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INTRODUCTION |
The sphingolipid metabolite sphingosine 1-phosphate
(SPP)1 is a bioactive lipid
that regulates diverse biological effects and signaling pathways
(reviewed in Ref. 1). SPP increases cell proliferation (2, 3) and
opposes ceramide-mediated apoptosis (4-7) through an intracellular
action (8, 9), yet some of its biological effects when added
exogenously are due to binding to cell surface receptors. Pertussis
toxin-sensitive G proteins are involved in some of the signaling
pathways activated by SPP (10-15), suggesting that it activates a
receptor coupled to a Gi/Go protein. In
agreement, low concentrations of SPP activate Gi
protein-gated inward rectifying K+ channels only when
applied at the extracellular face of atrial myocytes (16).
Several reports have demonstrated that SPP inhibits cell motility. SPP
inhibited chemotactic motility of mouse melanoma B16, mouse fibroblast
BALB/3T3 clone A31, and several tumor cell lines at nanomolar
concentrations (17-19). Moreover, SPP immobilized on glass beads
markedly inhibited melanoma cell motility. However, pertussis toxin
treatment did not block the effect of SPP, suggesting that in these
cells SPP acts through a cell surface receptor, independently of
pertussis toxin-sensitive G-proteins (20). In contrast, SPP inhibits
chemotaxis of human breast cancer cells only at high (micromolar)
concentrations, acting independently of EDG-1 (21).
We have recently identified SPP as a ligand for the G-protein-coupled
receptor, endothelial differentiation gene-1 (EDG-1) (22). EDG-1 binds
SPP with remarkable specificity and high affinity (KD = 8 nM) (9, 22). Binding of SPP to
EDG-1 resulted in inhibition of adenylate cyclase and activation of
mitogen-activated protein kinase (both Gi-mediated), but
did not mobilize calcium from internal stores (9, 23). In contrast,
Okamoto et al. (12) found that in HEL cells overexpressing
EDG-1, binding of SPP induced calcium mobilization (12).
Two other related G protein-coupled receptors, EDG-3 and EDG-5, have
recently been shown to bind SPP with similar high affinity (14, 24), to
confer responsiveness to SPP of a serum response element-driven
reporter gene when expressed in Jurkat cells, and to allow
SPP-stimulated 45Ca2+ efflux in
Xenopus oocytes (25). In agreement, overexpression of EDG-3
in Chinese hamster ovary cells led to phospholipase C activation and
calcium mobilization induced by SPP, which was significantly inhibited
by pertussis toxin (26). However, low concentrations of SPP mobilize
calcium from internal sources in BAEC in a pertussis toxin-sensitive
manner without activation of phospholipase C (27), suggesting the
involvement of novel, unidentified signaling pathways in SPP-induced
release of intracellular calcium.
Although the biological functions of the EDG family of GPCR are not
completely understood, the EDG-1 transcript was originally cloned as an
immediate-early gene induced during differentiation of HUVEC, cells of
the vessel wall accessible to platelet-derived ligands, into
capillary-like tubules (28). Moreover, SPP signaling in HEK293 cells
overexpressing EDG-1 leads, by a Rho-dependent mechanism,
to formation of a network of cell-cell aggregates resembling the
network formation of differentiated endothelial cells and P-cadherin
expression (22). Because SPP is stored and released from activated
platelets and serum concentrations of SPP are estimated to be
approximately 0.5 µM (29), about 60 times greater than the KD for binding to EDG-1, we suggested that SPP
might play an important role in angiogenesis acting through EDG-1
(22).
Angiogenesis, the process of new vessel formation from pre-existing
ones, or neovascularization, is a critical event for a variety of
physiological processes, such as wound healing, embryonic development,
corpus luteum formation, and menstruation. However, angiogenesis can be
activated in response to tissue damage and is important in certain
pathological conditions such as tumor growth and metastasis, rheumatoid
arthritis, diabetic retinopathy, psoriasis, and cardiovascular diseases
(30). Conversely, in states of inadequate tissue perfusion, such as
myocardial or limb ischemia, enhanced angiogenesis is essential and
beneficial (31). Endothelial cell migration and formation of new
capillary tubes are required events in the angiogenic response. In this
study, we investigated the potential role of SPP in angiogenesis by
examining regulation of endothelial cell motility, proliferation, and
tube formation through SPP receptors.
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EXPERIMENTAL PROCEDURES |
Materials--
IMEM, penicillin/streptomycin,
L-glutamine, amphotericin B, fetal calf serum, and fetal
bovine serum were from Biofluids (Rockville, MD). Medium 199 was from
Life Technologies (Gaithersburg, MD). Calf serum was from Colorado
Serum Co. (Denver, CO), and Matrigel, bFGF, endothelial cell growth
supplement, and rat tail type I collagen were from Collaborative
Biomedical Products (Bedford, MA). SPP, dihydro-SPP, and
sphingosylphosphorylcholine (SPC) were obtained from Biomol Research
Laboratory Inc. (Plymouth Meeting, PA).
