From the Department of Pharmacology, Boyer Center for
Molecular Medicine, Yale University School of Medicine, New Haven,
Connecticut 06536, the
Division of Cardiovascular Research, St.
Elizabeth's Medical Center of Boston, Boston, Massachusetts 02135, and
the § Center for Vascular Biology, Department of Physiology,
University of Connecticut, Farmington, Connecticut 06030-3501
Received for publication, November 2, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Sphingosine 1-phosphate (SPP) binds to members of
the endothelial differentiation gene family (EDG) of receptors and
leads to diverse signaling events including cell survival, growth,
migration and differentiation. However, the mechanisms of how SPP
activates these proangiogenic pathways are poorly understood. Here we
show that SPP signals through the EDG-1 receptor to the heterotrimeric G protein Gi, leading to activation of the
serine/threonine kinase Akt and phosphorylation of the Akt substrate,
endothelial nitric-oxide synthase (eNOS). Inhibition of Gi
signaling, and phosphoinositide 3-kinase (PI 3-kinase) activity
resulted in a decrease in SPP-induced endothelial cell chemotaxis. SPP
also stimulates eNOS phosphorylation and NO release and these effects
are also attenuated by inhibition of Gi signaling, PI
3-kinase, and Akt. However, inhibition of NO production did not
influence SPP-induced chemotaxis but effectively blocked the
chemotactic actions of vascular endothelial growth factor. Thus, SPP
signals through Gi and PI 3-kinase leading to Akt
activation and eNOS phosphorylation.
Sphingosine 1-phosphate
(SPP)1 is a bioactive lipid,
which can be stored and released from platelets upon their activation but can also be synthesized in response to extracellular stimuli by the
sequential action of sphingomyelinase, ceramidase, and sphingosine
kinase in many cell types (1). Five members of the endothelial
differentiation gene family (EDG) of G protein-coupled receptors
(EDG-1, EDG-3, EDG-5, EDG-6, and EDG-8) have been identified as SPP
receptors in a wide variety of cell types (2-5). These receptors
exhibit high affinity binding for SPP, and SPP is weakly displaced by
other sphingolipids, including lysophosphatidic acid. EDG receptors
differ in their association with the G protein family members. Both
EDG-3 and EDG-5 potently activate Gi, Gq,
G12, and G13, whereas EDG-1 and EDG-8 couples
to Gi but not Gq. In a similar manner, EDG-6
couples to Gi whereas a role for
Gq,G12, and G13 has yet to be
addressed (6).
SPP participates in a wide spectrum of angiogenic activities including
cell proliferation (7, 8), endothelial cell migration (9-11),
morphogenesis, and survival, and is involved in the formation of mature
neovessels in vivo (12, 13). All these angiogenesis signaling pathways involve the pertussis toxin (PTx)-sensitive G
protein, Gi. However, the downstream effectors of
Gi responsible for these effects are not well established.
It has been shown that SPP signaling via EDG-1 stimulates
mitogen-activated kinase family member ERK (ERK1/ERK2), an effect inhibited by two structurally distinct phosphoinositide 3-kinase (PI 3-kinase) inhibitors (LY294002 and wortmannin). Moreover, immunoprecipitation of Grb-2 from SPP treated cells resulted in the
recovery of PI 3-kinase activity, an effect blocked by pretreatment with pertussis toxin (14). PI 3-kinase catalyzes the phosphorylation of
the inositol ring of phosphatidylinositol lipids at the D-3 position producing phosphatidylinositol 3,4-bisphosphate and
phosphatidylinositol 3,4,5-trisphosphate. One downstream effector of PI
(3)-kinase is the serine/threonine kinase Akt (or protein kinase B)
(15). Upon receptor activation, Akt is recruited to the plasma membrane and binds to inositol lipids via its pleckstrin homology domain. Akt is
then phosphorylated by phosphoinositide-dependent kinases, and this phosphorylation enhances its catalytic activity toward a
variety of diverse substrates (16). Recently, we and others (17, 18)
have shown that Akt can phosphorylate endothelial nitric-oxide synthase
(eNOS) on serine 1179 (serine 1179 in bovine or serine 1177 in the
human ortholog, respectively) resulting in eNOS activation and nitric
oxide (NO) production (17-19). However, it is not known if SPP can
stimulate Akt, eNOS phosphorylation, and NO production in intact
endothelial cells.
