Fig. 4E), in

association with the parallel regulation of NF-nB activity

(Fig. 4D). Similar activity changes were observed for

ICAM-1 expression (data not shown).

Finally, the subtype of S1P receptors responsible for the

S1P-induced actions on adhesion molecule expression was

examined using S1P receptor subtype-selective antagonist

and agonist. Consistent with the results of the antisense

oligonucleotide (Fig. 2), the stimulatory action was antag-

onized clearly by an S1P3-selective antagonist CAY10444,

but only slightly, if any, by an S1P1-selective antagonist

VPC23019 (Fig. 5A). Furthermore, unlike S1P, an S1P1-

selecitve agonist SEW2871 alone did not exert a significant

stimulatory effect (Fig. 5B). On the other hand, as for the

inhibitory action of S1P, the S1P1-selective antagonist was

more effective than the S1P3-antagonist to reverse the S1P

action. Moreover, the S1P1-selecitve agonist clearly

inhibited the TNF-a-induced VCAM-1 expression to the

extent similar to S1P (Fig. 5B). Similar effects of S1P

tivation. (A and B), THP-1 monocytic cells were overlaid on the confluent

M), with TNF-a (60 pM), or with S1P plus TNF-a for 8 h. Fifteen minutes

graph from three separate experiments is shown in (A) and adhered THP-1

e indicated concentrations of S1P in the presence or absence of TNF-a (60

nel). (D and E) the cells were treated with (D) or without (E) pNFnB-Luc as(1 AM) or TNF-a (60 pM) in the presence or absence of l-NAME (1 AM) or

Fig. 5. Effects of the S1P receptor subtype-selective antagonist and agonist on VCAM-1 expression. HUVECs were incubated for 8 h with S1P (1 AM) and/or

TNF-a (60 pM) in the presence or absence of the indicated concentrations of VPC23019 or CAY10444 in (A), and incubated for 8 h with or without TNF-a (60

pM) in the presence of the indicated concentrations of SEW2871, to measure VCAM-1 expression.


receptor selective-agonist and antagonist were observed for

ICAM-1 expression (data not shown). These results further

suggest that the S1P3 receptor and S1P1 receptor are major

receptors critical for the stimulatory and inhibitory path-

ways, respectively, to adhesion molecule expression.

4. Discussion

S1P has been shown to exhibit a variety of anti-

atherogenic actions in endothelial cells and smooth muscle

cells, including regulation of proliferation, survival, and

migration [1–5]. However, the lipid mediator has also been

shown to stimulate the expression of pro-atherogenic

adhesion molecules, such as VCAM-1 and ICAM-1, in

endothelial cells, although the concentration of S1P required

for this action is higher than that required for anti-

atherogenic S1P actions including extracellular signal-

regulated kinase activation, cell survival, and migration

response. In the present study, we demonstrated that the

S1P-induced stimulation of adhesion molecule expression

was mediated through S1P receptors and NF-nB. More

importantly, our results indicated that S1P receptors are also

coupled to inhibitory pathways for adhesion molecule

expression through PI3-K and NOS activation. Consistent

with our results, a recent study also showed that S1P

prevented TNF-a-induced monocyte adhesion to vascular

endothelial cells, although S1P inhibition of VCAM-1 and

ICAM-1 expression was not observed by the study [33].

Although both S1P1 and S1P3 receptors may be involved in

both signaling pathways, the contribution of each receptor

seems to be somewhat different. Thus, the S1P3 receptor is

more important for the stimulatory pathway, and the S1P1receptor, for the inhibitory pathway.

Although it has been repeatedly shown that S1P

stimulates the expression of adhesion molecules, such as

VCAM-1 and ICAM-1 [18–22], the primary target(s) of

S1P is controversial. Xia et al. have observed that TNF-a-

induced adhesion molecule expression was inhibited by a

sphingosine kinase inhibitor, dimethylsphingosine [18].

Based on this result, the same authors suggested that

intracellular S1P synthesized by sphingosine kinase might

affect adhesion molecule expression through unidentified

intracellular target(s) [18]. In contrast, other reports have

shown that exogenous S1P-induced adhesion molecule

expression is inhibited by PTX, suggesting the mediation

by Gi/o-protein-coupled receptors [19–21]. Our results favor

the latter S1P receptor involvement, as evidenced by the

finding that the S1P actions were inhibited by the trans-

fection of antisense oligonucleotides specific to S1P1 and

S1P3 in association with the down-regulation of the

respective S1P receptor mRNA expression. The more

effective inhibition by the S1P3 antisense oligonucleotide

and an S1P3-selective antagonist compared with the S1P1antisense oligonucleotide (Fig. 2) and an S1P1-selective

antagonist (Fig. 5) suggests that the contribution of the S1P3receptor is greater than that of the S1P1 receptor. We also

confirmed that the S1P-induced actions were completely

inhibited by PTX. At present, we cannot clearly account for

the controversy concerning action sites of S1P, i.e., whether

they are intracellular targets or extracellular S1P receptors.

S1P can be synthesized extracellularly by exported sphin-

gosine kinase and act on S1P receptors [34]. If this was the

case, TNF-a-induced adhesion molecule expression should

have been inhibited by PTX. The cytokine effect, however,

was insensitive to the treatment with the toxin (Fig. 1D) and

antisense oligonucleotides against S1P receptors (Fig. 2B).