1-Oleoyl-2-hydroxy-sn-glycero-3-phosphate (LPA) was from Avanti Polar Lipids, Inc. (Alabaster, AB). The Diff-Quik kits were from
Sigma. [methyl-3H]Thymidine (55 Ci/mmol) was
purchased from Amersham Pharmacia Biotech.
Cell Culture--
Human embryonic kidney cells (HEK293, ATCC
CRL-1573) and HEK293-EDG-1 cells, kindly provided by Drs.
Menq-Jer Lee and Timothy Hla, were maintained in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin, with 1 mg/ml G418 sulfate for
HEK293-EDG-1, as described previously (9, 22). BAEC were
kindly provided by Dr. Luyuan Li and maintained in IMEM containing 10%
fetal bovine serum supplemented with penicillin (100 units/ml),
streptomycin (100 µg/ml), L-glutamine (2 mM),
and 1 ng/ml bFGF. HUVECs were isolated as previously reported (32), and
grown in medium 199 supplemented with gentamycin, 2 mM
glutamine, 500 units/dl sodium heparin, 2.5 mg/dl amphotericin B, and 2 mg/dl endothelial cell growth supplement.
SPP Binding Assay--
HUVEC or BAEC were washed with binding
buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 15 mM sodium fluoride, 2 mM deoxypyridoxine, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin and
leupeptin) and removed from dishes by scraping. Cells were then
pelleted and resuspended in binding buffer containing 4 mg/ml BSA.
105 cells were incubated with 0.2 nM
[32P]SPP, synthesized enzymatically using recombinant
sphingosine kinase (33) as described previously (9), in 0.2 ml of
binding buffer plus 4 mg/ml BSA for 30 min at 4 °C in the absence or
presence of 1000-fold excess unlabeled SPP or other lipid competitors, added as 4 mg/ml fatty acid-free BSA complexes. Cells were then pelleted at 8,000 rpm in a microcentrifuge, washed twice with binding
buffer containing 0.4 mg/ml fatty acid-free BSA, resuspended in binding
buffer without BSA and bound [32P]SPP quantitated by
scintillation counting. The phosphatase and protease inhibitors were
included in the binding assays as a precaution against the possibility
that cells which may have been damaged during scraping might leak
phosphatases or proteases which could cleave SPP or EDG receptors,
respectively. In addition, it has been proposed that cell surface lipid
phosphatases which can cleave exogenous SPP exist (34). Nevertheless,
identical specific binding of [32P]SPP was obtained in
the absence of the protease and phosphatase inhibitors. It should be
pointed out that SPP is not metabolized during the binding assay. When
[32P]SPP was incubated in the absence or presence of
endothelial cells under the same conditions as the binding assay, no
decrease in the amount of [32P]SPP was detected by TLC
nor did any additional bands appear.
Reverse Transcriptase (RT)-PCR--
The cDNA encoding the
open reading frame of EDG-1 was amplified with the Gene Amp RNA-PCR kit
(Perkin-Elmer) using RNA isolated with TRIzol Reagent (Life
Technologies) and digested with RNase-free DNase I (RQ-1,
Promega). The primers (Life Technologies) used for PCR
amplification were 5'-GATATCATCGTCCGGCATTAC and
5'-ACCCTTCCCAGTGCATTGTTC for EDG-1 (28);
5'-CACTCAGCAATGTACCTGTTCC and 5'-AACACCCAGTACGATGGTGAC for
EDG-5 (35, 36); and 5'-GACTGCTCTACCATCTTGCCC and
5'-GTAGATGACAGGGTTCATGGC for EDG-3 (37). PCR reactions
were performed for 30 cycles with denaturation at 95 °C for 45 s, annealing at 55 °C for 45 s, and elongation at 72 °C for
50 s. PCR products were analyzed by agarose gel electrophoresis
after staining with ethidium bromide.
Migration--
Chemotactic migration of cells in response to a
gradient of SPP was measured in a modified Boyden chamber as described
previously (21). In brief, polycarbonate filters (5 µm for BAEC and 8 µm for HUVEC) were coated with gelatin (0.1%) overnight. Cells were harvested by trypsinization, washed with serum-free IMEM containing 0.1% fatty acid-free BSA, and were added to the upper wells
(24-multiwell Boyden microchambers) at 1 × 105 cells
per well; the lower wells contained SPP diluted in serum-free IMEM
containing 0.1% BSA. After 2 h at 37 °C in 5%
CO2, non-migratory cells on the upper membrane surface were
removed with a cotton swab and the cells which traversed and spread on
the lower surface of the filter were fixed and stained with Diff-Quik.
The number of migratory cells per membrane was enumerated using a
microscope with a ×20 objective. Each data point is the average number
of cells in four random fields, each counted twice. Each determination represents the average ± S.D. of three individual wells.
