Sphingosine-1-phosphate promotes lymphangiogenesis by stimulating S1P1/G(i)/PLC/Ca2+ signaling pathways

Abstract

The lymphatic system plays pivotal roles in mediating tissue fluid homeostasis and immunity, and excessive lymphatic vessel formation is implicated in many pathological conditions, which include inflammation and tumor metastasis. However, the molecular mechanisms that regulate lymphatic vessel formation remain poorly characterized. Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that is implicated in a variety of biologic processes such as inflammatory responses and angiogenesis. Here, we first report that S1P acts as a lymphangiogenic mediator. S1P induced migration, capillary-like tube formation, and intracellular Ca(2+) mobilization, but not proliferation, in human lymphatic endothelial cells (HLECs) in vitro. Moreover, a Matrigel plug assay demonstrated that S1P promoted the outgrowth of new lymphatic vessels in vivo. HLECs expressed S1P1 and S1P3, and both RNA interference-mediated down-regulation of S1P1 and an S1P1 antagonist significantly blocked S1P-mediated lymphangiogenesis. Furthermore, pertussis toxin, U73122, and BAPTA-AM efficiently blocked S1P-induced in vitro lymphangiogenesis and intracellular Ca(2+) mobilization of HLECs, indicating that S1P promotes lymphangiogenesis by stimulating S1P1/G(i)/phospholipase C/Ca(2+) signaling pathways. Our results suggest that S1P is the first lymphangiogenic bioactive lipid to be identified, and that S1P and its receptors might serve as new therapeutic targets against inflammatory diseases and lymphatic metastasis in tumors.

Full text

doi:10.1182/blood-2007-11-125203
Prepublished online June 9, 2008;
2008 112: 1129-1138
Chi-Bom Chae and Yong Song Gho
Chang Min Yoon, Bok Sil Hong, Hyung Geun Moon, Seyoung Lim, Pann-Ghill Suh, Yoon-Keun Kim,
S1P1/Gi/PLC/Ca2+ signaling pathways
Sphingosine-1-phosphate promotes lymphangiogenesis by stimulating
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HEMOSTASIS, THROMBOSIS,AND VASCULAR BIOLOGY
Sphingosine-1-phosphate promotes lymphangiogenesis by stimulating
S1P1/Gi/PLC/Ca2⫹signaling pathways
Chang Min Yoon,1Bok Sil Hong,1Hyung Geun Moon,1Seyoung Lim,1Pann-Ghill Suh,1Yoon-Keun Kim,1Chi-Bom Chae,2
and Yong Song Gho1
1Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea; and 2Institute of Biomedical Science and
Technology, Konkuk University, Seoul, Republic of Korea
The lymphatic system plays pivotal roles in
mediating tissue fluid homeostasis and im-
munity, and excessive lymphatic vessel for-
mation is implicated in many pathological
conditions, which include inflammation and
tumor metastasis. However, the molecular
mechanisms that regulate lymphatic vessel
formation remain poorly characterized.
Sphingosine-1-phosphate (S1P) is a potent
bioactive lipid that is implicated in a variety
of biologic processes such as inflammatory
responses and angiogenesis. Here, we first
report that S1P acts as a lymphangiogenic
mediator. S1P induced migration, capillary-
like tube formation, and intracellular Ca2ⴙ
mobilization, but not proliferation, in human
lymphatic endothelial cells (HLECs) in vitro.
Moreover, a Matrigel plug assay demon-
strated that S1P promoted the outgrowth of
new lymphatic vessels in vivo. HLECs ex-
pressed S1P1 and S1P3, and both RNA
interference–mediated down-regulation of
S1P1 and an S1P1 antagonist significantly
blocked S1P-mediated lymphangiogenesis.
Furthermore, pertussis toxin, U73122, and
BAPTA-AM efficiently blocked S1P-induced
in vitro lymphangiogenesis and intracellular
Ca2ⴙmobilization of HLECs, indicating that
S1P promotes lymphangiogenesis by stimu-
lating S1P1/Gi/phospholipase C/Ca2ⴙsignal-
ing pathways. Our results suggest that S1P
is the first lymphangiogenic bioactive lipid
to be identified, and that S1P and its recep-
tors might serve as new therapeutic targets
against inflammatory diseases and lym-
phatic metastasis in tumors. (Blood. 2008;
112:1129-1138)
Introduction
Sphingosine-1-phosphate (S1P) has been implicated in a wide
spectrum of biologic processes, including the promotion of cell
growth and survival, migration and differentiation, platelet aggrega-
tion, inflammatory responses, and angiogenesis.1S1P is generated
by the phosphorylation of sphingosine through a process mediated
by sphingosine kinase 1 (SphK1) and SphK2. S1P acts both
intracellularly as a second messenger2and extracellularly as a
ligand for a family of G-protein–coupled S1P receptors.3S1P1
couples stringently to the Giprotein family, whereas S1P2 and
S1P3 couple to the Gi,G
q, and G12/13 protein families. Multiple
interconnections of S1P signaling through S1P1 and S1P3 induce
vascular endothelial cell proliferation, migration, morphogenesis,
cytoskeletal reorganization, and adherens junction assembly,
whereas signaling via S1P2 negatively regulates S1P-mediated
multiple responses of vascular endothelial cells.4The defective
vascular maturation observed in S1P1-deficient mice highlights a
fundamental role for S1P signaling on vasculogenesis.5Neutraliza-
tion of the action of extracellular S1P shows significant inhibition
of angiogenesis, tumor growth, metastasis, and lymphocyte trans-
migration, indicating that S1P is an important pathological regula-
tor of inflammation and angiogenesis.6-8
Lymphatic vessels play important roles in mediating tissue fluid
homeostasis and immunity.9Although lymphangiogenesis, the
formation of new lymphatic vessels from preexisting vessels,
occurs under physiological and pathological conditions, including
chronic inflammation10 and tumor metastasis,11 the molecular
mechanisms that regulate lymphatic vessel formation remain
largely uncharacterized. Vascular endothelial growth factor C
(VEGF-C), the first identified lymphangiogenic factor, is a se-
creted, proteolytically processed glycoprotein that activates VEGF
receptors 2 and 3 on lymphatic endothelial cells.12,13 VEGF-C is
overexpressed in many primary tumors and induces lymphangiogen-
esis and angiogenesis around tumor tissues, as well as promotes
tumor metastasis via newly formed lymphatic or blood vessels.14,15
In addition, VEGF-C is up-regulated in inflammatory cells and so
induces lymphangiogenesis and angiogenesis during chronic
inflammation.10
VEGF-A, hepatocyte growth factor (HGF), angiopoietin-1,
platelet-derived growth factor BB (PDGF-BB), and insulin-like
growth factor 1/2 (IGF1/2), all of which are proangiogenic factors,
can also give rise to in vivo lymphangiogenesis.16-19 However,
relatively less interest has been focused on the involvement of
bioactive lipid molecules compared with that of growth factors in
lymphangiogenesis. Because S1P is a potent proangiogenic and
inflammatory bioactive lipid molecule,20,21 we hypothesized that
S1P could affect lymphangiogenesis. In this report, we show that
S1P induced the migration and capillary-like tube formation of
lymphatic endothelial cells in vitro and lymphangiogenesis in vivo.