Adhesion molecule expression was associated with their

mRNA expression, suggesting a change at the transcrip-

tional levels. Recent studies suggested that NF-nB is

involved in the regulation of VCAM-1 and ICAM-1

expression elicited by cytokines [23]. Actually, we observed

that S1P stimulated NF-nB-dependent transcriptional activ-ity and that an inhibitor of NF-nB and the dominant-

negative mutant of an NF-nB regulatory protein, InBa,suppressed the S1P-induced adhesion molecule expression.


In addition to the stimulatory role, S1P receptors may be

also linked to the opposite signaling pathways on the

expression of adhesion molecules. The inhibitory pathway

seems to involve NOS activation and NO synthesis. Thus,

the NOS inhibitor l-NAME shifted the dose–response

curve of the S1P-induced adhesion molecule expression to

the left. We actually observed eNOS phosphorylation by

S1P, reflecting the activation of the enzyme. The concen-

tration of S1P required for ‘‘inhibitory’’ NOS activation is

lower than that required for ‘‘stimulatory’’ NF-nB activa-

tion, which may explain the shift of the dose–response

curve of the S1P-induced adhesion molecule expression by

the NOS inhibitor. Furthermore, we observed that NO donor

SNAP inhibited S1P-and TNF-a-induced adhesion mole-

cule expression and NF-nB activation. It is still unclear,

however, how NO inhibits the NF-nB-mediated adhesion

molecule expression [35].

We could detect the inhibitory activity of S1P more

clearly when the adhesion molecule expression was

stimulated by a potent stimulator, TNF-a, although the

S1P concentrations required for the inhibition were higher

than those for NOS activation probably due to the potent

ability of TNF-a on NF-nB activation. We have recently

shown that HDL is a carrier of S1P and some of anti-

atherogenic actions of the lipoprotein, such as survival and

migration, are mediated by S1P [4,5,7,9,36,37]. HDL has

previously been shown to inhibit TNF-a-induced adhesion

molecule expression [38]. These results suggest that the

inhibitory mechanism linked to S1P receptors for adhesion

molecule expression may in part account for the HDL-

induced inhibition of the TNF-a action. On the other hand,

Xia et al. proposed that S1P plays a second messenger role

of TNF-a and HDL inhibits TNF-a-induced action through

inhibition of S1P synthesis [18,38]. Although the present

study cannot completely rule out the possible second

messenger role of S1P in the TNF-a action, it would be

quite strange that intracellular S1P mediates the TNF-a-

induced cell adhesion, while extracellular S1P inhibits the

cytokine-induced action. Inhibitory action of S1P against

TNF-a-induced monocyte adhesion has recently been

reported by other group as well [33]. Further careful studies

are needed for the role of sphingosine kinase-synthesized

S1P in the TNF-a-induced action. Moreover, future studies

should be also addressed on the role of HDL-associated S1P

especially with respect to the lipoprotein action on the TNF-

a-induced adhesion molecule expression. NOS activation

seems to be mediated by Gi/o-proteins/PI3-K (Fig. 3A,B)

[8,11,15]. Antisense oligonucleotide experiments suggested

that both S1P1 and S1P3 receptors are involved in NOS

activation, although the S1P1 receptor antisense oligonu-

cleotide more effectively inhibited the enzyme activation

(Fig. 3C). The predominant role of the S1P1 receptor over

the S1P3 receptor in NO synthesis has been reported in the

same HUVECs by another research group [8]. A recent

study with S1P3 knock out mouse showed the involvement

of S1P3 in mouse artery NO synthesis. The divergence

might be explained by the difference in species between

human and mouse; however, in the mouse study, the

participation of the S1P1 receptor was not addressed [39].

In any event, both the stimulatory and inhibitory pathways

seem to involve Gi/o-proteins, even though the contribution

of the respective S1P receptor subtype in each pathway may

be somewhat different. Such an apparently strange obser-

vation may be explained by the different coupling of S1P

receptor subtypes to G-proteins other than Gi/o-proteins

[2,3,40,41]. The balance of the activity between the NF-nBsignaling pathways and the PI3-K/NOS pathways may

determine the threshold of S1P to stimulate adhesion

molecule expression.

In conclusion, S1P stimulates the expression of adhesion

molecules, such as VCAM-1 and ICAM-1, through S1P

receptors, especially S1P3, and NF-nB-involving pathways.

S1P receptors may be also coupled to an inhibitory

pathway involving PI3-K and NOS against adhesion

molecule expression. The inhibitory signaling pathway

may play a role in the S1P-induced suppression of adhesion

molecule expression by a potent stimulator, TNF-a. The

inhibitory action of S1P may be listed as a novel anti-

atherogenic action of S1P. In this inhibitory pathway, the

S1P1 receptor is more important than the S1P3 receptor.

The differential contribution of the respective receptor may

make it possible to control the pro-atherogenic expression

of adhesion molecules by targeting the respective S1P

receptor.

Acknowledgments

We are grateful to Mr. Masayuki Tobo, Ms. Mayumi

Komachi, and Ms. Chisuko Uchiyama for their technical

assistance. We thank to Prof. Kevin R. Lynch of

University of Virginia School of Medicine for a generous

gift of VPC23019. This work was supported by a Grants-

in-Aid for scientific research from the Japan Society for

the Promotion of Science, a grant of the 21st Century COE

Program from the Ministry of Education, Culture, Sports,

Science, and Technology of Japan, a Pusan National

University research grant and grants from ONO Medical

Research Foundation, The Nakatomi Foundation, Uehara

Memorial Foundation, and Charitable Trust Pathology

Foundation of Japan and Kurozumi Medical Foundation.

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