Checkerboard assays were carried out as described above except that
various dilutions of SPP in fatty acid-free BSA were placed in the top and/or bottom wells of the Boyden chamber. In most of the experiments, unless indicated otherwise, cells were serum starved for 2 h prior to the assays.
[3H]Thymidine Incorporation Assays--
Cells were
seeded at an initial density of 5 × 104 cells per
well in 24-well plates and allowed to attach overnight. Confluent BAEC
were growth arrested in culture media without bFGF for 48 h and
then treated for 24 h with SPP or bFGF. Since the sensitivity of
BAEC to stimulation by bFGF and SPP decreased with passage number, all
experiments were carried out with cells at less than passage 12. Confluent HUVEC were serum-starved for 24 h and then treated with
different concentration of SPP in medium 199 containing 1% FCS and
1000 units/dl heparin for 24 h. [3H]Thymidine (1 µCi/ml) was added 8 h before termination of the assay, and
[3H]thymidine incorporation into DNA was measured as
described (38). Values are the means of triplicate determinations and
standard deviations were routinely less than 10% of the mean.
In Vitro Angiogenesis--
Three-dimensional collagen gel plates
(24 well) were prepared by addition of 0.5 ml of a chilled solution of
0.7 mg/ml rat tail type I collagen in Dulbecco's modified Eagle's
medium adjusted to neutral pH with NaHCO3. After formation
of the collagen gel (about 1-2 mm thickness), BAEC were seeded at
50,000 cells/well. At 80% confluency, culture medium was changed to
media without bFGF and incubation was continued for 48 h, by which
time the cells formed a monolayer on the gel. The cells were then
treated with different concentrations of bFGF or SPP as indicated.
Cultures were maintained at 37 °C for 48 h and then fixed with
cold methanol. The gels were then soaked in phosphate-buffered
saline/glycerol (1:1) and transferred onto glass slides. The extent of
tube-like structures that formed in the gel was measured as total
length per field using computer-assisted imaging with a Hamamatsu C2400 video camera and a Zeiss Axioscope microscope to quantitate the extent
of tube formation. Three culture wells were used for each sample, and
three microscopic fields were examined for each well. Thus, each
experimental point represents results from examination of nine
microscopic fields.
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RESULTS |
Effect of SPP on Chemotaxis of EDG-1-overexpressing Cells--
SPP
was previously shown to inhibit chemotaxis of mouse melanoma cells by
binding to a putative cell surface receptor (20). Since we have
recently identified EDG-1 as a receptor for SPP (22), it was of
interest to examine the involvement of EDG-1 in SPP-regulated cell
motility. Human embryonic kidney 293 fibroblasts stably expressing
EDG-1 (HEK293-EDG-1) were selected for these studies since
they have high levels of specific SPP binding, whereas SPP binding is
nearly undetectable to parental and vector transfected cells (9, 22).
Surprisingly, low concentrations of SPP (10-100 nM) did
not inhibit, but rather enhanced chemotaxis of HEK293-EDG-1 cells by 1.5-1.9-fold, while chemotaxis of vector-transfected cells
was not altered by these concentrations of SPP (Fig.
1A). The concentrations of SPP
which stimulate chemotaxis of HEK293-EDG-1 cells are in the
same range as the measured affinity of EDG-1 for SPP
(KD = 8 nM) (22) and correlate closely
with binding and inhibition of forskolin-stimulated cAMP accumulation in these cells (9). These results suggest that low concentrations of
SPP may increase chemotaxis by binding to EDG-1. Higher concentration of SPP (1-10 µM), as previously reported in human breast
cancer cells (21), inhibited, rather than stimulated, chemotaxis of both vector and EDG-1-transfected cells.
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Fig. 1.
Effect of SPP on chemotaxis of HEK293 cells
overexpressing EDG-1. A, stable vector-transfected
(closed bars) or EDG-1-transfected (hatched
bars) HEK293 cells were treated with the indicated concentrations
of SPP, and chemotaxis was measured as described under "Experimental
Procedures." Data are mean ± S.D. of triplicate determinations.
Similar results were obtained in three independent experiments. The
asterisks indicate statistical significance determined by
Student's t test (p  0.01) compared with
untreated cells. B, competition by Lysosphingolipids for
[32P]SPP binding to HEK293 cells overexpressing EDG-1.
HEK293-EDG-1 cells were incubated with 0.2 nM
[32P]SPP in the absence or presence of the indicated
concentrations of unlabeled SPP, LPA, or SPC and specific binding was
measured as described under "Experimental Procedures."
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In contrast to undetectable levels of EDG-1 mRNA in
parental and vector transfected HEK293 cells (9, 39),
HEK293-EDG-1 cells express very high levels of
EDG-1 mRNA as detected by Northern analysis (9) and
RT-PCR (Fig. 2A).
Additionally, a low level of EDG-3 mRNA and
EDG-5 mRNA were detected by RT-PCR in these cells (Fig.
