 |
INTRODUCTION |
Sphingosine 1-phosphate
(SPP)1 is a bioactive lipid
produced by metabolism of the membrane phospholipid, sphingomyelin.
Activation of the sphingomyelinase enzyme followed by sequential
catalysis by ceramidase and sphingosine kinase results in the formation of SPP (1). Although the site of synthesis and mechanisms involved in
secretion of SPP are not well understood, it is clear that one
mechanism by which SPP acts is via the interaction with plasma membrane-localized G-protein-coupled receptors (GPCR) of the EDG family
(2, 3). To date, EDG-1, -3, -5, -6, and -8 were shown to bind to SPP
and transduce various intracellular signals (4-9). Signal transduction
mechanisms of EDG-1, -3, and -5 have been defined. These GPCRs are
stimulated by nanomolar concentrations of SPP and couple to different
G-proteins, which may form the basis for their differential signal
transduction properties (5, 6, 10). For example, EDG-1 couples to the
Gi protein, whereas EDG-3 and -5 couple to Gq,
G13, and Gi proteins (10). Downstream of the
heterotrimeric G-proteins, small GTPases of the Rho family, namely Rho
and Rac, are activated by SPP (11, 12). Rho and Rac regulate
cytoskeletal changes such as stress fiber assembly and cortical actin
formation, respectively (13). Based on changes in actin dynamics and
using antisense oligonucleotides to block the expression of EDG-1 and
-3, we proposed that EDG-1 is required for cortical actin assembly (a
Rac-regulated event) and EDG-3 for stress fiber formation (a
Rho-regulated event) (12). However, complex cross-regulatory mechanisms
appear to be involved since dominant negative Rac also blocks stress
fiber assembly in endothelial cells (12, 13). Direct measurements of
Rho and Rac activity induced by SPP in endothelial cells has not been reported.
SPP acts in a variety of cell systems to regulate cell proliferation,
migration, differentiation, and death (1-3, 14, 15). Although
controversy exists regarding its mode of action, i.e. whether SPP is a second messenger that acts intracellularly or a first
messenger that acts extracellularly, many of the aforementioned actions
are likely to be due to activation of EDG family of receptors (1-3,
14, 15). Furthermore, physiological functions of SPP are poorly understood.
EDG-1, the prototypical SPP receptor, was originally isolated from
human umbilical vein endothelial cells (HUVECs) as an inducible gene
(3, 16). SPP treatment of HUVECs results in activation of EDG-1 and -3 receptors, stimulation of the Gi-dependent cell survival pathway, Rac- and Rho-dependent adherens junction
assembly, and cytoskeletal rearrangement, which ultimately
results in the morphogenesis of HUVECs into capillary-like networks
(12). Indeed, SPP synergized with polypeptide angiogenic factors such
as fibroblast growth factor-2 and vascular endothelial cell growth
factor to induce mature neovessels in vivo (12). Recent data
on the deletion of the Edg-1 gene in the mouse indicate that
SPP/Edg-1 interaction is necessary for embryonic vascular
maturation (17). Interestingly, vasculogenesis and angiogenesis
occurred in the Edg-1
/
embryos, suggesting that Edg-1 is
dispensible for these processes during embryonic development.
Interestingly, genetic studies in zebrafish indicate that an EDG-5-like
receptor (termed as Miles Apart) controls myocardial progenitor cell
migration and heart development (18). These data suggest that a
physiological function of SPP is to regulate the development, growth,
and maintenance of the cardiovascular system.
Recently, several studies reported that SPP is a potent migration
inducer of vascular endothelial cells (19-24, 47). Previously, however, SPP has been shown to both stimulate and inhibit chemotactic responses in a variety of cell types. CHO cells expressing EDG-1 or
EDG-3 showed better migration toward SPP, whereas Edg-5-expressing cells did not (24). On the other hand, SPP inhibited chemoinvasiveness of MDA-MB-231, a human breast cancer cell line (25). Moreover, chemotactic motility of mouse melanoma has been shown to be inhibited by SPP in a pertussis toxin-sensitive G protein-independent way (26).
Molecular mechanisms of SPP-induced endothelial cell migration have not
been defined.
Here we investigated the mechanisms involved in SPP-induced migration
and morphogenesis of HUVECs. Data in this report show that SPP is
effective in stimulating the migration and lumen formation of HUVECs.
