From the Cardiovascular Division, Brigham and
Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 and the
Veterans Affairs Boston Healthcare System,
West Roxbury, Massachusetts 02132
Received for publication, September 13, 2000, and in revised form, November 22, 2000
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ABSTRACT |
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Sphingosine 1-phosphate (S1P) is a
platelet-derived sphingolipid that elicits numerous biological
responses in endothelial cells mediated by a family of G
protein-coupled EDG receptors. Stimulation of EDG receptors by
S1P has been shown to activate the endothelial isoform of nitric-oxide
synthase (eNOS) in heterologous expression systems (Igarashi, J., and
Michel, T. (2000) J. Biol. Chem. 275, 32363-32370).
However, the signaling pathways that modulate eNOS regulation by
S1P/EDG in vascular endothelial cells remain less well understood. We
now report that S1P treatment of bovine aortic endothelial cells (BAEC)
acutely increases eNOS enzyme activity; the EC50 for S1P
activation of eNOS is ~10 nM. The magnitude of eNOS
activation by S1P in BAEC is equivalent to that elicited by the agonist
bradykinin. S1P treatment activates Akt, a protein kinase implicated in
phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS
phosphorylation at Ser1179, a residue phosphorylated by
Akt; an eNOS mutant in which this Akt phosphorylation site
is inactivated shows attenuated S1P-induced eNOS activation.
S1P-induced activation both of Akt and of eNOS is inhibited by
pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin,
and by the intracellular calcium chelator BAPTA
(1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid). By contrast to S1P, activation of G protein-coupled
bradykinin B2 receptors neither activates kinase Akt nor promotes
Ser1179 eNOS phosphorylation despite robustly activating
eNOS enzyme activity. Understanding the differential regulation of
protein kinase pathways by S1P and bradykinin may lead to the
identification of new points for eNOS regulation in vascular
endothelial cells.
Vascular endothelial cells respond to intercellular messengers
that modulate processes as diverse as blood pressure homeostasis, platelet aggregation, and angiogenesis (for review, see Refs. 1 and 2).
Blood platelets secrete sphingosine 1-phosphate (S1P),1 a biologically active
sphingolipid that has been broadly implicated in angiogenesis, platelet
activation, and inhibition of apoptosis in vascular cells (3, 4). S1P
has also been characterized as a potent signal-transducing molecule
that may exert such diverse biological responses as cellular
differentiation (5, 6), hypertrophy (7), proliferation (8), and
migration (9-11). S1P is a potent ligand for the G protein-coupled
receptors termed EDG receptors (for review, see Ref. 12). The
EDG receptor family is comprised of at least eight independent subtypes
(EDG-1-8; see Refs. 12-15). EDG receptors are coupled via pertussis
toxin-sensitive G proteins to the activation of the mitogen-activated
protein kinase (MAP kinase) pathway (16) and to the elevation of the intracellular calcium concentration (10) in signal transduction pathways leading to angiogenesis (17). Additionally, S1P binding to the
EDG-1 receptor has recently been shown to activate the endothelial
isoform of nitric-oxide synthase (eNOS) (18), a key signaling protein
that plays a pivotal role in the maintenance of vascular homeostasis by
promoting vascular smooth muscle relaxation and inhibiting platelet
aggregation (1).
The EDG-1 receptor and eNOS are both localized to plasmalemmal
caveolae, which are specialized sphingolipid-enriched domains in the
plasma membrane that serve as sites for the sequestration of diverse
signaling proteins (18, 19). However, the specific intracellular
signaling pathways that couple S1P/EDG receptor stimulation to eNOS
activation remain less well understood. eNOS is a
calcium/calmodulin-dependent enzyme: in vascular
endothelial cells, eNOS is activated in response to the transient
increases in intracellular calcium initiated by the activation of
diverse G protein-coupled receptors, including the bradykinin B2 receptor.
eNOS is also activated by phosphorylation by protein kinase Akt (20,
21), and eNOS is inhibited by the MAP kinases ERK1/2 (22). eNOS
phosphorylation by protein kinase Akt is promoted by vascular
endothelial growth factor (20, 23) and by fluid shear stress (21, 24),
but Akt activation has not been implicated previously in eNOS
regulation by G protein-coupled receptors. The protein kinase Akt
(known also as protein kinase B; for review, see Ref. 25) functions as
a downstream effector of phosphoinositide 3-kinase
(PI3-K)-dependent signaling pathways (for review, see Ref.