2A). Similar to our previous results (9), both unlabeled SPP
and dihydro-SPP effectively competed with [32P]SPP for
binding to EDG-1 (data not shown), whereas, LPA and SPC were completely
ineffective (Fig. 1B). Moreover, LPA, even at concentrations
as high as 10 µM, and for prolonged incubations, had no
significant effect on SPP binding to HEK293-EDG-1 cells.
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Fig. 2.
Expression of EDG receptor mRNAs in
endothelial cells. RT-PCR analysis of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), EDG-1, EDG-3,
and EDG-5 expression was carried out with RNA isolated from
HEK293-EDG-1 cells (A), HUVEC
(B), or BAEC (C) as described under
"Experimental Procedures" in the absence ( ) or presence (+) of
MULV-RT (RT). Similar results were found in two independent
experiments. bp, base pair(s).
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Expression of EDG Receptors in Endothelial Cells--
Previously,
it has been demonstrated that human endothelial cells express high
levels of EDG-1 mRNA (28). Thus, it was of interest to
examine whether the other SPP receptors, EDG-3 and EDG-5 (24, 25, 40),
are also expressed in HUVEC as well as BAEC which, based on indirect
evidence, have been proposed to have putative Gi-coupled
SPP receptors linked to calcium mobilization (27). Consistent with
previous studies, RT-PCR analysis clearly demonstrated an
EDG-1 PCR amplification product of about 1300 base pairs, in
agreement with the predicted size (1284 base pairs), in HUVEC and BAEC
(Fig. 2, B and C). HUVEC and BAEC also apparently expressed somewhat lower levels of EDG-3 and barely
detectable EDG-5 mRNA. It should be noted that the
bovine SPP receptor cDNAs have not yet been cloned and sequenced;
however, our PCR primers were designed to cover highly conserved
regions. Restriction analysis of the HUVEC RT-PCR products yielded
fragments of the expected sizes confirming their identity (data not
shown). The entire open reading frame of each of the three SPP
receptors is encoded within a single exon (40, 41), and RT-PCR primers
which span an intron junction cannot be used to evaluate genomic DNA
contamination. Therefore, controls without reverse transcriptase were
performed in all cases (Fig. 2).
Effects of SPP on Chemotaxis of Endothelial Cells--
Since SPP
increased chemotaxis of HEK293 cells by apparently acting through
EDG-1, the effect of SPP on chemotaxis of HUVEC and BAEC, which express
EDG-1 and -3, was examined (Fig. 3,
A and B). SPP stimulated chemotaxis of both HUVEC
and BAEC, reaching a maximum effect at 1 µM (7- and
10-fold, respectively). Similar to the results with
HEK293-EDG-1 cells, concentrations of SPP greater than 1 µM were less effective, although significant enhancement of chemotaxis was still evident at higher concentrations.
Interestingly, bFGF (20 ng/ml), a potent angiogenic factor (42),
increased chemotaxis of BAEC to the same extent as did 1 µM SPP.
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Fig. 3.
Effect of SPP on chemotaxis of HUVEC and
BAEC. A, HUVEC were allowed to migrate toward the
indicated concentrations of SPP and chemotaxis was measured as
described under "Experimental Procedures." B, BAEC were
allowed to migrate toward different concentrations of SPP or 20 ng/ml
bFGF as indicated, and chemotaxis was measured. Data are mean ± S.D. of triplicate determinations. Similar results were obtained in at
least three independent experiments. C, HUVEC were
pretreated with vehicle or 200 ng/ml pertussis toxin for 3 h, and
then allowed to migrate toward vehicle or 100 nM SPP as
indicated. Asterisks indicate statistical significance
determined by Student's t test (p 0.01).
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It has recently been shown that directional migration toward
appropriate agonist ligands can be triggered via receptors coupled to
Gi but not by agonists for receptors coupled to two other G proteins, Gs and Gq (43). Because biochemical
evidence and the yeast two-hybrid system indicate that EDG-1 is capable
of interaction with G
i1 and Gi3 (44), we
investigated the possibility that Gi proteins may be
involved in the chemotactic response induced by SPP. To this end, HUVEC
were treated with pertussis toxin, which ADP-ribosylates and
inactivates Gi and Go proteins, prior to
addition of SPP. Pertussis toxin pretreatment completely
abolished SPP-induced chemotaxis (Fig. 3C).
SPP Stimulates Directional Migration--
We next determined
whether the effect of SPP was mediated by enhanced directed migration
in response to the gradient of chemoattractant (chemotaxis) or by
increased random motility due to the presence of the chemoattractant
itself (chemokinesis). Checkerboard assays were performed with various
concentration of SPP in the top, bottom, or both chambers of the Boyden
apparatus. The greatest numbers of cells were found to migrate either
along the chemotactic gradient, i.e. toward increasing
concentrations of SPP in the bottom chamber (Table
I, bold), and also in the
direction of the increasing chemokinetic gradient, i.e. when
the concentration of SPP was the same in both the top and bottom
chambers (Table I, italic), indicating that SPP stimulates
both chemokinetic and chemotactic responses.