This involves rapid assembly of the initial focal contacts through
v
3 and
1 family of
integrins. Furthermore, we demonstrate that activation of Rho by SPP
through EDG-1 and EDG-3 in HUVECs is required for this process.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
HUVECs (Clonetics) were
cultured in M199 medium (Cellgro, Inc.) supplemented with 10% fetal
bovine serum (Hyclone) and heparin-stabilized endothelial cell growth
factor, as previously described (16). CHO cell transfectants
(pCDNA, EDG-1, EDG-3) were maintained in F-12 medium with 10%
fetal bovine serum and 1 mg/ml G418. Transfection of antisense
oligonucleotides to block the expression of EDG-1 and EDG-3 was
performed using NovaFECTORTM reagent (VennNova, LLC, Pompano Beach,
FL). HUVECs were plated the day before transfection in complete
growth medium. 18-mer phosphothioate oligonucleotides (PTO; antisense
EDG-1, 5'-GAC GCT GGT GGG CC C CAT-3'; sense EDG-1, 5'-ATG GGG CCC ACC
AGC GTC-3'; antisense EDG-3, 5'-CGG GAG GGC AGT TGC CAT-3'; sense
EDG-3, 5'-ATG GCA ACT GCC CTC CCG-3') were mixed with NovaFECTORTM in
200 µl of OPTI-MEM® I and left for 5 min to allow DNA-lipid complex
formation. Cells were rinsed three times with pre-warmed OPTI-MEM®1,
and the DNA-lipid complex was layered over cells at the final
concentration of 400 nM. After 4 h of transfection,
medium was removed and replaced with complete growth medium.
Northern Blot Analysis--
Total RNA was isolated from HUVECs
using the RNA-STAT 60 (Tel-test) following the manufacturer's
instructions. Fifteen micrograms of total RNA were resolved on 1%
agarose-formaldehyde gels, and the integrity of the RNA was monitored
by ethidium bromide staining. RNA was capillary-transferred onto
Zeta-Probe membrane (Bio-Rad), UV-cross-linked by Stratalinker
(Stratagene), and hybridized with radiolabeled cDNA probes. The
human EDG-1, EDG-3, and glyceraldehyde-3-phosphate dehydrogenase
cDNAs were radioactively labeled using [
-32P]dCTP
(Amersham Pharmacia Biotech) by a random primer labeling system (Roche
Molecular Biochemicals). The hybridized membranes were washed as
described (16) and visualized by autoradiography. The bands were
quantified using Image Quant (Molecular Dynamics).
Microinjection--
HUVECs were grown on fibronectin-coated
glass bottom dishes and microinjected using an Eppendorf
Micromanipulator 5171 and Transjector 5246. Approximately 500 cells
were microinjected cytoplasmically with 0.1 µg/µl C3 exoenzyme
(Calbiochem) or 0.8 µg/µl dominant-negative N17Rac protein (12)
with Femtotips (Eppendorf) at 100 hectopascals/0.2 s. Injected cells
were marked by coinjecting of 5 mg/ml Texas Red®-labeled dextran
(Molecular Probes) (12).
Migration Assay--
Cell migration assays were performed using
24-well chemotaxis chambers (Nalgene). Polycarbonate filters with a
pore size of 8 µm (Nalgene) were coated with 5 µg/ml fibronectin,
200 µg/ml Matrigel (Becton Dikinson), or 5 µg/ml bovine vitronectin
(Life Technologies) at 4 °C overnight in phosphate-buffered saline
(PBS) and dried under sterile air. SPP and/or inhibitors were diluted to appropriate concentrations in M199 supplemented with 0.5% fatty acid free bovine serum albumin (Sigma), and 600 µl of the final dilution was placed in the lower chamber of a modified Boyden chamber.
HUVECs were washed with PBS and trypsinized for the minimum time
required to achieve cell detachment. Approximately 5 × 104 cells suspended in 100 µl of M199 with 0.5% bovine
serum albumin were placed in the upper compartment. The cells were
allowed to migrate for 3 h at 37 °C in a humidified chamber
with 5% CO2. After the incubation period, the filter was
removed, and the nonmigrated cells on the upper side of the filter were
removed with a cotton swab. The filters were fixed with 4%
formaldehyde and stained with hematoxylin. Migration was quantified by
counting cells in three random high power fields (100×) for each
filter. Alternatively, filters were stained in 0.1% crystal violet and
eluted with 10% acetic acid in 96-well plates. Quantification was done
based on absorbance (A) at 575 nm by a Spectramax 340 (Molecular Devices) plate reader.