Several reports have implicated Akt and/or NO as downstream effectors
of angiogenic growth factors that can promote endothelial cell survival
(20-23) and migration (24-27). These findings, in addition to reports
demonstrating a role for Akt and NO promoting angiogenesis "in
vivo" (28, 29), suggested that PI 3-kinase/Akt/eNOS pathway may
function as a downstream target for the angiogenesis properties of SPP.
Therefore, in this study we assessed whether the PI 3-kinase/Akt/eNOS
pathway is activated upon SPP stimulation in endothelial cells, thus
contributing to the angiogenic properties of this bioactive lipid.
Cell Culture and Reagents--
Bovine lung microvascular
endothelial cells (BLMVECs, Vec Technologies) were cultured as
described previously (30, 31).
Cell Migration Assay--
Migration assays were performed using
a modified Boyden chamber (Neuroprobe, Cabin John, MD). Briefly,
exponentially growing cells were harvested with trypsin (0.05%, v/v)
and EDTA (0.53 mM), counted, and resuspended at a density
of 0.4 × 106 in chemotaxis medium (Dulbecco's
modified Eagle's medium with 1% fatty acid-free bovine serum albumin)
before being placed in the upper well of a 48-well chemotaxis chamber
(Neuroprobe). The lower wells of the chemotaxis chamber contained
chemotaxis medium without (controls) or with VEGF (10 ng/ml) or SPP
(10-500 nM). Upper and lower wells were separated by a
polyvinilpyrrolidone-free polycarbonate filter with 8-µm pores
(Poretics Corp., Livermore, CA) coated with 100 µg/ml type I collagen
(Collaborative Biomedical Products, Bedford, MA). The chamber was
incubated for 4 h at 37 °C. After incubation, cells were fixed
with ethanol (70%) and nonmigrating cells on the upper surface of the
filter removed. Migrated cells were stained with Giemsa and counted
(magnification, ×400) in three random fields per well. Each experiment
was performed in triplicate, and migration was expressed as the number
of total cells counted per well. In some experiments, BLMVECs were
pre-incubated with or without L-NAME (2 mM) or
LY294002 (10 µM) for 30 min or 1 h, respectively, or
with or without PTx (200 ng/ml) for 6 h at 37 °C. In previous
experiments, this concentration of L-NAME, but not
D-NAME, completely blocked VEGF- or calcium
ionophore-stimulated NO production or cGMP accumulation in a reporter
bioassay system as described (30, 32). In addition, the concentration
of LY294002 completely abolished VEGF- or serum-stimulated Akt
phosphorylation (24).
Measurement of NO Release--
For measurement of NO, we
analyzed the release of NO Western Blotting--
BLMVECs treated with or without SPP (500 nM), PTx (200 ng/ml), or LY294002 (10 µM)
were washed twice with ice-cold phosphate-buffered saline, and total
cell lysates were prepared by scraping the cells in lysis buffer (50 mM Tris-HCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1% (v/v) Nonidet P-40, 0.1% SDS, 0.1% deoxycholic acid, 20 mM NaF, 1 mM NaPP, 1 mM sodium
vanadate, 1 mM Pefabloc, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Lysates were rotated for 1 h at 4 °C and
the insoluble material removed by centrifugation at 12,000 × g for 10 min at 4 °C. Equal amounts of the denatured
proteins were separated on a 7.5% sodium dodecyl
sulfate-polyacrylamide gel (Mini Protean II, Bio-Rad) and transferred
to a nitrocellulose membrane. Membranes were blocked by incubation in
Tris-buffered saline (10 mM Tris, pH 7.5, 100 mM NaCl) containing 0.1% (v/v) Tween 20 and 5% (v/v)
non-fat dry milk for 2 h, followed by a 2-h incubation, at room
temperature, with rabbit polyclonal anti-phospho-Akt-Ser473
or anti-Akt antibodies (New England Biolabs, Beverly, MA) or with
rabbit polyclonal anti-Phospho-eNOS-Ser1177 (New England
Biolabs) or mouse monoclonal anti-eNOS (Transduction Laboratories,
Lexington, KY), The filters were washed extensively in Tris-buffered
saline, containing 0.1% (v/v) Tween, before incubation for 1 h
with goat anti-mouse or donkey anti-rabbit horseradish peroxidase-conjugated secondary antibody. Membranes were then washed
and developed using enhanced chemiluminescence substrate (ECL, Amersham
Pharmacia Biotech).