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Table I
Checkerboard analyses of HUVEC migration induced by SPP
The checkerboard assay was arranged with increasing concentrations of
SPP in the lower chamber (bold indicates migration due to
chemoattaction) or increasing concentrations of SPP in both upper and
lower chambers, and cell migration assays were performed as described
under "Materials and Methods." Data are mean ± S.D. of
triplicate determinations.
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Binding of SPP and Dihydro-SPP Correlates with Stimulation of Cell
Migration--
It was important to determine whether
[32P]SPP was specifically bound to the endogenous SPP
receptors on HUVEC. As shown in Fig.
4A, in HUVEC there was
significant specific binding of both SPP and sphinganine 1-phosphate
(dihydro-SPP), which lacks the double bond at the 4-position. Moreover,
dihydro-SPP also markedly enhanced chemotaxis (Fig. 4B). The
structure of SPP is similar to that of LPA, another serum-borne
lysolipid that binds and signals through the related
Gi-coupled receptors, EDG-2 and EDG-4 (45-47). However,
excess LPA did not compete with [32P]SPP for binding to
HUVEC, nor did it have a significant stimulatory effect on chemotaxis.
SPC had no significant effect on specific [32P]SPP
binding and had a small but not statistically significant effect on
chemotaxis (Fig. 4, A and B). Similar results
were obtained with BAEC, where only SPP and dihydro-SPP, albeit less
potently, competed with labeled SPP for binding, whereas LPA and SPC
had no significant effect. These results are consistent with our
previous observation that dihydro-SPP blocked binding to HEK293 cells
overexpressing EDG-1 in a dose-dependent manner similar to
unlabeled SPP (9, 24), while neither LPA nor SPC had a significant
effect on SPP binding (Fig. 1B). It should be pointed out
that in these studies, all lysosphingolipids were added to cells as
0.4% BSA complexes, using conditions which were found to be optimal
for binding of SPP to its receptor (9, 24), whereas these might not be
optimal conditions for binding of LPA to EDG-2 and EDG-4. For example, LPA prepared as a 1% BSA complex promoted survival of Schwann cells,
while SPP as a 0.01% complex was ineffective (48). We also examined
the binding affinity of SPP for its putative receptor on endothelial
cells by displacing bound [32P]SPP with increasing
concentrations of unlabeled SPP. 50% of the bound
[32P]SPP was competed at 10 nM unlabeled SPP
in BAEC. Thus, binding of SPP to endothelial cells is also of high
affinity and in agreement with the Kd of EDG-1 (8.6 nM). These results indicate that there is an excellent
correlation between the Kd and the
concentration-dependent effect of SPP on cell
migration.
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Fig. 4.
SPP and dihydro-SPP, but not LPA,
specifically compete with [32P]SPP binding and stimulate
chemotaxis in HUVEC. A, effects of SPP and other
lysophospholipids on binding of [32P]SPP to HUVEC. HUVEC
were incubated in the presence of 0.2 nM
[32P]SPP for 30 min at 4 °C in the absence or presence
of 300 nM unlabeled SPP, dihydro-SPP (DH-SPP), SPC, or LPA
and binding was measured as described under "Experimental
Procedures." B, HUVEC were allowed to migrate
toward the indicated lysophospholipids (100 nM) and
chemotaxis was measured. Asterisks indicate statistical
significance determined by Student's t test
(p 0.01).
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SPP Stimulates Proliferation of HUVEC and BAEC--
Many
angiogenic factors, in addition to enhancing chemotaxis, stimulate
in vitro proliferation of endothelial cells (49-51). Since
SPP increased chemotaxis of endothelial cells, and has previously been
shown to be a potent mitogen for diverse cell types (1, 2, 52), it was
of interest to examine the effects of SPP on proliferation of
endothelial cells. SPP treatment of HUVEC induced a
dose-dependent increase of DNA synthesis as measured by
[3H]thymidine incorporation with a maximum effect at
0.1-1 µM (Fig. 5A). Similar to SPP, 1 µM dihydro-SPP also stimulated DNA synthesis in HUVEC by
1.83 ± 0.1-fold, whereas LPA at a concentration as high as 10 µM had no significant effect. In agreement with our previous reports in various cell types (53-55), 10 µM
SPC stimulated DNA synthesis by 1.95 ± 0.1-fold.
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Fig. 5.
The effect of SPP on DNA synthesis of HUVEC
and BAEC. Quiescent HUVEC (A) or BAEC (B)
were treated with the indicated concentrations of SPP or 20 ng/ml bFGF
for 24 h and DNA synthesis as measured by
[3H]thymidine incorporation was determined as described
under "Experimental Procedures." Data are mean ± S.D. of
triplicate determinations and are representative of at least three
independent experiments. Asterisks indicate statistical
significance determined by Student's t test
(p 0.01). C, quiescent HUVEC and BAEC
were incubated in the absence (filled bars) or presence of
20 ng/ml pertussis toxin (open or hatched bars).