Real-time polymerase chain reaction (PCR) analysis revealed that
human lymphatic endothelial cells (HLECs) expressed S1P1 and
S1P3. S1P-induced lymphangiogenesis was significantly inhibited
by pertussis toxin (PTX), RNA interference–mediated down-
regulation of S1P1, and an S1P1 antagonist, indicating the involve-
ment of Giprotein activation coupled with S1P1. In addition, the
inhibition of S1P-induced in vitro lymphangiogenesis by U73122
and BAPTA-AM demonstrated that phospholipase C (PLC)/Ca2⫹
Submitted November 20, 2007; accepted May 27, 2008. Prepublished online as
Blood First Edition paper, June 9, 2008; DOI 10.1182/blood-2007-11-125203.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2008 by The American Society of Hematology
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was an important signaling pathway. Moreover, we found that S1P
induced intracellular Ca2⫹mobilization and formation of actin
stress fibers in a PTX/PLC-dependent manner in HLECs. Our
findings strongly suggest that S1P is the first lymphangiogenic
bioactive lipid and that S1P secreted from inflammatory cells or
tumor cells may induce lymphangiogenesis during tumor growth,
metastasis, and inflammation.
Methods
Materials
S1P, heparin, FITC-dextran (2000 kDa) and sulfinipyrazone were pur-
chased from Sigma-Aldrich (St Louis, MO); S1P1 selective antagonist
(R-isomer) and S1P1 control molecule (S-isomer) were acquired from
Avanti Polar Lipids (Alabaster, AL); L-NAME, LY294002, and BAPTA-AM
were obtained from BioMol Research Laboratories (Plymouth Meeting,
PA); PTX was purchased from Calbiochem (La Jolla, CA); PD98059,
SB203580, and U73122 were acquired from Alexis Biochemicals (San
Diego, CA); recombinant human VEGF-C and basic fibroblast growth
factor (bFGF) were obtained from R&D Systems (Minneapolis, MN);
human fibronectin, growth factor–reduced (GFR)–Matrigel, and Matrigel
were purchased from BD Biosciences (San Jose, CA); medium 199, fetal
bovine serum (FBS), penicillin/streptomycin, human endothelial serum–
free medium (HE-SFM), fura-2/AM, lipofectamine 2000, AlexaFluor594-
streptavidin, AlexaFluor488 donkey anti–mouse and anti–rabbit, and Alex-
aFluor594 donkey anti–rat IgG antibodies were obtained from Invitrogen
(Carlsbad, CA). Diff-Quick solution was purchased from Baxter Healthcare
(McGraw Park, IL); One Step SYBR reverse-transcription (RT)–PCR kit
was acquired from TaKaRa Bio (Tokyo, Japan); hamster antibody 8.1.1 was
purchased from Developmental Studies Hybridoma Bank (Iowa City, IA);
biotin-labeled anti–hamster IgG antibody, normal mouse, rat, rabbit IgGs
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit
anti–Prox-1 antibody was acquired from RELIATech (Braunschweig,
Germany); rat anti–lymphatic vascular endothelial hyaluronan receptor-1
(LYVE-1) antibody was kindly gifted by Dr Gou Young Koh (Korea
Advanced Institute of Science and Technology, Daejeon, Republic of
Korea); mouse anti–human podoplanin antibody (D2-40) was obtained
from Signet Laboratories (Dedham, MA); rabbit anti–phospho-histone-H3
(PH3) antibody was obtained from Upstate Biotechnology (Lake Placid,
NY); shRNAs in pRS plasmid against S1P1 and S1P3 were acquired from
OriGene (Rockville, MD); HLECs and human dermal lymphatic microvas-
cular endothelial cells (HMVECs-dLy) were purchased from ScienCell
(San Diego, CA) and Lonza (Walkersville, MD), respectively.
Cell culture
Human umbilical vein endothelial cells (HUVECs) were isolated from
freshly delivered umbilical cords as described previously22 and main-
tained in medium 199 supplemented with 20% FBS, 3 ng/mL bFGF,
5 U/mL heparin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin.
HLECs were maintained in endothelial cell medium supplemented with
5% FBS, endothelial cell growth supplement (ScienCell), 100 U/mL
penicillin, and 0.1 mg/mL streptomycin. HMVECs-dLy were cultured in
EGM-2 MV medium (Lonza). Cells were maintained at 37°C in a
humidified 5% CO2atmosphere. Cells from 4 to 5 passages were used
for the experiments in this study.