Northern Blotting--
Twenty micrograms of BLMVEC total RNA and
280 pg of the EDG-1, EDG-3, EDG-5, and EDG-8 in vitro
transcripts were loaded, and Northern analysis was conducted as
described previously (2).
Expression of EDG Receptors in BLMVECs--
It has been shown that
endothelial cells express high levels of EDG-1 transcript (2).
Accordingly, we first examined whether other SPP receptors are
expressed in BLMVECs. Consistent with previous studies, Northern blot
analysis clearly demonstrated abundant expression of EDG-1 mRNA.
BLMVECs also expressed a lower level EDG-5 transcript and barely
detectable EDG-3 mRNA. However, the EDG-8 mRNA was undetectable
in BLMVECs (Fig. 1). In vitro transcripts for EDG-1, -3, -5, and -8 were also loaded as positive controls.
SPP Stimulates Chemotaxis through a PTx-sensitive G Protein in
BLMVECs--
Since activation of the EDG-1 receptor is important for
angiogenesis, we first investigated the involvement of EDG-1 in
SPP-induced chemotaxis in endothelial cells by employing a modified
Boyden chamber assay. BLMVECs were subjected to a migration assay in the absence or presence of SPP (10-500 nM) as a
chemoattractant. As shown in Fig. 2, SPP
dose-dependently increased endothelial cell migration and
the migratory activity at 500 nM was about 5-6 times that
of the control. SPP induced greater cell migration than typically seen
with VEGF as a chemoattractant (see Fig. 6). To investigate the
possibility that a Gi-coupled receptor may be involved in
chemotaxis induced by SPP, BLMVECs were treated with pertussis toxin
(200 ng/ml) for 6 h prior to addition of SPP. Pertussis toxin
pretreatment, which ADP-ribosylates and inactivates Gi
proteins, completely abolished SPP-induced chemotaxis (Fig. 2),
suggesting that SPP binding to EDG-1 increases chemotaxis in a
Gi-dependent manner.
SPP-stimulated Akt Activation and Endothelial Cell Migration Is
Blocked by Pertussis Toxin and Inhibitors of PI 3-Kinase--
It has
been shown that the SPP binding to EDG-1 triggers activation of
ERK1/ERK2, an effect inhibited by PI 3-kinase inhibitors (14). Recent
work from us and others has shown that inhibitors of PI 3-kinase, as
well as dominant-negative suppression of Akt activity, attenuate
growth factor-induced migration (24, 33). To directly test the role of
PI 3-kinase in SPP-induced cell migration, we first examined if SPP can
activate Akt phosphorylation. As seen in Fig.
3A, SPP stimulated Akt
phosphorylation in a time-dependent manner with maximal
activation occurring within 5 min and sustained phosphorylation lasting
for up to 15 min. SPP-stimulated phosphorylation of Akt on
Ser473 was antagonized by preincubation of BLMVECs with
LY294002 and PTx. Next, we examined the effects of the LY294002 on
basal and SPP-stimulated cell migration in endothelial cells. As shown
previously, SPP stimulated cell migration (lane
4) and this effect was completely inhibited by PTx
pretreatment (lane 6) whereas PTx did not
influence basal migration (lane 3). Furthermore,
SPP-stimulated cell migration was blocked by ~50% by LY294002 (from
90.9 ± 4.9 to 43.9 ± 7.0 migrated cells/field for cells
treated with SPP and cells treated with SPP and LY294002, respectively)
(lane 5), although basal migration (without
stimulation) was not affected by this drug (lane
2). Collectively, these data indicate that SPP signals via Gi to PI 3-kinase and Akt and the PI 3-kinase pathway is in
part responsible for endothelial cell migration initiated by this
bioactive lipid.
SPP Stimulates NO Production through Akt Activation in a
PTx-sensitive, PI 3-Kinase, Akt-dependent
Manner--
Recently, we have shown that Akt can phosphorylate bovine
eNOS on serine 1179, resulting in eNOS activation and NO production (17). To examine whether SPP-induced Akt activation is coupled to NO
production, we examined the effects of SPP on eNOS phosphorylation and
NO release. SPP increased NO production in a dose-dependent manner in BLMVECs (Fig. 4A),
an effect blocked by the NOS inhibitor, L-NMMA (data
not shown). Maximal SPP-induced NO release was abrogated by PTx
(lane 5), whereas PTx did not influence basal NO
synthesis (lane 6). As previously reported for
VEGF-stimulated NO production, LY294002 attenuated SPP-induced NO
release (from 1.0 ± 0.1 to 0.4 ± 0.1 pmol/µg of protein
for cells treated with SPP and cells treated with SPP and LY294002,
respectively).