26) and plays pivotal roles in numerous cellular responses including
angiogenesis (27). Although G protein-coupled receptors may regulate
the PI3-K/Akt pathway, the activation of Akt by S1P-induced EDG
receptor stimulation has not been described previously.
In the present study, we provide evidence that S1P potently and
robustly activates eNOS in cultured vascular endothelial cells via EDG
receptors in a pathway that involves G protein-dependent activation of kinase Akt. eNOS activation by the S1P/EDG pathway stands
in contrast to the activation of eNOS by bradykinin B2 receptors, in
which activation of eNOS appears to proceed independently of
Akt-mediated phosphorylation.
Materials--
Fetal bovine serum (FBS) was from Hyclone (Logan,
UT); all other cell culture reagents, media, and LipofectAMINE were
from Life Technologies Inc. S1P and dihydro-S1P (sphinganine
1-phosphate) were from BioMol (Plymouth Meeting, PA). PD98059 and BAPTA
were from Calbiochem. Anti-phospho-eNOS antibody (phosphoserine 1177 in
the human eNOS sequence, corresponding to Ser1179 in bovine
eNOS), anti-phospho-Akt antibody (Ser473), anti-Akt
antibody, Akt enzyme activity kit, and ERK enzyme activity kit were
from Cell Signaling Technologies (Beverly, MA). Anti-phospho-ERK1/2
antibody (Thr183/Tyr185) was from
Promega (Madison, WI). Anti-eNOS monoclonal antibody was from
Transduction Laboratories (Lexington, KY). Super Signal substrate for
chemiluminescence detection and secondary antibodies conjugated with
horseradish peroxidase were from Pierce.
L-[3H]Arginine was from Amersham
Pharmacia Biotech. Protein determinations were made with the
Bio-Rad protein assay kit. All other reagents, including anti-FLAG
monoclonal antibody, were from Sigma.
Plasmid Construction--
cDNA encoding full-length human
EDG-1 receptor epitope-tagged with FLAG peptide (FLAG/EDG-1, described
in (16), was provided by Timothy Hla, University of Connecticut) was
subcloned into pcDNA3 (Invitrogen) as described previously (18).
Full-length wild-type bovine eNOS cDNA subcloned into pBK-CMV was
described previously (28). eNOS S1179A mutant cDNA was made with
polymerase chain reaction-based mutagenesis and verified with standard
DNA sequencing technique (22).
Cell Culture, Transfection, and Drug Treatment--
Bovine
aortic endothelial cells (BAEC) were obtained from Cell Systems
(Kirkland, WA) and maintained in culture in Dulbecco's modified
Eagle's medium supplemented with FBS (10%, v/v) as described (29).
Cells were plated onto gelatin-coated culture dishes and studied prior
to cell confluence between passages 5 and 9.
COS-7 cells were maintained in culture as described previously (30).
The day before transfection, cells were split at a ratio of 1:8 in
Dulbecco's modified Eagle's medium containing 10% FBS. They were
then cotransfected with cDNAs encoding 2 µg of FLAG/EDG-1 and/or
0.03 µg of eNOS by using LipofectAMINE, as described previously (18).
The total DNA amount was normalized using "empty" (no insert)
vector plasmid DNA. For both BAEC and transfected COS-7 cells, the
culture medium was changed to Dulbecco's modified Eagle's medium
without FBS, and incubation proceeded overnight prior to all
experiments (10) to exclude the effects of S1P contained in FBS.
S1P and dihydro-S1P were solubilized in methanol and stored at
Western Blot Analyses--
Protein expression and the degree of
protein phosphorylation were assayed by the Western blot analysis.