Migration assay
A migration assay was performed in a 48-well microchemotaxis chamber
(Neuro Probe, Cabin John, MD) as described previously.23 Polycarbonate
membranes with 12-␮m pores (Neuro Probe) were coated with 1 ␮g/mL
human fibronectin in double-distilled water and then dried for 1 hour. Cells
were harvested and resuspended in HE-SFM containing medium only or
with 100 ng/mL PTX, 10 ␮M LY294002, 1 mM L-NAME, 10 ␮M PD98059,
25 ␮M SB203580, 30 ␮M BAPTA-AM, 5 ␮M U73122, S1P1 antagonist,
or S1P1 control molecule. The bottom chamber was loaded with
3⫻104cells, and the filter membrane was laid over the cells. The
microchamber was then inverted and incubated at 37°C for 2 hours. After
reinverting the chamber to its upright position, the upper wells were then
loaded with HE-SFM containing the indicated concentration of S1P or
100 ng/mL S1P with 100 ng/mL PTX, 10 ␮M LY294002, 1 mM L-NAME,
10 ␮M PD98059, 25 ␮M SB203580, 30 ␮M BAPTA-AM, 5 ␮M U73122,
S1P1 antagonist, or S1P1 control molecule. The chamber was then
reincubated at 37°C for 4 hours, and the filter membrane was fixed and
stained using Diff-Quick solution. The cells that migrated through the filter
membrane were quantified by counting 3 random fields of each well using a
Nikon Eclipse TS100 microscope (Nikon, Tokyo, Japan) equipped with a
Plan Fluor 20⫻/0.50 DIC M/N2 objective lens and COOLPIX 995 digital
camera (Nikon). Experiments were carried out in triplicate and repeated
independently 3 times.
Proliferation assay
HLECs and HMVECs-dLy were plated at 2.5 ⫻104cells/well on a
fibronectin-coated 48-well plate. After starvation in HE-SFM for 6 hours,
the cells were incubated with S1P or VEGF-C for 24 hours. The cells were
treated with 0.5 ␮Ci (0.0185 MBq) [3H]-thymidine per well and further
incubated for 12 hours. The radioactivity of incorporated [3H]-thymidine
was determined in a liquid scintillation counter. Experiments were carried
out in triplicate and repeated 3 times.
Capillary-like tube formation assay
The tube formation assay was performed as described previously.23 Cells
(2 ⫻104cells/well) in 0.4 mL HE-SFM with the indicated concentration of
S1P, 500 ng/mL VEGF-C, or 100 ng/mL S1P with 100 ng/mL PTX, 10 ␮M
LY294002, 1 mM L-NAME, 10 ␮M PD98059, 25 ␮M SB203580, 30 ␮M
BAPTA-AM, 5 ␮M U73122, S1P1 antagonist, or S1P1 control molecule
were plated on a GFR Matrigel-coated 24-well plate. After a 6-hour
incubation, the cells were fixed with Diff-Quick solution, and 2 randomly
chosen fields per well were visualized and acquired using a Nikon Eclipse
TS100 microscope equipped with a 10⫻/0.25 Ph1 ADL objective lens and
COOLPIX 995 digital camera, and processed using Adobe Photoshop 7.0
(Adobe Systems, San Jose, CA). Total tube area was analyzed using Scion
Image software (Frederick, MD). Analyses of the test samples were
performed in duplicate and independent experiments were repeated 3 times.
Immunocytochemistry
HLECs and HMVECs-dLy were fixed with 4% paraformaldehyde and
incubated with anti–Prox-1, anti–LYVE-1, or anti-podoplanin antibody. For
immunostaining of Prox-1, fixed cells were permeabilized with 0.2% Triton
X-100. Isotype-matched control IgGs were used for negative staining. After
treatment of AlexaFluor-conjugated secondary antibodies, cells were coun-
terstained with Hoechst. Five randomly chosen fields were visualized using
AxioSkop2 Plus Fluorescent microscope (Carl Zeiss, Jena, Germany)
equipped with an Achroplan 20⫻/0.45 objective lens and images were
acquired using an AxioVision 4.0 software (Carl Zeiss) and processed using
Adobe Photoshop 7.0.
Real-time RT-PCR
PCR primers for human S1P1, S1P2, S1P3, and ␤-actin were designed
using the Primer3 program (Whitehead Institute, http://biotools.umass-
med.edu/bioapps/primer3_www.cgi). The primers used are shown in Table
1. After phosphate-buffered saline washing, subconfluent HLECs were
trypsinized and centrifuged. Cell pellets were used for isolation of total
RNA via RNease Mini Kit (QIAGEN, Valencia, CA). For real-time
RT-PCR, total RNA (100 ng) was amplified with a One Step SYBR RT-PCR
kit using a LightCycler 2.0 PCR system (Roche Diagnostics, Mannheim,
Germany). This experiment was repeated twice.
Matrigel plug assay, immunostaining, and lymphangiography
C57BL/6 (male, 5 weeks old, 5 mice per group) mice were subcutaneously
injected with 0.5 mL Matrigel containing 0.4 ␮g S1P or 1 ␮g VEGF-C. The
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mice were killed at 10 days after implantation, and the Matrigel was
removed, fixed in 4% paraformaldehyde, and embedded in paraffin.
Cross-sections of paraffin-embedded Matrigel were stained with hematoxy-
lin and eosin (H&E) and images were visualized using an Olympus BX51
microscope (Olympus, Tokyo, Japan) equipped with an UPlanFL N
100⫻/1.30 oil objective lens and acquired using analysis LS Research
software (Soft Imaging System, Munster, Germany). Lymphatic vessels
were immunostained with anti–Prox-1, anti–LYVE-1, or anti-podoplanin
antibody. Proliferating nuclei in lymphatic vessels were immunostained
with rabbit anti-PH3 antibody. For lymphangiography, at 10 days after
implantation, FITC-dextran (2000 kDa, 10 mg/mL) was injected intrader-
mally apart from Matrigel, and lymphatic vessel networks were analyzed.
All images were visualized using an FV1000 Olympus Confocal micro-
scope (Olympus) equipped with an UPlanSApo 20⫻/0.75 objective lens
and acquired using FV1000-ASW 1.5 software (Olympus). This experiment
was independently repeated twice. Animal study protocols were approved
by the Institutional Animal Care and Use Committee at Pohang University
of Science and Technology.