Next, we documented the phosphorylation state of eNOS by immunoblot
analysis (Fig. 4B). As predicted, SPP rapidly stimulated eNOS phosphorylation on serine 1179 (upper panel)
without changing total eNOS levels (bottom
panel). Maximal phosphorylation was seen at 5 min, which
decreased over 20 min to levels slightly above basal values.
SPP-stimulated phosphorylation of eNOS was antagonized by preincubation
of BLMVECs with PTx and LY294002. These findings correlate well with
the results in Figs. 3A and 4A, respectively.
Finally, to more firmly establish a link between SPP signaling to Akt,
we infected endothelial cells with adenoviruses expressing either a
dominant negative form of Akt (AAA-Akt) or GFP and assessed NO release
and eNOS phosphorylation. As seen in Fig.
5A, SPP-induced NO release was
abrogated by expression of AAA-Akt. In addition, SPP-induced
phosphorylation of eNOS was markedly suppressed (Fig. 5B).
Collectively, our results indicate that SPP binding to EDG-1 signals
through Gi, leading to PI 3-kinase-dependent
Akt stimulation, eNOS phosphorylation, and NO production.
Activation of eNOS and NO Are Not Involved in SPP-stimulated
Chemotaxis of BLMVECs--
As a downstream signal of PI 3-kinase/Akt,
we examined the involvement of NO in SPP-induced chemotaxis in BLMEC
because VEGF-stimulated endothelial cell migration can be blocked by
L-arginine-substituted analogues that inhibit NOS. Notably,
the NOS inhibitor L-NAME (2 mM) had no effect
on the chemotaxis produced by SPP (Fig.
6A), even though the same
concentration of L-NAME blocked VEGF-induced cell migration
(Fig. 6B), as shown previously (24). Therefore, SPP
activates chemotaxis without requiring activation of eNOS.
The central findings of this study are that SPP activates Akt,
eNOS phosphorylation, and NO production through the PTx sensitive G
protein Gi. In addition, we show that PI 3-kinase is a
downstream effector of Gi that participates in the
chemoattractant actions of SPP in endothelial cells; however,
SPP-stimulated NO production does not influence the tractional forces
leading to cell migration. Since vasodilation and increases in
endothelial cell permeability also accompany an angiogenic response, it
is possible that SPP activation of Gi-dependent
PI 3-kinase/Akt/eNOS leading to NO production may regulate local blood
flow and permeability during angiogenesis.
SPP has been implicated in both inhibition and stimulation of
chemotactic responses. SPP inhibits B16/F10 melanoma cell motility through a pertussis toxin-insensitive pathway (34) and inhibits chemoinvasiveness and motility of breast cancer cell lines (35). In
contrast, SPP induces endothelial cell chemotaxis, and this effect is
blocked by PTx (9, 11, 36). Similarly, SPP also stimulates the
migration of HEK 293 and Chinese hamster ovary cells overexpressing
EDG-1 or EDG-3 in a PTx-sensitive manner. However, EDG-5 transfection
into Chinese hamster ovary cells does not correlate with induction of
cell migration (11, 37). Our results are consistent with these latter
studies showing that SPP-induced chemotaxis in endothelial cells is
blocked by PTx, suggesting an important role for Gi-protein
coupled SPP receptors to cell migration. Furthermore, the EDG-1
mRNA is the most prominently expressed SPP receptor in BLMVECs in
contrast to the lower levels of EDG-5 mRNA and the barely
detectable EDG-3 mRNA. Together, these data suggest that the
activation of Gi coupled to the EDG-1 receptor is necessary
for SPP-induced chemotaxis in BLMVECs.