Attachment Assay--
Antibodies against integrins were
purchased from Chemicon. Ninety-six well plates were coated with FN (5 µg/ml), VN (5 µg/ml, Life Technologies), or MG (200 µg/ml,
Beckton Dickinson) at 4 °C overnight. Plates were blocked with 10 mg/ml heat-denatured fatty acid free bovine serum albumin for 30 min.
HUVECs (5 × 105 cells/ml in M199 with 0.1% fatty
acid free bovine serum albumin) were incubated with function-inhibiting
anti-integrin antibodies (
1,
1, LM609,
Chemicon) or other reagents for 10 min before plating. Then 100 µl of
the cell suspension/well was added onto plates and incubated for 30 min
at 37 °C under 5% CO2 with the lid off. After
incubation, nonspecifically attached and unattached cells were removed
by rinsing with PBS. Attached cells were fixed in 5% glutaraldehyde
for 20 min and stained with 0.1% crystal violet. Stains were dissolved
in 10% acetic acid, and plates were read at
A575 nm by a Spectramax 340 plate reader.
Immunocytochemistry--
Cells were rinsed with ice-cold PBS and
fixed in 3.7% formaldehyde for 15 min at room temperature followed by
permeabilization with 0.1% Triton X-100. Anti-paxillin antibody (1:
100, Transduction Laboratories) in PBS was incubated for 90 min at room
temperature. The primary antibody staining was visualized with
FITC-conjugated goat anti-mouse (1:200, Jackson Laboratories).
Actin cytoskeleton was stained with either FITC- or
tetramethylrhodamine B isothiocyanate-phalloidin (0.05 µg/ml, Sigma)
for 30 min at room temperature. Images were observed with a Zeiss
Axiovert fluorescence microscope and captured by a SPOTTM digital
camera (Diagnostic Instruments, Inc).
Affinity Precipitation of Rho-GTP/Rac-GTP and
Immunoblotting--
HUVECs were washed with ice-cold PBS and lysed in
radioimmune precipitation buffer (50 mM Tris, pH 7.5, 1%
Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM
NaCl, 10 mM MgCl2, 1× protease inhibitor
mixture), and cell lysates were cleared by centrifugation at
20,000 × g at 4 °C for 10 min. Equal amounts of
lysates were incubated with GST-C21 (Rho binding domain of
Rhotekin quanine nucleotide exchange factor, kindly provided by
Dr. Shuh Narumiya, Kyoto University, Japan) or GST-PAK (Rac binding
domain of p21 activated kinase, kindly provided by Dr. Martin Schwartz,
The Scripps Research Institute, La Jolla, CA) beads at 4 °C for
1 h. The beads were washed three times with wash buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 150 mM
NaCl, 5 mM MgCl2, 10% glycerol, 1 × protease inhibitor mixture). Bound proteins were resolved on 12%
SDS-polyacrylamide gel electrophoresis and detected by Western blot
analysis using a monoclonal antibody against Rho (Santa Cruz
Biotechnology) or Rac1 (Transduction Labs). Equal loading was confirmed
by blotting against Rho, Rac, or
-actin (Sigma).
Endothelial Cell Morphogenesis Assay in Three-dimensional Fibrin
Gel--
This assay was performed as described in Bayless et
al. (27) with minor modifications. HUVECs were rinsed with PBS,
trypsinized, and resuspended in Dulbecco's minimum essential medium
(Cellgro) with 5 µg/ml soybean trypsin inhibitor (Sigma). Cells were
rinsed twice with Dulbecco's minimum essential medium and resuspended at a density of 3 × 106/ml. Fibrinogen was added as a
final concentration of 10 mg/ml, and polymerization was achieved by
adding 1unit/ml thrombin. Fibrin gel was covered with a volume of
Dulbecco's minimum essential medium containing 20% charcoal-stripped
serum, heparin-stabilized endothelial cell growth factor, and different
concentrations of SPP along with integrin antagonists. After an 18-h
incubation at 37 °C with 5% CO2, medium was removed,
and gels were fixed with 3% glutaraldehyde in PBS. Pictures were taken
from several random fields of each well. Gels were embedded into
paraffin block and sectioned for hematoxylin staining.
 |
RESULTS |
EDG-1 and EDG-3 Are Required for SPP-stimulated Migration of
HUVECs--
SPP was previously shown to induce endothelial cell
migration; however, mechanisms involved are not clearly defined
(19-24). We and others have shown that HUVECs express EDG-1 and -3 subtypes of GPCRs for SPP (12, 19-24). To define the role of SPP
receptors in endothelial cell migration, we utilized the antisense
PTOs, which block the expression of EDG-1 and -3 GPCRs (12). HUVECs were transfected with sense or antisense PTOs against EDG-1 or -3 using
the lipid-mediated method. Expression of EDG-1 and -3 receptors was
assayed by Northern analysis. As shown in Fig.