After drug treatments, cells in a 60-mm dish were washed with ice-cold
phosphate-buffered saline and incubated for 10 min on ice with 500 µl
of lysis buffer containing 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 150 mM NaCl, 1 mM Na3VO4,
2.5 mM sodium pyrophosphate, 1 mM
Immunokinase Assay--
The enzyme activity of Akt or ERK1/2 in
BAEC was determined in vitro by using commercially available
kinase activity kits following the supplier's protocol. Briefly, an
equal amount (70 µg) from each cell lysate described above was
adjusted to 200 µl with cell lysis buffer. Akt or ERK1/2 was
immunoprecipitated by using an immobilized anti-Akt or anti-ERK1/2
antibody conjugated to agarose beads for 2 h at 4 °C. After
being washed extensively, the beads were incubated with 40 µl of
kinase buffer comprising 25 mM Tris, pH 7.5, 5 mM Quantitation of Intracellular NO Generation--
eNOS enzyme
activity was quantified as the formation of
L-[3H]citrulline from
L-[3H]arginine, essentially as described
previously (18, 35). Briefly, cells in a given well of a six-well plate
were incubated in 800 µl of buffer containing 25 mM
HEPES, pH 7.3, 109 mM NaCl, 5.4 mM KCl, 0.9 mM CaCl2, 1 mM MgSO4,
and 25 mM glucose for 1 h at 37 °C. eNOS activity
was assayed by adding a mixture of unlabeled 10 µM
L-arginine, 10 µCi/ml L-[3H]arginine,
and various concentrations of S1P or vehicle to the culture (each
treatment was performed in triplicate cultures, and then each was
assayed in duplicate). Following incubation at 37 °C for 10 min,
cells were washed with ice-cold phosphate-buffered saline, scraped into
2 ml of solution containing 20 mM sodium acetate, 1 mM L-citrulline, 2 mM EDTA, and 2 mM EGTA, pH 5.5, followed by sonication. An aliquot was
withdrawn for determination of the total protein content and total
cellular 3H incorporation, and the remaining sample was
applied to Dowex 50W-X8 400 column to separate
L-[3H]citrulline. The flow-through fraction
was analyzed by liquid scintillation counting;
L-[3H]citrulline formation in the cells was
expressed as fmol of L-[3H]citrulline
produced/mg of cellular protein/min.
Other Methods--
All experiments were performed at least three
times. Mean values for individual experiments are expressed as
mean ± S.E. Statistical differences were assessed by analysis of
variance followed by Scheffe's F test using STAT VIEW II
(Abacus Concepts). A p value less than 0.05 was considered
statistically significant.
We first studied the responses of BAEC to S1P by analyzing the
effects of S1P on activation of protein kinases Akt and ERK1/2 (Fig.
1). We exploited two independent
experimental approaches to assess Akt or ERK activation in BAEC: we
analyzed immunoblots probed with antibodies that specifically detect
the phosphorylated (activated) forms of these kinases, and we also
performed in vitro kinase activity assays using protein
substrates specific for kinases Akt and ERK. Note that the activation
of these kinases is accompanied by the phosphorylation of specific
amino acid residues in these proteins (25, 36). After the addition of
100 nM S1P to BAEC, we detected increases in Akt
phosphorylation as well as Akt enzyme activity within 1 min, reaching a
maximum ~4-fold increase in activity by 2-5 min, with a gradual
return to basal levels seen at 120 min following drug addition (Fig.
1). Immunoblot analyses probed with an anti-Akt antibody were used to
verify that these cell lysates contain equivalent amounts of Akt
protein. The MAP kinases ERK1/2 also undergo robust and rapid
phosphorylation and kinase activation after the addition of S1P to
BAEC; ERK1/2 activity and phosphorylation levels returned to the basal
within 20 min following S1P addition (Fig. 1). In these same cell
lysates from S1P-treated BAEC, we analyzed immunoblots probed with an
antibody raised against a phosphopeptide comprising the eNOS amino acid sequence around Ser1179, the site at which eNOS
undergoes phosphorylation by kinase Akt (20, 21). As shown in Fig. 1,
S1P treatment of BAEC markedly increased eNOS phosphorylation at
Ser1179, with a time course similar to that seen for Akt
activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C; the same volume of methanol was used as a vehicle-control, and the final concentration of methanol did not exceed 0.4% (v/v) in
any experiment. PD98059, wortmannin, and BAPTA were solubilized in
dimethyl sulfoxide and kept at
20 °C; where indicated, dimethyl sulfoxide (0.1% v/v) was used as a vehicle-control.