Directional cell motility is driven by chemoattractants that bind to G
protein coupled receptors receptors (interleukin-8 and fMLP) or growth
factors that signal through receptor tyrosine kinases (VEGF, basic
fibroblast growth factor, and platelet-derived growth factor). In both
cases, many studies have shown that PI 3-kinase is an important
mediator of these chemotactic responses. Recently, three different
groups have shown that neutrophils or peritoneal macrophages deficient
in p110 It has recently been demonstrated that overexpression of EDG-1 receptor
and eNOS in COS-7 cells led to SPP-dependent eNOS activation (42); however, the mechanism was not explored. We and others
(17, 18, 43, 44) have shown that VEGF, fluid shear stress, estrogen, or
IGF-1 can stimulate Akt and subsequent eNOS phosphorylation on serine
1179, resulting in eNOS activation and NO production. Moreover,
substitution of serine 1179 with aspartate leads to constitutive
activation of eNOS and NO production due to enhanced electron flux from
the reductase domain to the oxygenase domain (45). In agreement with
these studies, here we show that SPP induces eNOS phosphorylation on
serine 1179 and NO production in endothelial cells. SPP-induced eNOS
phosphorylation and NO release were inhibited by PTx, LY294002, and
dominant negative Akt, suggesting that SPP binding to EDG-1 signals via
Gi to PI 3-kinase/Akt leading to eNOS phosphorylation and
NO production. It should be noted that the PI 3-kinase inhibitor
LY294002 was not able to completely block SPP-stimulated NO production
at a concentration that abrogates Akt activation, suggesting other pathways of eNOS activation exist. Furthermore, similar results were
obtained with dominant negative Akt. Indeed, SPP increases cytoplasmic
calcium in endothelial cells via a PTx-sensitive mechanism (12), thus
providing calcium to activate calmodulin and the binding of calmodulin
to eNOS leading to an increase in eNOS activity (46).
The present study also shows that production of NO is not required for
endothelial cell migration mediated by SPP. In contrast, previous
reports have pointed to an important role of NO in VEGF-induced endothelial cell migration (24-27). These apparently contradictory observations most likely reflect the diversity of mechanisms activated in endothelial cells in response to different chemoattractants. However, it is feasible that the increase in NO production mediated by
SPP may be involved in other angiogenic functions promoted by this
bioactive lipid, including cell survival through
inactivation of caspase-3 (23) and enhancement of blood flow and permeability.
Thus, our findings define two additional proangiogenic actions of SPP:
activation of Akt and NO release. Further elucidation of the precise
roles of Akt and NO in the context of angiogenesis will increase our
understanding of the important role of SPP during vascular development,
angiogenesis, and vessel homeostasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
BLMVECs express primarily EDG-1. Total
RNA isolated from BLMVECs (lanes 1) was probed
with EDG-1, -3, -5, and -8 cDNAs. Positive controls
(lanes 2) are in vitro transcripts for
EDG-1, -3, -5, and -8. A picture of the 18 and 28 S rRNA bands on the
formaldehyde/agarose gels is depicted to show equal loading of RNA in
all the blots (lower panel).
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Fig. 2.
SPP-stimulated chemotaxis of BLMVECs is
mediated via a PTx-sensitive G protein. BLMVECs were pretreated
without or with PTx (200 ng/ml) for 6 h, trypsinized, and
resuspended in chemotaxis medium. 20,000 cells were then added on a
polycarbonate membrane (8-µm pore size) coated with type I collagen
in a modified Boyden chamber, and were exposed for 4 h to varying
concentrations of SPP with or without PTx (100 ng/ml) added in the
upper and lower chambers. At the end of the treatments, migrated cells
were stained with Giemsa stain and counted in three random fields
(original magnification, ×400). Data points represent the mean number
of migrating cells/field (± S.D.) calculated in three different wells.
Representative results from three separate experiments are shown.
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Fig. 3.
SPP mediates Akt activation and regulates
endothelial cell migration through the PTx-sensitive Gi
protein and PI 3-kinase. A, serum-starved BLMVECs were
pre-treated without or with LY294002 (10 µM) for 1 h
or PTx (200 ng/ml) for 6 h and then stimulated with SPP (500 nM) for different times. Lysates were analyzed by Western
blotting (40 µg) with antibodies to phospho-Akt (upper
panel) or Akt (lower panel). Similar
results were obtained in three additional experiments.
D.units reflects relative densitometric units of p-Akt to
total Akt. In B, BLMVECs were pretreated without or with
LY294002 (10 µM) for 1 h or PTx (200 ng/m) for
6 h, and then trypsinized and resuspended in chemotaxis medium.
Directional migration was assessed as above. Results are expressed as
mean number of migrating cells/field (± S.D.) calculated in three
different wells. Representative results from three separate experiments
are shown.
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Fig. 4.