RNA interference–mediated down-regulation of S1P1 and S1P3
The shRNA retroviral pRS plasmids for S1P1 and S1P3 were transfected
into mouse packaging PT67 cells (Clontech, Palo Alto, CA) using
lipofectamine 2000 and cultured with puromycin (Clontech). For negative
control, empty pRS plasmid was used. After infection with retrovirus,
HLECs were cultured with puromycin. The shRNA sequences against S1P1
and S1P3 are as follows: S1P1, 5⬘-GTACTTCCTGGTGTTAGCTGTGCT-
CAACT-3⬘; S1P3, 5⬘-TCACCACCGTGCTCTTCTTGGTCATCTGC-3⬘.
Measurement of intracellular free calcium mobilization
Intracellular Ca2⫹mobilization in HLECs was determined with the
fluorescent Ca2⫹indicator fura-2/AM as described previously.24 Briefly,
HLECs were incubated with fura-2/AM at a final concentration of 3 ␮Min
HE-SFM at 37°C for 30 minutes. After loading, the cells were washed twice
with Ca2⫹-free Locke solution (158.4 mM NaCl, 5.6 mM KCl, 1.2 mM
MgCl2, 0.2 mM EGTA, 5 mM HEPES, and 10 mM glucose, pH 7.3) to
remove extracellular dye. Sulfinipyrazone was added to both the loading
medium and the washing solution to a final concentration of 250 ␮Mto
prevent dye leakage. Approximately 1.5 ⫻105cells of the cell suspension
were transferred to a quartz cuvette and placed in a thermostatically
controlled cell holder at 37°C, in which the cell suspension was continu-
ously stirred, and exposed to 100 ng/mL S1P with 100 ng/mL PTX, 10 ␮M
LY294002, 1 mM L-NAME, 10 ␮M PD98059, 25 ␮M SB203580, 30 ␮M
BAPTA-AM, or 5 ␮M U73122. LY294002, L-NAME, PD98059, SB203580,
BAPTA-AM, and U73122 were preincubated with the cells for 30 minutes
prior to exposure to S1P, and PTX was preincubated for 6 hours.
Fluorescence ratios were taken by dual excitation at 340 and 380 nm, and
emission at 500 nm by the alternative wavelength time scanning method.
Experiments were repeated 3 times.
Statistical analyses
Student ttest was used to calculate Pvalues based on comparisons with the
appropriate control samples tested at the same time.
Results
S1P induced in vitro lymphangiogenesis
Lymphangiogenesis is a complex cellular process that occurs via
proliferation, migration, and differentiation of lymphatic endothe-
lial cells. To investigate whether S1P had in vitro lymphangiogenic
activity, we performed migration, proliferation, and capillary-like
tube formation assays in HLECs, lymphatic endothelial cells
derived from lymph nodes. S1P significantly induced the migration
of HLECs in a dose-dependent manner over the migration in the
presence of medium alone (Figure 1A), whereas the presence of
S1P at concentrations up to 200 ng/mL did not show any effect on
the proliferation of HLECs (Figure 1B). VEGF-C, a potent
lymphangiogenic factor, also induced the migration and prolifera-
tion of HLECs (Figure 1A,B). Although S1P had no mitogenic
activity on HLECs, the migratory activity of S1P (100 ng/mL) was
much higher than that of VEGF-C (500 ng/mL). We next examined
the ability of S1P to promote the capillary-like tube formation of
HLECs on GFR Matrigel. In HLECs, the presence of S1P at
20 ng/mL, 100 ng/mL, and 200 ng/mL caused 1.7 plus or minus
0.3-fold, 2.1 plus or minus 0.2-fold, and 2.2 plus or minus 0.1-fold
increases in tube area, respectively, compared with the negative
control containing only the medium (Figure 1C). VEGF-C
(500 ng/mL) also stimulated tube formation (2.4 ⫾0.3-fold
increase).
To further confirm that S1P has in vitro lymphangiogenic
activity, we examined the ability of S1P to promote the migration
and capillary-like tube formation of HMVECs-dLy, lymphatic
endothelial cells derived from dermal skins. S1P significantly
promoted the migration and tube formation of HMVECs-dLy in a
dose-dependent manner (Figure 1D,E), whereas the presence of
S1P at concentrations up to 200 ng/mL did not show any effect on
the proliferation of HMVECs-dLy (data not shown). We next
characterized HLECs and HMVECs-dLy with lymphatic endothe-
lial cell–specific marker proteins by immunocytochemistry. More
than 95% of HLECs and HMVECs-dLy expressed Prox-1, LYVE-1,
and podoplanin (Figure 1F). These results suggest that S1P exerts
in vitro lymphangiogenic activity by promoting migration and
differentiation, but not proliferation, of human lymphatic endothe-
lial cells.
S1P promoted in vivo lymphangiogenesis
Since S1P stimulated migration and differentiation of HLECs and
HMVECs-dLy in vitro, we performed a Matrigel plug assay to
investigate whether S1P had in vivo lymphangiogenic activity.
Matrigels containing S1P (0.4 ␮g) or VEGF-C (1 ␮g) were subcu-
taneously injected into C57BL/6 mice; after 10 days, the mice were
killed and Matrigels were extracted. H&E staining and immunostain-
ing of podoplanin revealed that both S1P and VEGF-C induced
pronounced lymphatic vessel formation (Figure 2A-C), whereas
lower numbers of lymphatic vessels were observed in the control.