After 2 h, cells were washed and exposed to the indicated
concentrations of SPP or bFGF for 24 h and
[3H]thymidine incorporation was measured. Data are
expressed as fold increases compared with non-stimulated cells.
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SPP was also mitogenic for BAEC; however, a maximal effect in these
cells required higher concentrations (1-10 µM). bFGF has been reported to be a potent endothelial cell mitogen (56). Surprisingly, although bFGF stimulated proliferation of BAEC, at
optimal concentrations it was only as effective as 1 µM
SPP and less effective than 10 µM SPP (Fig.
5B). It should be noted that sensitivity of BAEC to SPP
decreased with increasing passage number, similar to a previous report
on the effect of passage number on bFGF responses (57). In addition to
SPP, dihydro-SPP stimulated DNA synthesis, whereas we found that LPA at
a concentration up to 10 µM was not mitogenic, in fair
agreement with previous studies where LPA only stimulated DNA synthesis
in BAEC at concentrations around 30 µM (57).
To investigate the possibility that a Gi-coupled receptor
may be involved in the proliferative response induced by SPP,
endothelial cells were treated with pertussis toxin prior to addition
of SPP. Both HUVEC and BAEC are more sensitive to pertussis toxin than Swiss 3T3 fibroblasts. In contrast with our previous studies with quiescent Swiss 3T3 fibroblasts (10, 11), where half of the stimulated
DNA synthesis was still evident even at the highest effective
concentration of pertussis toxin, pertussis toxin pretreatment of HUVEC
and BAEC completely inhibited the SPP-induced mitogenic response, while
it had no significant effect on DNA synthesis induced by bFGF (Fig.
5C).
SPP Induces Capillary-like Tube Formation in Vitro--
Later
stages of angiogenesis require morphological alterations of endothelial
cells, which result in lumen formation (31). Critical steps in
angiogenesis, such as migration and differentiation, have been studied
using an in vitro model of angiogenesis in which cultured
endothelial cells are induced to invade a three-dimensional collagen
gel where they form a network of capillary-like structures or tubes
when stimulated by angiogenic factors (58, 59). This phenomenon is
thought to mimic the formation of new blood vessels in vivo.
In agreement with previous studies (59), confluent monolayers of BAEC
treated with bFGF (50 ng/ml) formed networks of capillary-like tubular
structures within the gel (Fig.
6A). In contrast, little
invasion or network cord formation and only a few short capillary
sprouts originating from untreated BAEC embedded in collagen gels were
detected. SPP evoked a dose-dependent increase in the
formation of capillary-like tubes of BAEC invading the collagen gel.
Apparently thinner tubes were formed in response to lower doses of SPP
than those formed in response to bFGF. Quantitative evaluation of tube
formation revealed that SPP, similar to bFGF, markedly increased the
length of the endothelial tubular structures (Fig. 6B).
There was an additive effect when SPP was applied together with bFGF,
suggesting that SPP can potentiate the effect of bFGF on in
vitro angiogenesis. Similar to SPP, dihydro-SPP, also markedly enhanced capillary-like tube formation of BAEC, whereas LPA was completely inactive and SPC had a small but significant effect (Fig.
6C). Pertussis toxin not only inhibited endothelial cell migration and proliferation induced by SPP, it also markedly decreased the SPP-induced tube formation (Fig. 6C). In sharp contrast,
pertussis toxin had no effect on tube formation induced by bFGF.
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Fig. 6.
Induction of in vitro
angiogenesis in collagen gels by SPP is attenuated by pertussis
toxin. BAEC were treated with the indicated concentration of SPP
in the absence or presence of bFGF (50 ng/ml) and capillary tube
formation on collagen gels was examined as described under
"Experimental Procedures." A, representative
phase-contrast micrographs of BAEC after 4 days of incubation in normal
medium (upper panels) or in the presence of 50 ng/ml bFGF
(lower panels) and the indicated concentrations of SPP.
B, quantitative analysis of capillary tube formation. Data
are expressed as length of tubes per square millimeter
(n = 4 pairs of duplicates). Asterisks
indicate statistical significance determined by Student's t
test (p 0.01). C, BAEC were treated in
the absence or presence of pertussis toxin (PT, 20 ng/ml),
without or with the indicated lysophospholipids (1 µM) or
bFGF (50 ng/ml) and capillary tube formation on collagen gels was
determined as described under "Experimental Procedures." Data are
expressed relative to the maximum response elicited by bFGF.
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DISCUSSION |
Previously, many studies have shown that SPP inhibits chemotaxis
of diverse cell types (17, 19, 20, 60, 61). Although in human breast
cancer cells, SPP inhibited chemotaxis independently of EDG-1 (21), a
wealth of evidence suggests that in many other cell lines, SPP inhibits
chemotaxis through unidentified cell surface receptors (17, 20, 62).