1A, treatment with EDG-1
antisense PTO specifically reduced the steady-state mRNA levels for
EDG-1 and not EDG-3. Likewise, antisense EDG-3 PTO blocked expression
of EDG-3 mRNA in a specific manner. Quantitative analysis indicated
that antisense PTO for EDG-1 (
s1) showed >90% suppression of EDG-1 mRNA levels. Antisense PTO for EDG-3
(
s3) had almost 60% reduction of EDG-3 mRNA levels
(Fig. 1A), whereas sense counterparts for
s1 and
s3
had no effect. These data indicate that the antisense EDG-1 and -3 PTOs
are efficacious and selective in inhibiting the expression of
respective receptors.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
SPP-induced HUVEC migration is mediated via
EDG-1 and EDG-3. HUVECs were pretreated with 400 nM
antisense PTO against EDG-1 ( s1), EDG-3
( s3), sense counterpart for s1 (s1), or
sense counterpart for s3 (s3) as described. A, After
12 h, cells were harvested, and 15 µg of total RNA were analyzed
by Nothern blot for EDG-1, -3, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Quantitation was done based on the
ratio of EDG-1/glyceraldehyde-3-phosphate dehydrogenase mRNA or
EDG-3/glyceraldehyde-3-phosphate dehydrogenase mRNA. Data represent
mRNA levels from 4 to 5 independent experiments. B,
after 18 h, cells were trypsinized and applied for migration assay
in the presence or absence of SPP (60 nM). Data are
expressed as the number of cells per field from four independent
fields. Results represent the mean ± S.E. of four independent
experiments.
|
|
When treated with SPP, HUVECs migrated on different extracellular
matrices (ECMs) such as FN, VN, and MG. SPP induced ~6-fold (FN), ~ 22 fold (VN), ~25 fold (MG) stimulation of migration above the
base-line rate in all three matrices (Fig. 1B). However, the basal rate of cell migration was higher in FN-coated dishes, even though SPP stimulated further chemotaxis. SPP-induced HUVEC migration was dramatically inhibited by the antisense EDG-1 and -3 PTOs, suggesting that each of these GPCRs for SPP is required for chemotaxis.
SPP induced a dose-dependent increase of HUVEC migration
over the range of 10 nM~0.5 µM (data not
shown). Cell migration responses were inhibited by pretreatment with C3
exotoxin (4 days, 5 µg/ml), which inhibits the small GTPase Rho (Fig.
2A). These data suggest that
EDG receptor signaling via the Rho pathway is essential for SPP-induced
endothelial cell migration.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 2.
SPP-induced HUVEC migration and adhesion
requires Rho activity. A, HUVECs were allowed to
migrate in the presence or absence of 60 nM SPP in the
lower chamber on FN (5 µg/ml), VN (5 µg/ml), and MG (200 µg/ml)
as described. For pretreatment, HUVECs were incubated with C3 (5 µg/ml, 4 days). Data are expressed as A575 nm
(OD575), which corresponds to the number of cells
migrated. Results represent the mean ± S.E. of two independent
experiments. B, HUVECs or C3-pretreated (5 µg/ml, 4 days)
HUVECs were trypsinized and kept in suspension in the absence or
presence of SPP (100 nM). After 10 min, cells were replated
on VN and further incubated. After 20 min, cells were fixed and stained
with tetramethylrhodamine B isothiocyanate-phalloidin and anti-paxillin
antibodies (FITC channel) (bar, 10 µm). Regions indicated
by the large arrows are shown at higher magnification in the
lower panels. Small arrowheads in the lower
panels indicate focal contact sites (green) that
are frequently observed at the tips of stress fibers (red).
The green signal indicates immunoreactivity for paxillin
(FITC), and the red signal indicates the reactivity for
F-actin (tetramethylrhodamine B isothiocyanate-phalloidin).
C, HUVECs were microinjected with Texas Red®-labeled
dextran alone (5 µg/ml), dextran with C3 (0.1 µg/µl), or dextran
with N17Rac (0.8 µg/µl). Injected cells are marked with
arrowheads. After 4 h, cells were replated on VN-coated
dishes in the presence of SPP (100 nM) and further
incubated for 20 min. Cells were fixed, stained with anti-paxillin and
FITC-conjugated secondary antibody, and visualized under two different
fluorescence channels (upper panels, FITC to label paxillin
in focal adhesions; lower panels, Texas Red to mark
microinjected cells; bar, 10 µm). Injected cells without
solid focal contacts were scored (Texas Red, 2.9%; C3, 100%; N17Rac,
5.1%; n > 100 cells for each category).
|
|
The Rho GTPase is activated by extracellular mediators and regulates
the formation of actin stress fibers and focal adhesion sites (28, 13).