Treatment of endothelial cells with SPP
induces NO production through PI 3-kinase activation in a PTx-sensitive
manner. A, BLMVECs plated on 60-mm2 dishes
were pre-treated without or with LY294002 (10 µM) for
1 h or PTx (200 ng/ml) for 6 h. To stimulate NO release,
varying concentrations of SPP were added for 30 min. Supernatant were
collected, and NO was quantified by chemiluminescence. Results are
expressed as mean (± S.D.) of net NO
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Fig. 5.
Dominant negative Akt prevents SPP-induced NO
production and eNOS phosphorylation in endothelial cells.
A, endothelial cells were infected with adenoviruses
(multiplicity of infection of 25) for GFP and HA-tagged AAA-Akt as
described, and cells were stimulated with SPP (500 nM) for
30 min. NO production was quantified as above. *, p < 0.05 compared with AAA-Akt with or without SPP and GFP without SPP. In
B, BLMVECs were infected with adenoviruses for GFP, and
AAA-Akt. After 12 h in complete medium, cells were serum-starved
and treated with or without SPP (500 nM) for 5 min. Cells
were harvested, and cell lysates were subjected to immunoblot analysis
with antibodies specific for phospho-eNOS, total eNOS, and HA.
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Fig. 6.
NO is not involved in SPP-stimulated
chemotaxis of BLMVECs. A, BLMVECs were pretreated
without or with PTx (200 ng/ml) for 6 h or L-NAME (2 mM) for 30 min, trypsinized, and resuspended in chemotaxis
medium. Then, a migration experiment was performed as above. Data
points represent the mean number of migrating cells/field (± S.D.)
calculated in three different wells. Representative results from two
separate experiments are shown. B, BLMVECs were pretreated
without or with L-NAME for 30 min, trypsinized, and
resuspended in chemotaxis medium and migration assays performed in
response to VEGF (10 ng/ml). Data points represent the mean number of
migrating cells/field (± S.D.) calculated in three different wells.
Representative results from two separate experiments are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a catalytic subunit of PI 3-kinase, show a reduction in
movement toward chemoattractants such as fMLP (38-40). Indeed, we and
others have shown that inhibition of PI 3-kinase or its downstream
target Akt blocked VEGF-stimulated migration in endothelial cells (24,
26) and constitutively active Akt is sufficient to stimulate
chemokinesis. A recent study in prostate cancer cells has demonstrated
that
v
3
integrin/ matrix-dependent cell migration was also linked to
the PI 3-kinase/Akt pathway (41). Accordingly, here we show that
binding of SPP to the Gi-coupled receptor, presumably
EDG-1, activates the PI 3-kinase/Akt signaling pathway. Importantly, we
also demonstrate that inhibition of PI 3-kinase attenuates SPP-induced
chemotaxis in BLMVECs, in agreement with studies that established a
crucial role of the PI 3-kinase/Akt signaling pathway in chemotaxis.
The precise mechanism of how activation of Akt leads to chemotaxis is
not known but is likely though direct modulation of actin
polymerization/depolymerization pathways.
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ACKNOWLEDGEMENT |
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We thank Genentech for the generous supply of VEGF.
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FOOTNOTES |
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* This work was supported in part by National Institute of Health Grants RO1 HL57665, HL61371, and HL64793 (all to W. C. S.) and DK45659 (to T. H.); by American Heart Association national grant-in-aid (to W. C. S.); by Ministerio de Educación y Cultura Grant EX99-38446345 (to M. M.-R.); and by an American Heart Association Northeast Chapter scientist development grant (to M.-J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported in part by fellowships from the Heart and Stroke Foundation of Canada, the Fonds pour la Formation de Chercheurs et de l'Aide à la Recherche, and from the Canadian Institutes of Health Research.
** Established Investigator of the American Heart Association.
Established Investigator of the American Heart Association. To
whom all correspondence should be addressed. Tel.: 203-737-2291; Fax:
203-737-2290; E-mail: william.sessa@yale.edu.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M009993200
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ABBREVIATIONS |
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The abbreviations used are: SPP, sphingosine 1-phosphate; PTx, pertussis toxin; EDG, endothelial differentiation gene family; eNOS, endothelial nitric-oxide synthase; NOS, nitric-oxide synthase; PI 3-kinase, phosphoinositide 3-kinase; HA, hemagglutinin; VEGF, vascular endothelial growth factor; L-NAME, L-nitro-arginine methylester; GFP, green fluorescent protein; BLMVEC, bovine lung microvascular endothelial cell; ERK, extracellular signal-regulated kinase.
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