-glycerophosphate, and a mixture of protease inhibitors (as
described in Ref. 31). Cells were harvested by scraping and centrifuged
at 4 °C for 10 min in a microfuge. The supernatant was analyzed for
protein concentration, and an equal amount of cellular proteins (20 µg/lane) was separated by SDS-PAGE and transferred to a
nitrocellulose membrane. The membrane was then probed with the
appropriate primary antibody. Membrane-bound primary antibodies were
visualized by using secondary antibodies conjugated with horseradish
peroxidase and Super Signal substrate, as described previously (32).
Densitometric analyses of Western blots were performed using a
ChemiImager 4000 (Alpha-Innotech).
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM
MgCl2, 200 µM ATP, and 0.5 µg of
recombinant glycogen synthase kinase-3 protein (for the Akt
assay) or 1 µg of Elk-1 protein (for ERK assay) for 30 min at
30 °C. Note that glycogen synthase kinase-3 and Elk-1 are well
established substrates for Akt (33) and for ERK (34), respectively, and
serve here as substrates in the BAEC-derived protein kinase reaction.
The reaction was terminated by adding 13 µl of 4 × SDS sample
buffer and boiling. The resulting reaction mixture was then separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed for
the phosphorylated form of glycogen synthase kinase-3 or Elk-1 by
Western blot analysis, as described above. The degree of enzyme activation was determined as a fold increase of phosphorylation in each
substrate protein relative to basal kinase activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
S1P-induced activation of Akt, ERK1/2, and
Ser1179 eNOS phosphorylation.
A shows the results of immunoblots and immunokinase assays
analyzed in BAEC lysates prepared from cells treated with 100 nM S1P for the indicated times. Cell lysates (20 µg/lane)
were resolved by SDS-PAGE and probed using antibodies directed against
phospho-Akt, Akt, phospho-ERK1/2, and phospho-eNOS
(Ser1179) antibodies. Signals corresponding to the proteins
indicated on the left of each panel are shown. The same cell
lysates (70 µg/lane) were also subjected to immunokinase assays as
described in detail under "Experimental Procedures." Akt or ERK1/2
was immunoprecipitated from BAEC lysates and incubated with their
corresponding substrate proteins in the presence of ATP. The reaction
mixture was then resolved by SDS-PAGE, transferred to a nitrocellulose
membrane, and probed with the antibodies specific to phosphorylated
forms of the substrate proteins. Signals corresponding to
phosphoglycogen synthase kinase-3 (Akt substrate) and phospho-Elk-1
(ERK1/2 substrate) are shown as Akt activity and ERK
activity, respectively. Shown are the representative data of four
independent experiments. B shows the results of
densitometric analyses from pooled data, plotting the fold increase of
the degree of phosphorylation of Akt, ERK1/2, and eNOS at the time
indicated, relative to the signals present at t = 0. Each data point represents the mean ± S.E. derived from four
independent experiments.
We next analyzed the dose response to S1P for Akt and ERK1/2
activation as well as Ser1179-phospho-eNOS formation in
BAEC treated for 5 min with increasing concentrations of S1P. Fig.
2A shows immunoblots of cell
lysates probed with antibodies directed against phospho-Akt (top
panel), phospho-ERK1/2 (middle panel) or
Ser1179-phospho-eNOS (lower panel). S1P-induced
activation of both Akt and ERK1/2, as well as
Ser1179-phospho-eNOS formation, all showed similar
EC50 values of ~10 nM (Fig. 2B).
Treatment of BAEC with the S1P analog dihydro-S1P (which acts solely
via EDG receptors (15)) led to Akt phosphorylation as well as
Ser1179-phospho-eNOS formation (data not shown). Together,
these data indicate that S1P induces the reversible receptor-mediated
activation of Akt and ERK1/2 and also promotes the synthesis of
Ser1179-phospho-eNOS, with EC50 values in a
physiologic range.
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To characterize further the S1P-induced Akt and ERK activation as well
as Ser1179-phospho-eNOS formation, BAEC were treated with
various inhibitors prior to the addition of S1P. As shown in Fig.