When HUVECs were plated on FN, VN, or MG and treated with SPP for 15 min, both focal adhesion sites and stress fibers were induced (Fig.
2B and data not shown). Thus, cell matrix adhesion and
spreading were induced by SPP. FN supported basal adhesion of HUVECs in
the absence of stimulation and further enhanced adhesion, stress fiber
formation, and focal contact site assembly after SPP addition (data not
shown). Although VN did not support basal adhesion in the absence of
the ligand, SPP strongly induced adhesion, spreading, and focal contact
assembly on cells plated on VN (Fig. 2B). Although these
events occurred on Matrigel, a complex extracellular matrix enriched in
laminin (29), only small focal contact sites and stress fibers were
induced by SPP (data not shown). Treatment of cells with C3 exotoxin
strongly inhibited SPP-induced focal contact site assembly and stress
fiber formation (Fig. 2B). Similarly, microinjection of C3
exotoxin also blocked focal contact site assembly (Fig. 2C).
Interestingly, microinjection of dominant negative Rac (N17Rac) protein
did not inhibit SPP-induced focal contact formation and cell spreading (Fig. 2C). These data suggest that SPP/EDG receptor
signaling induces a Rho signal that is essential for initial focal
contact assembly and ultimately for cell migration.
Regulation of Rho and Rac by SPP in HUVECs--
To directly
measure the effect of SPP on Rho and Rac activation, we utilized the
GST pull-down assays for Rho and Rac GTPases (28, 30, 31). As shown in
Fig. 3A, strong activation of Rho by SPP was observed in HUVECs plated on both VN and FN. Rac was
also induced, although the magnitude of stimulation was less than that
of Rho. Interestingly, basal Rac activity was enhanced in FN, which
supports better HUVEC adhesion under basal conditions. SPP effect was
more pronounced in HUVECs plated on VN than FN. These data indicate
that SPP directly stimulates Rho and Rac GTPases in HUVECs.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
SPP modulates the activity of Rho and Rac in
HUVECs. A, HUVECs were grown on VN- or FN-coated dishes
to confluency. Cells were serum-starved in plain M199 for 5 h and
stimulated with 100 nM SPP for 3 min. B, HUVECs
were trypsinized and kept in suspension in the presence of SPP (100 nM) for 60 min. At each time point, cells were harvested,
and cell lysates were analyzed for active Rho or Rac by affinity
precipitation assay as described. A representative example from two
independent experiments is shown.
|
|
Since SPP induced focal contact formation in nonadherent HUVECs in a
C3-inhibitable manner, we tested the effect of SPP on Rho and Rac
activation in nonadherent conditions. HUVECs were detached from the
culture dish and held in suspension with 100 nM SPP for
0-60 min at 37 °C. At each time point, cells were harvested and
analyzed for the level of active Rho and Rac. As shown in Fig.
3B, SPP strongly stimulated Rho activity of HUVECs in
suspension within 10-20 min (upper panel). In contrast, SPP
did not significantly affect Rac activity (Fig. 3B,
lower panel) of nonadherent HUVECs. These data indicate that
SPP induces Rho activity in an adherence-independent manner, which is
required for initial focal contact assembly.
Regulation of Rho and Rac by EDG-1 and EDG-3--
We next
addressed the involvement of EDG-1 and -3 receptors in the activation
of Rho and Rac by SPP. Cells were pretreated with the antisense PTO of
EDG-1 or EDG-3 and then stimulated with 100 nM SPP. As
shown in Fig. 4, antisense PTO against
EDG-3 strongly inhibited the SPP-induced Rho activation. However,
antisense PTO against EDG-1 also inhibited Rho activation, suggesting
that both receptors contribute to Rho activation. Similarly, antisense
EDG-1 and EDG-3 blocked Rac. These data suggest that both EDG-3 and -1 receptors contribute to SPP-induced Rho and Rac activation.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
SPP activates Rho and Rac via EDG-1 and
EDG-3. HUVECs were pretreated with 400 nM indicated
PTOs. After 12 h recovery, cells were serum-starved for 5 h
and stimulated with 100 nM SPP for 3 min. Cell lysates were
analyzed for active Rho (A) or Rac (B) by an
affinity precipitation assay as described. C, quantitation
of A and B was done by densitometry. Results
represent the mean ± S.E. of 3-4 independent experiments (*,
p < 0.05, t test). D, CHO cells
expressing EDG-1-FLAG or EDG-3-FLAG were serum-starved for 48 h
and stimulated with 100 nM SPP for 2 min. Lysates were
analyzed for active Rho or Rac by the affinity precipitation assay as
described. Expression levels of EDG-1-FLAG or EDG-3-FLAG were measured
by anti-FLAG immunoprecipitation followed by anti-FLAG
immunoblotting.