The podoplanin-positive lymphatic vessels were also positive for
Prox-1, another lymphatic endothelial cell–specific marker, and
sprouting lymphatic vessels were observed in S1P- and VEGF-C–
treated Matrigel (Figure 2D). In the VEGF-C– and S1P-treated
mice, respectively, 70.6% plus or minus 12.5% and 53.8% plus or
Table 1. Primer sets of human S1P receptors and ␤-actin
Gene Sequence Product size, bp
S1P1
Forward 5⬘-TGCGGGAAGGGAGTATGTTT-3⬘
60Reverse 5⬘-CGATGGCGAGGAGACTGAAC-3⬘
S1P2
Forward 5⬘-GCCTCTCTACGCCAAGCATTA-3⬘
107Reverse 5⬘-TTGAGCGGACCACGCAGTA-3⬘
S1P3
Forward 5⬘-TGATTGTGGTGAGCGTGTTCA-3⬘
68Reverse 5⬘-GGCCACATCAATGAGGAAGAG-3⬘
␤
-actin
Forward 5⬘-TCTACAATGAGCTGCGTGTG-3⬘
127Reverse 5⬘-ATGGCTGGGGTGTTGAAG-3⬘
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minus 6.8% of LYVE-1–positive lymphatic vessels were stained by
PH3, a marker of cell proliferation (Figure 2E,F). Furthermore,
FITC-dextran microlymphography revealed that both S1P and
VEGF-C were able to induce the growth of functional lymphatic
vessels (Figure 2G). These observations suggest that S1P is a
potent lymphangiogenic lipid molecule in vivo and that the
lymphangiogenic activity of S1P is comparable to that of VEGF-C,
a potent lymphangiogenic growth factor.
S1P stimulated lymphangiogenesis via the S1P1/Gipathway
S1P1, S1P2, and S1P3 are widely expressed subtypes in vascular
endothelial cells,25-27 and S1P stimulates proliferation and migra-
tion of vascular endothelial cells via S1P1 and S1P3.20,28 To
investigate whether S1P-induced lymphangiogenesis was mediated
by the activation of S1P receptors, we performed real-time PCR
analysis to identify the expression pattern of S1P receptors in
HLECs. Real-time PCR analysis revealed that both HLECs and
HUVECs expressed S1P1 and S1P3, but not S1P2 (Figure 3A).
Although it has been reported that VEGF induces S1P1 receptors in
endothelial cells,29 real-time PCR analysis revealed that VEGF-C
did not affect the expression of S1P1 and S1P3 in HLECs (data not
shown). Because 2 distinctive S1P receptors were expressed on
HLECs, we next carried out migration and tube formation assays to
determine which receptor was mainly involved in S1P-induced
lymphangiogenesis. S1P-induced migration and tube formation of
HLECs were almost completely blocked by treatment with 100 ng/
mL PTX, a Giprotein–specific inhibitor, whereas treatment with
PTX in the control did not affect HLECs (Figure 3B,C). Both S1P1
Figure 1. S1P induced migration and the formation
of capillary-like tube structure of human lymphatic
endothelial cells. (A) After 4 hours of incubation with
S1P or VEGF-C, migrated HLECs were stained and
counted in 3 random fields. (B) S1P or VEGF-C was
added to serum-starved HLECs for 24 hours, followed
by additional incubation for 12 hours with 0.5 ␮Ci
(0.0185 MBq) [3H]-thymidine in HE-SFM. Results are
expressed as the percentage [3H]-thymidine incorpora-
tion of the control versus S1P- or VEGF-C–treated
HLECs. (C) HLECs were laid on a 24-well, GFR
Matrigel-coated plate and incubated with S1P or
VEGF-C for 6 hours. Two randomly chosen fields per
well were photographed and the total tube area was
analyzed using Scion Image. (D,E) Effect of S1P on the
migration (D) and capillary-like tube formation (E) of
HMVECs-dLy, lymphatic endothelial cells derived from
dermal skins, was examined as described in panels A
and C, respectively. (F) Cultured HLECs and HMVECs-
dLy were fixed, and immunostained using antibodies
against Prox-1 (green), LYVE-1 (red), and podoplanin
(green). The nuclei were counterstained by Hoechst
(blue). Scale bars represent 50 ␮m. Note that HLECs
and HMVECs-dLy were not immunolabeled by isotype-
matched control IgG antibodies (data not shown). All
values are expressed as means (⫾SD). Data are
representative of 3 independent experiments with simi-
lar results. * and ** indicate statistically significant
differences (P⬍.05 and P⬍.01, respectively).
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and S1P3 convert external signals into intracellular signals via
heterotrimeric G proteins coupled to them; S1P1 is coupled with
the Giprotein, but S1P3 is coupled with the Gqand G12/13 proteins
as well as the Giprotein.4Therefore, our results suggest that the
activation of Giproteins coupled to S1P1 may be involved in
S1P-induced migration and differentiation of HLECs.
We next examined the effect of specific knockdown of S1P1 and
S1P3 by RNA interference on S1P-induced migration and differen-
tiation of HLECs. Down-regulation of S1P1 significantly inhibited
S1P-induced migration and tube formation of HLECs, whereas
down-regulation of S1P3 did not (Figure 4A). To further confirm
that S1P promotes lymphangiogenesis by stimulating S1P1, we
investigated the effect of an S1P1 antagonist30 on S1P-induced in
vitro and in vivo lymphangiogenesis. The S1P1 antagonist blocked
S1P-induced migration and tube formation of HLECs in a dose-
dependent manner, whereas an inactive S1P1 control molecule did
not (Figure 4B,C). Furthermore, Matrigel plug assays showed that
the S1P1 antagonist almost completely blocked S1P-induced
lymphatic vessel formation, whereas the inactive S1P1 control
molecule did not (Figure 4D,E). These results strongly suggest that
the activation of Giproteins coupled to S1P1 may be involved in
S1P-induced migration and differentiation of HLECs in vitro and
lymphangiogenesis in vivo.
S1P-induced lymphangiogenesis was mediated via the
PLC/Ca2ⴙpathway
After activation of the Giprotein complex by an upstream receptor
such as S1P1, the Giprotein complex is dissociated into Gi␣and
Gi␤␥ subunits to activate downstream signaling molecules. To
investigate downstream signaling events involved in the S1P-
induced lymphangiogenesis of HLECs, we performed migration
and tube formation assays with LY294002, L-NAME, PD98059,
SB203580, BAPTA-AM, and U73122, which are specific inhibi-
tors for phosphatidylinositol 3-kinase (PI3K), nitric oxide synthase
(NOS), p44/42 mitogen-activated protein kinase (MAPK), p38
MAPK, intracellular Ca2⫹chelator, and PLC, respectively. Approxi-
mately 78.3% and 85.2% of S1P-induced migration of HLECs was
efficiently blocked when 100 ng/mL S1P was applied to HLECs
with 30 ␮M BAPTA-AM and 5 ␮M U73122, respectively (Figure
Figure 2. S1P promoted in vivo lymphangiogen-
esis. Ten days after subcutaneous injection of Matrigel
containing none, VEGF-C (1 ␮g), or S1P (0.4 ␮g), in
C57BL/6 mice (5 mice per group), the Matrigel was
removed, fixed, embedded in paraffin, sectioned at
4␮m, and immunostained. (A) Cross-sections of Matri-
gel were stained by H&E. Scale bars represent 50 ␮m.