3, pretreatment with pertussis toxin
completely abolished S1P-induced Akt and ERK1/2 activation as well as
Ser1179-phospho-eNOS synthesis, indicating that the effects
of S1P are dependent upon the activation of pertussis toxin-sensitive G
protein pathways. Interestingly, the intracellular calcium chelator
BAPTA completely inhibited Akt activation and
Ser1179-phospho-eNOS synthesis, suggesting that activation
of the PI3-K/Akt pathway by S1P is dependent upon the elevation of
intracellular calcium concentration (Fig. 3). In contrast, BAPTA did
not block S1P-induced ERK1/2 activation; rather, calcium chelation with BAPTA led to a marked increase in the phosphorylation of
these MAP kinase pathway proteins. Wortmannin, an inhibitor of PI3-K (an upstream activator of Akt), blocked S1P-mediated Akt activation and
Ser1179-phospho-eNOS formation but did not inhibit
S1P-mediated activation of ERK1/2 (Fig. 3). In contrast to the effects
of wortmannin, the kinase inhibitor PD98059 (which inhibits the MAP
kinase kinase MEK, which in turn phosphorylates ERK1/2), abolished
ERK1/2 activation by S1P, but blocked neither S1P-mediated activation
of Akt nor Ser1179-phospho-eNOS synthesis (Fig. 3). These
experiments demonstrate that the S1P-mediated activation of Akt and
ERK1/2 and the formation of Ser1179-phospho-eNOS are
mediated by pertussis toxin-sensitive G proteins and that Akt
activation and Ser1179-phospho-eNOS formation are
calcium-dependent, whereas ERK1/2 activation is not.
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We next examined features of S1P-mediated eNOS activation in BAEC. Fig.
4A shows the results of a S1P
dose-response experiment in which cellular eNOS activity was
quantitated by measuring the formation of
L-[3H]citrulline in BAEC loaded with
L-[3H]arginine, applying a well characterized
assay for cellular NO synthesis (35). The NOS inhibitor
L-N-nitroarginine completely
abolishes S1P-induced NO synthesis in BAEC (Fig. 4B).
S1P-mediated eNOS activation is also blocked by the calcium chelator
BAPTA and by pertussis toxin treatment, as shown in Fig. 4. The PI3-K
inhibitor wortmannin, which completely abolishes eNOS phosphorylation
(Fig. 3), attenuates but does not completely abrogate S1P-stimulated
eNOS activation (Fig. 4B); S1P-mediated eNOS activation is
reduced by 70%, but residual S1P-augmented enzyme activity remains
above basal (p < 0.05). PD98059 is without effect,
suggesting that S1P-induced ERK1/2 activation (Fig. 3) does not
modulate eNOS in these experimental settings (Fig. 4B).
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We explored the relationships between S1P-induced kinase activation and
S1P-modulated eNOS activation further by performing transient
transfection experiments using cDNA constructs encoding eNOS and
EDG-1 in COS-7 cells, following a cotransfection protocol described
previously (18). It is of note that COS-7 cells do not express
endogenous EDG-1 receptors (37). We studied wild-type eNOS cDNA as
well as an eNOS mutant in which the putative kinase Akt phosphorylation
site Ser1179 is changed to Ala (S1179A) (20-22) in
cotransfection experiments with a cDNA construct encoding the EDG-1
receptor epitope-tagged with FLAG peptide (FLAG/EDG-1). Note that this
epitope-tagged receptor construct retains essential features of
wild-type EDG-1 receptor (5, 16, 18, 38). COS-7 cells transiently
expressing FLAG/EDG-1 and/or eNOS cDNAs were treated with 100 nM S1P for 5 min; cell lysates were probed by Western blot
analysis with various antibodies, as shown in Fig.
5. The overall expression of endogenous
kinase Akt protein was unaffected by cDNA transfections; expression
levels of the variously transfected cDNAs were concordant with the
specific cDNA constructs used in these experiments (Fig. 5A). S1P treatment induces the phosphorylation (activation)
of Akt only in cells transfected with the EDG-1 cDNA. Similarly, Ser1179-phospho-eNOS formation is observed only when S1P is
added to cells cotransfected with EDG-1 and wild-type eNOS cDNAs.
Importantly, when cells are cotransfected with EDG-1 and the S1179A
mutant eNOS, S1P-induced Ser1179-phospho-eNOS formation was
not detected despite robust phosphorylation of Akt (Fig.
5A). We next examined the effects of S1P on eNOS activation
in these cells. COS-7 cells transiently expressing FLAG/EDG-1 and eNOS
cDNAs (wild-type or S1179A) were incubated with
L-[3H]arginine and treated with S1P or
vehicle; cells were harvested, and lysates were analyzed for
L-[3H]citrulline formation as described
above. As we have observed previously (18), S1P activates eNOS in cells
cotransfected with FLAG/EDG-1 and wild-type eNOS, with an
EC50 of ~20 nM (Fig. 5B). In cells
cotransfected with the S1179A eNOS mutant plus EDG-1, there is a
markedly attenuated response to S1P compared with wild-type eNOS (Fig.