|
|
To confirm the antisense PTO blockage data, we utilized the CHO cell
system, which exhibited a very low endogenous SPP receptor activity and
expression (23, 32). CHO cells stably transfected with EDG-1 or -3 receptors or vector-transfected controls were stimulated with
SPP, and Rho and Rac activity was directly measured. As
shown in Fig. 4D, both Rho and Rac were activated in both
EDG-1 and -3-transfected CHO cells. These data confirm the antisense PTO inhibition experiments in HUVECs and suggest that both EDG-1 and -3 are capable of activating Rho as well as Rac-dependent signaling pathways.
SPP-induced HUVEC Migration Is Inhibited by Integrin
Inhibitors--
Focal contact sites are formed by activation and
clustering of various integrins and associated molecules (33). It is
known that
3- and
1-containing integrins,
namely,
v
3,
5
1, and
2
1
are critical for endothelial cell adhesion, migration, and morphogenesis (34-36). To assess the role of different integrins in
SPP-induced HUVEC migration, we utilized blocking antibodies for
1 and
3
v
3
as well as echistatin, a disintegrin antagonist of
v
3 integrin (37). As shown in Fig.
5A, HUVEC adhesion was stimulated by SPP potently in VN and MG and less potently in FN. These
data are consistent with the finding that FN supports basal adhesion to
HUVECs more than VN and MG. Antibodies to
1 integrins strongly inhibited basal and SPP-induced adhesion in FN and MG. This is
consistent with the knowledge that HUVECs adhere to FN via the
5
1 integrin and adhere to MG via the
laminin receptor
2
1 (35, 36). Blockage of
3 integrins with anti-
3 antibody, LM609,
or echistatin did not influence basal or SPP-induced cell adhesion on
FN and MG. In contrast, HUVEC adhesion to VN is strongly inhibited by
anti-
3 antibody, LM609, and echistatin, suggesting that
the
v
3 integrin is involved. These data
suggest that SPP activates the
1- as well as
3-type of integrins, particularly
v
3, to induce cell adhesion. SPP-induced
cell adhesion and not basal adhesion was strongly inhibited by the C3
exotoxin, suggesting the involvement of Rho activation.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5.
SPP induces HUVEC migration on a different
matrix through
v 3
and 1 integrins. A,
attachment of HUVECs to FN, VN, and MG were challenged with SPP (100 nM), blocking antibodies to 1 (30 µg/ml),
3 (30 µg/ml), LM609 (20 µg/ml), control antibody
(IgG1, 30 µg/ml), echistatin (Ech, 100 nM), or C3 (5 µg/ml, 4 days). Attached cells were fixed
and stained with crystal violet as described. Data represent the
mean ± S.E. of absorbance values at 575 nm corresponding to the
number of attached cells (n = 3). B, HUVECs
were allowed to migrate on mixed matrix of FN, VN, and MG (5, 5, 200 µg/ml, respectively) with or without SPP (100 nM).
Migration was challenged with function-blocking antibodies to
1 integrin (30 µg/ml), 3 integrin (30 µg/ml), v 3 integrin (LM, 20 µg/ml), control antibody (IgG1, 30 µg/ml), or
echistatin (Ech, 100 nM). Data represent
mean ± S.E. of duplicate determinations. NT, no
treatment.
|
|
We next conducted HUVEC migration assays on the mixed matrix of FN, VN,
and MG along with the integrin-blocking reagents. As depicted in Fig.
5B, the addition of anti-integrin
1,
3, and
v
3 antibodies to
the migration medium suppressed the SPP-induced HUVEC migration on the
mixture of FN, VN, and MG. Echistatin, a disintegrin known to inhibit
the binding of
v
3 to VN and rather weakly
5
1 to FN (37), abolished the SPP-induced
HUVEC migration on the mixed matrix. These data indicate that
SPP-induced activation of
1-and
3-type
integrins, especially
v
3, are critical
for the stimulation of HUVEC migration.