(B,C) Lymphatic vessels in Matrigel were immuno-
stained for podoplanin (red). Representative photo-
graphs and the number of podoplanin-positive lym-
phatic vessels per field are shown in panels B and C,
respectively. Scale bars represent 200 ␮m. (D) Lym-
phatic vessels in Matrigel were immunostained for
Prox-1 (green) and podoplanin (red). Arrows show
sprouting lymphatic vessels. Scale bars represent
50 ␮m. (E,F) Proliferating lymphatic endothelial cells in
Matrigel were double immunostained for PH3 (green)
and LYVE-1 (red). Representative photographs are
shown in panel E. Scale bars represent 20 ␮m. The
number of LYVE-1⫹(䡺) or LYVE-1⫹/PH3⫹(f) vessels
per field were counted (F). (G) Uptake of injected
FITC-dextran (2000 kDa) into newly formed lymphatic
vessels was visualized using confocal microscope.
Scale bars represent 100 ␮m. All values are expressed
as means (⫾SEM). ** indicates statistically significant
difference (P⬍.01).
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5A). Furthermore, S1P-induced capillary-like tube formation of
HLECs was completely inhibited by treatment with 30 ␮M
BAPTA-AM and 5 ␮M U73122 (Figure 5B). However, treatment
with the inhibitors for PI3K, NOS, p44/42 MAPK, and p38 MAPK
did not show any inhibitory effect on S1P-induced migration and
tube formation of HLECs. All these results suggest that PLC and
intracellular Ca2⫹mobilization, but not NOS, PI3K, p44/42
MAPK, and p38 MAPK, are major signaling pathways involved in
S1P-induced lymphangiogenesis.
Increased mobilization of intracellular Ca2ⴙby S1P was
mediated through the Gi/PLC/Ca2ⴙpathway
The activation of PLC by extracellular S1P evokes the induction of
robust Ca2⫹mobilization in HUVECs, which is important to the
S1P-induced migration of vascular endothelial cells.31,32 Since we
demonstrated the involvement of S1P-mediated PLC activation and
intracellular Ca2⫹release in the migration and capillary-like tube
formation of HLECs, we investigated whether the S1P-mediated
increase in intracellular Ca2⫹release was dependent on the Gi/PLC
pathway in HLECs. The application of S1P to fura-2/AM-loaded
HLECs caused a dramatic increase in Ca2⫹release into the cytosol
in a dose-dependent manner (data not shown). The increase in Ca2⫹
influx by 100 ng/mL S1P was significantly inhibited by treatment
with 100 ng/mL PTX (78.9% ⫾14.0%), 30 ␮M BAPTA-AM
(80.3% ⫾7.3%), and 5 ␮M U73122 (81.3% ⫾4.7%), but treat-
ment with the signaling inhibitors for PI3K, NOS, p44/42 MAPK,
and p38 MAPK did not show any inhibitory effect on Ca2⫹
mobilization (Figure 5C). These results suggest that S1P-induced
migration and differentiation of HLECs may be mediated by
intracellular Ca2⫹release following Gi/PLC activation.
Discussion
The lymphatic system has important roles in regulating tissue fluid
balance for homeostasis and facilitating interstitial protein trans-
port and immunologic function through lymph nodes. Clinical
evidence suggests that the dissemination of malignant tumors to
regional lymph nodes via the lymphatic vessels is important in
tumor metastasis and that chronic inflammation causes lymphangio-
genesis and lymphedema, although the molecular mechanisms
regulating lymphangiogenesis are largely unclear. In addition to
VEGF-C, a potent lymphangiogenic growth factor, VEGF-A,
bFGF, HGF, angiopoietin-1, IGF-1/2, and PDGF-BB, previously
known as proangiogenic factors, have lymphangiogenic activity.
However, no previous studies have examined the effects of
bioactive lipid molecules, including S1P, on lymphangiogenesis. In
this report, we provide evidence that S1P induces in vitro and in
vivo lymphangiogenesis by stimulating the migration and differen-
tiation of lymphatic endothelial cells via a S1P1/Gi/PLC/Ca2⫹
signaling pathway. To the best of our knowledge, this study is the
first to establish that S1P is a lymphangiogenic lipid mediator.
During angiogenesis and lymphangiogenesis, the migration and
proliferation of endothelial cells are important procedures to
maintain net vascular structure by replenishing new endothelial
cells in the empty space caused by the migration of endothelial
cells toward stimuli. Several reports have supported the conclusion
that S1P has potent migratory effects on vascular endothelial
cells.21,33 In our study, the S1P (100 ng/mL)–induced migration of
HLECs was approximately 5.3-fold higher than that induced by
VEGF-C (500 ng/mL). This result indicates that the activity of S1P
to induce migration of lymphatic endothelial cells was similar to
that for vascular endothelial cells, in which S1P has 3- to 10-fold
greater effects than induction by VEGF-A or bFGF.34 Furthermore,
we also demonstrated that S1P (0.4 ␮g/0.5 mL in Matrigel) can
promote pronounced lymphangiogenesis in vivo, similar to VEGF-C
Figure 3. S1P-induced in vitro lymphangiogenesis was mediated through the
S1P1/Giprotein. (A) Total RNA (100 ng) from HUVECs (䡺) or HLECs (f) was
amplified using primers for S1P receptors and ␤-actin. For quantification, the targets
were normalized to ␤-actin as an internal standard. (B) After a 2-hour preincubation
with 100 ng/mL PTX, HLECs were treated with 100 ng/mL S1P and 100 ng/mL PTX
for an additional 4 hours. The migrated HLECs were stained and counted in 3 random
fields. (C) HLECs were laid on a GFR Matrigel-coated 24-well plate and incubated
with 100 ng/mL S1P and 100 ng/mL PTX for 6 hours. Two randomly chosen fields per
well were photographed and the total tube area was analyzed using Scion Image. All
values are expressed as means (⫾SD). Data are representative of 3 independent
experiments with similar results. NS and ** indicate no significant difference and a
statistically significant difference (P⬍.01), respectively.