Unexpectedly, we found in this study that binding of SPP to its
Gi protein-coupled receptor EDG-1 markedly increased cell
motility. In accord with its affinity for EDG-1, SPP at nanomolar
concentrations increased chemotaxis of EDG-1-transfected but not
vector-transfected HEK293 cells, as well as HUVEC and BAEC which
constitutively express EDG-1.
Although a recent study demonstrated that LPA can bind to EDG-1 leading
to receptor phosphorylation, ERK activation, as well as
Rho-dependent morphogenesis and P-cadherin expression (39), in our study, LPA, even at a concentration as high as 10 µM, did not compete for binding of radiolabeled SPP to
HEK293-EDG-1 cells. In agreement, LPA did not displace bound
[32P]SPP from endothelial cells nor did it stimulate
chemotaxis of HUVEC. In contrast, dihydro-SPP, which binds to EDG-1
(9), EDG-3, and EDG-5 (24), was as potent as SPP in induction of chemotaxis. However, LPA, similarly to SPP, can mimic serum in inducing
invasion of carcinoma and hepatoma cells into monolayers of mesothelial
cells (63). Although the underlying mechanism of this effect is not
clear, it may involve increased cell adhesion rather than enhanced cell
motility. Moreover, in most other cell types, LPA stimulates
chemokinesis and chemotaxis (62, 64-66), which might be due to binding
to its specific receptors, EDG-2 and EDG-4 (45-48).
We have previously shown that the SPP-induced cAMP decrease (9) and
ERK2 activation in EDG-1-transfected cells (22) were completely blocked
by pretreatment with pertussis toxin, which uncouples Gi
from GPCR. Similarly, preincubation with pertussis toxin abolished the
effect of SPP on migration of endothelial cells. Collectively, these
findings suggest that binding of SPP to the serpentine receptor EDG-1
on the endothelial cell surface activates a pertussis toxin-sensitive
Gi protein crucial for chemotaxis. In agreement, it has
recently been demonstrated that activation of Gi-coupled
receptors and the subsequent release of G
dimers is
required to initiate signal transduction leading to directed cell
migration (43, 67). It should be noted that the receptors for all known
leukocyte chemoattractants, including the chemokines, are members of a
seven-transmembrane domain superfamily and coupled to a variety of
G
subunits (68, 69). Moreover, pertussis toxin inhibits
chemotaxis by preventing chemoattractant receptors from activating
trimeric G proteins of the Gi subfamily (67).
Endothelial cells are accessible to platelet-derived ligands in the
serum, and might be the target of serum-borne SPP which regulates their
proliferation, migration, and differentiation into capillary-like
tubules, important aspects of angiogenesis (31). SPP is well
established as a potent mitogen for diverse cell types (reviewed in
Ref. 1). Indeed, we found that SPP also stimulated DNA synthesis in
endothelial cells and this was completely blocked by pertussis toxin,
suggesting a role for Gi in this process. In agreement, SPP
was recently reported to stimulate HUVEC proliferation (70), albeit at
somewhat higher concentrations which resembled the dose-response curve
that we found for BAEC. It is possible that differences in sensitivity
to SPP might arise from differences in passage numbers as has been
shown for responses to bFGF (57). Interestingly, SPP stimulated DNA
synthesis in Swiss 3T3 fibroblasts only at high concentrations and in
contrast to endothelial cells, was only partially inhibited by
pertussis toxin (10). Moreover, in these cells, DNA synthesis was
significantly and specifically increased by microinjection of SPP (9).
Pertussis toxin reduced the level of DNA synthesis caused by exogenous
SPP to approximately the same level as that induced by microinjected SPP (9). Thus it is likely that in fibroblasts, both intracellular as
well as receptor-mediated responses to SPP are involved in its
mitogenic effect. It is possible that there is a complex interplay between cell surface receptor signaling and intracellular targets for
SPP, which can contribute to its mitogenic response in certain cell
types. Thus, SPP, may act in a similar manner to other bioactive lipids, such as leukotriene B4 (LTB4), which act through cell surface
receptors and also have intracellular targets. LTB4 is a potent
chemoattractant that is primarily involved in activation of
inflammatory cells by binding to its GPCR. However, it can also bind
and activate the intranuclear transcription factor PPAR, resulting
in the activation of genes that terminate inflammatory processes
(71).
In this study, we also observed that SPP in addition to stimulating
both random, nondirectional migration (chemokinesis) and directional
migration (chemotaxis) of endothelial cells, also markedly enhanced
morphogenetic differentiation of endothelial cells in vitro.
BAEC cultured on collagen showed increased tube formation in the
presence of SPP, demonstrating that SPP stimulates endothelial cell
differentiation as well as migration. Moreover, pertussis toxin not
only inhibited endothelial cell migration and proliferation induced by
SPP, it also completely inhibited SPP-induced, but not bFGF-induced,
formation of capillary-like tubes. Taken together, these results
demonstrate that SPP has angiogenic activity in vitro acting
through a Gi-coupled cell surface receptor.