SPP-induced Endothelial Cell Morphogenesis in Three-dimensional
Fibrin Matrix Requires
v
3,
1 Integrins, as Well as the Activity of
Rho--
Bayless et al. (27) demonstrate that HUVECs
undergo morphogenetic differentiation into lumenal structures in a
three-dimensional fibrin matirx in the presence of phorbol 12-myristate
13-acetate. Recently it has been shown that SPP can induce endothelial
morphogenesis on MG (12) and collagen (21). We next determined the
requirement for integrins in SPP-induced morphogenesis of HUVECs in the
fibrin matrix. HUVECs were set to differentiate into tubular structures in the absence (Fig. 6A) or
presence (Fig. 6, B-H) of 500 nM SPP along with
a combination of anti-integrin antibodies or C3 (5 µg/ml, 4 days
pretreatment). Cells given SPP alone (Fig. 6B) or SPP with
control IgG1 (Fig. 6C) formed lumenal
structures. On the other hand, function-blocking antibodies against
1 (Fig. 6F),
3 (Fig.
6D),
v
3 (Fig. 6 E) as well as
C3 (Fig. 6G) and echistatin (Fig. 6H) suppressed
SPP-induced morphogenesis. These data suggest that SPP-induced
Rho-dependent focal contact assembly, integrin activation,
and migration are required for HUVEC morphogenesis into lumenal
structures, an essential step in angiogenesis.

View larger version (138K):
[in this window]
[in a new window]
|
Fig. 6.
Inhibition of SPP-induced HUVEC morphogenesis
by antagonists of
v 3
or 1. HUVECs were
pre-incubated with 30 µg/ml IgG1 (C), 30 µg/ml 3 antibody (D), 25 µg/ml LM609
(E), 30 µg/ml anti- 1 antibody
(F), 5 µg/ml C3 (G), or 100 nM
echistatin (H). Cells were mixed with unpolymerized fibrin
solution, allowed to gel, and treated with 500 nM SPP
(B-G) or not (A) for 18 h. Cells were
fixed, and cellular morphology was photographed. Arrows
denote a tubular structure (white arrows) or regressed
structure (black arrows). A sectioned structure of a vacuole
stained by hematoxylin is shown in A and B
(insets). Bar, 10 µ m.
|
|
 |
DISCUSSION |
Angiogenesis or new blood vessel formation is a critical process
required for a variety of physiological processes such as wound
healing, embryonic development, and maintenance of the reproductive system (38). Coordinated regulation of endothelial cell migration, proliferation, and assembly is essential in angiogenesis. For coordinated migration, both soluble factors and ECM-derived cues are
thought to be important (39). Growth factors such as platelet-derived growth factor, vascular endothelial cell growth factor, and fibroblast growth factor as well as ECM molecules including FN, collagen, fibrin,
VN, and laminin are important in regulating different aspects of
angiogenesis (40). ECM molecules interact with integrins, which are
heterodimeric receptors required for cell adhesion, migration, and
morphogenesis. Endothelial integrins have been attractive targets for
the inhibition of angiogenesis since
v
3 (41) as well as
1-containing integrins (42) are
implicated in angiogenic responses.
1-Containing
integrins are important in endothelial cell tubule formation in
three-dimensional collagen matrix (35). In addition,
v
3 has been shown to be up-regulated in
angiogenic blood vessels (41). Moreover,
v
3 functions in a synergistic manner with
receptor tyrosine kinases such as platelet-derived growth factor
receptor and vascular endothelial cell growth factor receptor for cell
migration and proliferation (43, 44). However, gene deletion studies
indicate that
v
3 is dispensable for
embryonic angiogenesis (45).
Results from our lab (12) as well as others (19) implicate that SPP is
a regulator of angiogenesis. SPP released during platelet activation
accounts for the majority of the angiogenic activity in serum (48). Of
interest, numerous groups report that SPP induces the migration of
endothelial cells in vitro (19-24). However, molecular
mechanisms involved are not well defined. Our data indicate that
antisense PTO-mediated suppression of EDG-1 and -3, both, attenuated
the SPP-induced migration of HUVECs, suggesting that both receptors are
involved. SPP was proposed to be a dual messenger, i.e. an
extracellular messenger that interacts with cell surface receptors and
an intracellular second messenger that regulates intracellular events
(1-3, 14, 15). Data in this study suggest that HUVEC migration is
mediated by EDG-1 and -3 GPCRs.