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(1 ␮g/0.5 mL in Matrigel), suggesting that S1P might be an
important regulator of lymphangiogenesis. Although the physiolog-
ical concentration of S1P has been reported to be 0.2 ␮g/mL in
serum, levels of S1P are elevated in pathological conditions:
0.4 ␮g/mL in synovial fluids from rheumatoid arthritis patients and
0.9 ␮g/mL in ascites of ovarian cancer patients.35,36 However, we
do not know the local concentration of S1P in tissues.
Although S1P directly induces the proliferation of human aortic
endothelial cells (HAECs) and bovine aortic endothelial cells,21,28,33
S1P (40 ng/mL) showed less potent mitogenic activities on these
cells (approximately 1.3-fold increase of proliferation in HAECs
and bovine aortic endothelial cells), suggesting that the mitogenic
activity of S1P is less potent than that of VEGF-A (10 ng/mL). In
this study, we found that S1P, up to 200 ng/mL, did not induce the
Figure 4. S1P-induced lymphangiogenesis was mediated through the S1P1. (A) HLECs were infected with retroviruses carrying S1P1 and S1P3 shRNA expression
vectors in pRS plasmid, after which stably transfected cells were obtained by selection with puromycin. pRS plasmid was used as control. After a 4-hour incubation with
100 ng/mL S1P,migrated HLECs were stained and counted in 3 random fields (䡺). HLECs were laid on a GFR Matrigel-coated 24-well plate and incubated with 100 ng/mL S1P
for 6 hours. Two randomly chosen fields per well were photographed and the total tube area was analyzed using Scion Image (f). (B,C) Effect of S1P1 antagonist or S1P1
control molecule on the S1P-induced migration (B) and capillary-like tube formation (C) of HLECs was examined as described in panel A. (D,E) Ten days after subcutaneous
injection of Matrigel in C57BL/6 mice (5 mice per group), the Matrigel was removed, fixed, embedded in paraffin, sectioned at 4 ␮m, and immunostained using antibodies
specific for Prox-1 (green) and podoplanin (red). (D) Representative photographs of untreated control mice, and mice treated with S1P (0.4 ␮g) in the absence or presence of
S1P1 antagonist (100 ␮M) or S1P1 control molecule (100 ␮M). Scale bars represent 50 ␮m. (E) The number of Prox-1⫹/podoplanin⫹lymphatic vessels per field was counted.
All values are expressed as means plus or minus SD (A-C) and means plus or minus SEM (E). In panels A-C, data are representative of 3 independent experiments with similar
results. ** indicates a statistically significant difference (P⬍.01).
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proliferation of HLECs and HMVECs-dLy, but lymphatic vessels
formed by S1P were proliferative in in vivo Matrigel plug assay.
The discrepancies in in vitro and in vivo results might be explained
as follows. During angiogenesis and lymphangiogenesis, migration
is very earlier cellular response than proliferation by angiogenic or
lymphangiogenic stimuli, and is induced by lower concentrations
than those required for cell proliferation.37 Lymphatic endothelial
cells can be dedifferentiated and stimulated to proliferate when
contact inhibition is broken by migration. It is well known that
soluble forms of E-selectin, vascular cell adhesion molecule-1, and
intercellular adhesion molecule-1 show angiogenic responses in
vascular endothelial cells by induction of migration and differentia-
tion without any in vitro mitogenic activity of vascular endothelial
cells.22,38 Another possible explanation is that lymphatic endothe-
lial cells can be proliferative by lymphangiogenic factors secreted
from leukocytes recruited by S1P-induced migration. It has been
known that S1P promotes trafficking of various kinds of leukocytes
that secret lymphangiogenic factors such as VEGF-C/-D. However,
the precise mechanisms by which S1P induces in vivo proliferation
of lymphatic endothelial cells remain to be elucidated.
Although S1P receptors are coupled with various G proteins,
such as Gi,G
q, and G12/13, S1P1 is coupled with only the Giprotein;
other S1P receptors can bind to various G proteins. We identified 2
kinds of S1P receptors, S1P1 and S1P3, in HLECs. S1P-mediated
migration and tube formation were completely blocked by the
application of PTX. Since PTX specifically binds to the Giprotein
and inhibits the dissociation of the Giheterotrimer protein complex
into 2 subunits, our results suggest that the Giprotein is an
important signaling mediator of lymphangiogenesis induced by
extracellular S1P. Although S1P3 can also bind to the Giprotein,
PTX shows partial inhibition of the responsiveness mediated by
S1P3/Gi, but complete inhibition of that mediated by S1P1/Gi.We
further showed that shRNA-mediated down-regulation of S1P1
significantly blocked S1P-induced migration and differentiation of
HLECs, whereas down-regulation of S1P3 did not. Moreover, an
S1P1 selective antagonist significantly blocked S1P-induced in
vitro and in vivo lymphangiogenesis, whereas an inactive S1P1
control molecule did not. These results strongly suggest that
S1P-induced lymphangiogenesis is mediated mainly by activation
of the Giprotein coupled to S1P1.