In contrast to its effect on HEK293-EDG-1 cells, SPP did not
stimulate chemotaxis (Fig. 1), proliferation (9), or morphogenetic differentiation in vector-transfected HEK293 cells (22), further supporting the notion that the effects that we observed on chemotaxis, proliferation, and tube formation of endothelial cells are mediated by
EDG-1. In contrast, parental and vector-transfected HEK293 cells do
show activation of ERK mitogen-activated protein kinases in response to
SPP which might be attributable to endogenous EDG-3 or EDG-5. Taken
together with our results, these findings suggest that EDG-1 may
mediate these stimulatory effects of SPP on endothelial cells. However,
as the other known SPP receptor EDG-3, which is also expressed in
HUVEC, has been shown in some cases to couple to pertussis
toxin-sensitive G proteins as well as to Rho and phospholipase C
signaling pathways (14, 26), this receptor might also contribute to the
effects of SPP on tube formation. Recently, the polycyclic anionic
compound suramin has been shown to selectively antagonize SPP-activated
calcium transients in EDG-3, but not in EDG-1 or EDG-5 expressing
oocytes, with an IC50 of ~22 µM, suggesting
that it is an antagonist selective for the EDG-3 GPCR isotype (72).
However, addition of 100 µM suramin did not abrogate the
ability of SPP to induce tube formation in BAEC.2 In contrast, suramin
inhibited Rho-dependent neurite retraction induced by SPP
in N1E-115 neuronal cells (73) and SPP-induced invasion of T-lymphoma
cells (74). However, in agreement with its lack of effect on
SPP-induced tube formation, suramin had no significant effect on
proliferation, stress fiber formation, and focal adhesion kinase
phosphorylation induced by SPP in Swiss 3T3 fibroblasts (75).
Similar to SPP, dihydro-SPP also markedly enhanced cell migration and
capillary-like tube formation of BAEC, whereas LPA was completely
inactive. Another structurally related analog of SPP, SPC, although it
had no significant effect on binding of labeled SPP to endothelial
cells, it had a small but significant stimulatory effect on tube
formation, and at high concentrations, it also stimulated
proliferation. These effects might be related to the potent action of
SPC as a wound healing agent (55). Interestingly, high micromolar
concentrations of SPC activated calcium transients in EDG-1, -3, and -5 expressing oocytes (72) and SRE-driven gene transcription in Jurkat T
cells (25). While these observations suggest that SPC might be a very
low affinity ligand for these EDG receptors, it is also possible that
these effects were not mediated by SPC itself as it was recently found
that commercial preparations of SPC are contaminated with highly potent
alkenyl glycero-3-phosphates (76).
Some of the downstream effects of SPP signaling through EDG-1, such as
decreased cAMP (9, 23) and activation of ERK2 (22), are pertussis
toxin-sensitive, while others, such as morphogenetic differentiation,
are pertussis toxin-insensitive but inhibited by the C3 exoenzyme (22)
which blocks signaling through the small GTPase Rho (77). Thus it
appears that EDG-1 can couple to Gi proteins as previously
reported (44), as well as to G12/13 proteins which are
thought to regulate Rho (78, 79), and thus might also be important for
SPP-induced chemotaxis and angiogenesis. Recently, it has been
demonstrated that disruption of the gene encoding G13
impaired the ability of endothelial cells to organize into a vascular
system and greatly impaired migratory responses (80), suggesting that
in addition to Gi, other proteins including G13, might be required for regulation of cell movement.
In agreement, T-lymphoma cell invasion is dependent on SPP
receptor-mediated RhoA and phospholipase C signaling pathways which
lead to pseudopodia formation (74).
In summary, in this study we have demonstrated that SPP has
appropriate properties to be considered as a bona fide
angiogenic factor, i.e. it stimulates chemokinetic and
chemotactic motility, proliferation of vascular endothelial cells, and
stimulates angiogenesis in vitro, similarly to the known
angiogenic factor bFGF. Because bFGF and SPP have an additive effect on
formation of capillary-like tubes by endothelial cells invading
collagen gels, SPP may be a specific type of angiogenic factor. It is
possible that SPP plays a role in normal blood vessel formation or in
injury, when local production of SPP could be increased by activated
platelets, and extravasation of intravascular fluid could also present
SPP into tissues at concentrations sufficient to promote angiogenesis and wound healing. Elucidation of the molecular mechanisms by which SPP
stimulates cell migration and angiogenesis might provide clues for
development of new therapeutic agents to either promote or block these
processes by targeting the EDG family of GPCR.
,
, and
TOP
ABSTRACT
INTRODUCTION
Supported by Predoctoral Fellowship BC961968 from the United
States Army Medical Research and Material Command, Breast Cancer Research Program.