HUVEC migration on a variety of ECM surfaces were induced by SPP,
suggesting that SPP regulates the activity of several types of
integrins. HUVECs express a variety of integrins including
v
3 (VN receptor),
2
1 (laminin receptor), and
5
1 (FN receptor) (34-36, 41, 42).
v
3 and
1 integrins have
been implicated as critical factors of angiogenesis (41, 42). These
data support the notion that SPP-induced signaling pathways induce
integrin activation. Indeed, SPP induced focal contact site assembly on all three ECM surfaces in a Rho-dependent manner.
Interestingly, Rac activity was not required for initial focal contact
assembly and cell spreading.
The Rho family of GTPases are critical regulators of cell motility,
cytoskeletal dynamics, cell-cell adhesion, and cell-matrix adhesion
(46). SPP induced changes in focal adhesion assembly, and actin changes
in fibroblasts and endothelial cells require the activity of the Rho
GTPases. Specifically, we showed that SPP-induced actin stress fibers
were blocked by the C3 exotoxin, and both cortical actin formation as
well as stress fibers were blocked by the dominant negative Rac protein
(12). Although these studies indirectly implicate the role of Rho and
Rac in SPP-induced events, direct measurement of Rho and Rac activity has not been reported. In this study we show that SPP induces both Rho
and Rac GTPases in HUVECs in a rapid manner. SPP induced Rho in
nonadherent HUVECs, whereas Rac activation required cell attachment on
ECM. This is consistent with the finding that SPP induced cell
attachment and spreading to ECM surfaces by inducing focal contact site
assembly that was inhibited by the C3 exotoxin. These data are
consistent with the work on Rho regulation by serum, lysophosphatidic
acid, and SPP in murine fibroblasts, in which the authors showed that
serum factors, cell adhesion, and the cytoskeletal structures all
contribute to Rho activity (28).
Interestingly, inhibition of EDG-1 and -3 expression with antisense
PTOs resulted in the attenuation of SPP-induced Rho and Rac activity.
These data are consistent with the fact that both EDG-1 and -3 antisense PTOs inhibited SPP-induced migration and suggest that
cooperative signaling of EDG-1 and -3 receptors occurs in HUVECs.
Surprisingly, each of the receptors was capable of activating Rho and
Rac in transfected CHO cells. Indeed SPP induced migration of CHO cells
expressing EDG-1 or EDG-3 (data not shown). This apparent discrepancy
between HUVECs and CHO transfectants may be due to cell type-specific
differences in signaling pathways. The issue of cooperative signaling
between EDG-1 and -3 needs to be further explored. A second issue that
needs to be further dissected is the induction of Rac and Rho by both
EDG-1 and -3. Previous studies that monitored actin changes suggest
that EDG-1 preferentially couples to the Rac pathway, whereas EDG-3
couples to the Rho pathway (12). It is important to stress that such conclusions were based on indirect measurements of Rac and Rho activity. However, direct measurement of Rac and Rho activity by
biochemical assays in this report indicates that both receptors are
capable of activating both Rac and Rho in both HUVECs (by antisense
inhibition studies) and in CHO cell transfectants. Further studies
should define the intermediate steps between EDG-1/-3 and Rac/Rho
GTPases in different cell types.
SPP-induced focal contact assembly is required for cell spreading and
migration. Our data show that
1 integrins are used to
attach and spread on FN and MG, whereas
v
3 was responsible on the VN ECM surface.
Activation of both types of integrins are important in HUVEC migration
induced by SPP since blocking antibodies to
1 and
v
3 as well as echistatin attenuated
migration on mixed ECM surface. Furthermore, antagonists to
v
3 and
1 integrins inhibited SPP-induced endothelial cell morphogenesis in a
three-dimensional fibrin matrix. These data strongly suggest that
SPP/EDG receptors cooperate with
v
3 and
1 integrins to induce HUVEC migration and morphogenesis.
Previous work indicates that SPP induces VE-cadherin assembly into
adherens junctions, which was necessary for HUVEC morphogenesis on
Matrigel (12). Relationships between SPP-induced cell-cell junctions
and integrin-based focal contact site assembly need to be investigated
in the future.
In summary, data in this report demonstrate that
v
3 and
1 integrins are
required for SPP-induced migration of HUVECs, and SPP-induced
activation of Rho through EDG-1 and EDG-3 is required for this process.
SPP signaling via the EDG-1 receptor is required for embryonic vascular
system maturation in vivo (17), and SPP modulates
angiogenesis in the adult (12). SPP-induced activation of integrins may
be critical in such events.