S1P effectively activates PI3K, p44/42 MAPK, p38 MAPK, and
PLC in vascular endothelial cells, and these activations are
dramatically inhibited by treatment with PTX.21,39 However, PI3K
and p44/42 MAPK signaling pathway are less important to
S1P-induced vascular endothelial cell migration.21,28,40-42 Further-
more, whether the involvement of the p38 MAPK pathway is
important to S1P-induced vascular endothelial cell migration
remains unclear.21,28 In this study, we found that inhibition of PLC
activation and chelation of intracellular Ca2⫹ions by treatment
with U73122 and BAPTA-AM resulted in a significant reduction in
S1P-induced migration and tube formation, as well as Ca2⫹influx
to lymphatic endothelial cells. This suggests that Ca2⫹ions
generated by active PLC are an important second messenger in
S1P-induced migration and differentiation of lymphatic endothelial
cells. Furthermore, application of inhibitors for PI3K, p44/42
MAPK, and p38 MAPK did not affect S1P-induced migration and
differentiation of HLECs, suggesting that PI3K, p44/42 MAPK,
and p38 MAPK are not involved in S1P-induced lymphangiogenesis.
Our present data strongly suggest that extracellular S1P pro-
motes new lymphatic vessel formation with the following mecha-
nisms (Figure 6). Extracellular S1P binds to and stimulates its
receptor S1P1, which is expressed on HLECs, resulting in the
Figure 5. S1P-induced in vitro lymphangiogenesis was mediated through the
PLC/Ca2ⴙpathway. (A)After a 4-hour incubation with 100 ng/mL S1P containing 10 ␮M
LY294002, 1 mM L-NAME, 10 ␮M PD98059, 25 ␮M SB203580, 30 ␮M BAPTA-AM, or
5␮M U73122, the migrated HLECs were stained and counted in 3 random fields. (B) HLECs
were laid on a GFR Matrigel-coated 24-well plate and incubated with 100 ng/mL S1P
containing 10 ␮M LY294002, 1 mM L-NAME, 10 ␮M PD98059, 25 ␮M SB203580, 30 ␮M
BAPTA-AM, or 5 ␮M U73122 for 6 hours. Two randomly chosen fields per well were
photographed and the total tube area was analyzed using Scion Image. (C) HLECs were
loaded with fura-2/AM for 30 minutes. The cells were resuspended in Ca2⫹-free Locke
solution, transferred to a quartz cuvette, and exposed to 100 ng/mL S1P with 100 ng/mL
PTX, 10 ␮M LY294002, 1 mM L-NAME, 10 ␮M PD98059, 25 ␮M SB203580, 30 ␮M
BAPTA-AM, or 5 ␮M U73122. Relative intracellular Ca2⫹influx was calculated from the
tracing. All values are expressed as means (⫾SD). Data are representative of 3 independent
experiments with similar results. ** indicates a statistically significant difference (P⬍.01).
1136 YOON et al BLOOD, 15 AUGUST 2008 䡠VOLUME 112, NUMBER 4
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activation of coupled Giproteins. Dissociation of active heterotri-
meric Giproteins from activated S1P1 stimulates PLC. Activated
PLC stimulates the release of intracellular Ca2⫹that causes the
migration and differentiation of HLECs, resulting in lymphangio-
genesis. S1P is generated by the phosphorylation of sphingosine,
which is mediated by SphK1 and SphK2. In many primary tumor
cells or inflammatory cells, the expression or activity of SphK1,
also known as oncogenic kinase,43 is up-regulated by many
angiogenic and inflammatory cytokines such as PDGF, epidermal
growth factor, tumor necrosis factor-␣, and interleukin-1␤.44 Al-
though it remains unclear how increased intracellular S1P is
secreted to the extracellular milieu in tumor-associated cells or
inflammatory cells, extracellular S1P can be generated by exported
SphK1 in HUVECs.45,46 Hence, the possibility exists that extracel-
lular S1P secreted from tumor-surrounded stromal cells, platelets,
or inflammatory cells,47 as well as tumor cells, can activate vascular
and lymphatic endothelial cells; this activation induces angiogen-
esis and lymphangiogenesis for tumor cell survival and tumor
metastasis to regional lymph nodes.
In this study, we demonstrated that S1P has in vitro and in vivo
lymphangiogenic activity and identified S1P as the first lipid
lymphangiogenic factor. This report may allow us to categorize
S1P, a bioactive lipid molecule, as a new lymphangiogenic factor.
Because several bioactive lipids, including lysophosphatidic acid,
sphingosylphosphorylcholine, gangliosides, and sphingomyelin,
have proangiogenic activity,48 investigations of other bioactive
lymphangiogenic lipids may be warranted to clarify the mecha-
nisms of lymphangiogenesis. In addition, because chronic inflam-
mation and cancer are highly related to each other, coordinated
regulation of lymphangiogenesis and inflammation by S1P sug-
gests that they may be pharmacological targets for development of
novel anti-inflammatory and antitumor metastatic therapies.
Acknowledgments
We thank Mr Hee-Yeoul Park and Ms Kyung Young Ji (Pohang
University of Science and Technology, Pohang, Republic of Korea)
for immunohistologic and confocal analysis, respectively.
This work was supported by the Korea Science and Engineering
Foundation (KOSEF, Daejeon, Republic of Korea) grant funded by the
Korea government (MOST, no. R15-2004-033-05001-0), supported by
a grant of the National R&D Program for Cancer Control (Goyang,
Republic of Korea), Ministry of Health & Welfare, Republic of Korea
(0320380-2), and supported by the Korea Basic Science Institute
(Daejeon, Republic of Korea) K-MeP (T27021; Y.S.G.). B.S.H., H.G.M.,
and S.L. were recipients of Brain Korea 21 fellowships.
Authorship
Contribution: C.M.Y. designed research, performed research, ana-
lyzed data, and drafted the paper; B.S.H. performed research,
analyzed data, and drafted the paper; H.G.M. and S.L. performed
research and analyzed data; P.-G.S. and Y.-K.K. designed and
advised research; C.-B.C. designed research, advised research, and
reviewed the paper; Y.S.G. designed research, advised research,
and drafted the paper.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Yong Song Gho, Division of Molecular and
Life Sciences, Pohang University of Science and Technology,
Pohang 790-784, Republic of Korea; e-mail: ysgho@postech.ac.kr.
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