5B), despite the fact that these cells express eNOS protein
at equivalent levels (Fig. 5A).
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We explored next the differential regulation of S1P-mediated responses
by comparing directly the signaling pathways elicited by S1P with those
activated by bradykinin, a well known activator of eNOS in BAEC (39).
As shown in Fig. 6A, 1 µM bradykinin induces the activation (phosphorylation) of
ERK1/2 in BAEC, as we have reported previously (Fig. 6A
(22)). However, bradykinin does not induce the activation of
Akt or formation of Ser1179-phospho-eNOS, although the same
cell preparations respond to S1P (Fig. 6A). We compared
bradykinin and S1P-mediated eNOS activation in BAEC, as shown in Fig.
6B. Although bradykinin and S1P both activate eNOS to a
similar extent, bradykinin-induced eNOS activation is not inhibited by
pertussis toxin nor by wortmannin, in contrast to the inhibitory
effects of these agents on S1P-mediated eNOS activation. PD98059 has no
substantive effect on bradykinin-dependent eNOS activation
in this experimental system. It is notable that bradykinin and S1P
activate eNOS to a similar magnitude (Fig. 6B). When cells
are treated with both bradykinin and S1P in combination, there is no
additive increase in eNOS activity compared with cells treated
individually with bradykinin or S1P.
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DISCUSSION |
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These studies have provided several lines of evidence demonstrating that S1P induces eNOS activation in cultured endothelial cells, at least in part mediated by Ser1179 eNOS phosphorylation and involving the PI3-K/Akt pathway. As shown in Fig. 1, the addition of S1P rapidly activates Akt kinase and ERK1/2, concomitant with eNOS phosphorylation on serine 1179. S1P-mediated Akt activation and Ser1179 eNOS phosphorylation persist for up to 2 h, whereas ERK1/2 phosphorylation returns to basal levels within 20 min, suggesting that activation of the MAP kinase pathway may be involved in shorter term regulation of eNOS than the PI3-K/Akt pathway (Fig. 1). Dose-response experiments (Fig. 2) show that the effects of S1P on activation of Akt, ERK1/2, and eNOS 1179Ser phosphorylation all show a similar EC50 of ~10 nM. Our observation of an EC50 for S1P-mediated eNOS activation in the low nanomolar range is consistent with values observed previously for other S1P-mediated responses (5). Concentrations of S1P in human blood have been measured in the range of several hundred nanomolar (40). However, the extent to which extracellular S1P may be protein-bound (or otherwise complexed) has not been rigorously determined, such that the concentration of free "biologically active" S1P available for interaction with EDG receptors may be considerably lower. Moreover, local blood concentrations of S1P may increase acutely when the compound is released from activated platelets (4, 40), suggesting that levels of S1P may be subject to physiological regulation. Thus, the potency of S1P in modulating eNOS signaling pathways in endothelial cells indicates that this compound may dynamically regulate NO-dependent signal transduction pathways in the vascular wall in physiological or pathophysiological states.
S1P-mediated activation of Akt, ERK1/2 and 1179Ser eNOS
phosphorylation alike are completely blocked by pertussis toxin (Fig. 3), indicating that these signaling responses are all mediated by
pertussis toxin-sensitive G protein pathways in endothelial cells.
These results demonstrate that the responses to S1P are mediated by G
protein-coupled cell surface (EDG) receptors rather than via
intracellular mechanisms. Many of EDG receptor-mediated signaling
events involve the activation of pertussis toxin-sensitive G proteins,
but it has not been reported previously that S1P activates the
PI3-K/Akt pathway. Other G protein-coupled receptors have been observed
to involve the PI3-K/Akt pathway (41-43), and recent studies have
provided insight into the mechanisms whereby G protein subunits
modulate PI3-K activity. For example, the PI3-K catalytic subunit
P110, together with its regulatory subunit P101, can be activated by
G protein
subunits, leading to PI3-K activation (44). Other
investigators have shown more recently that PI3-K catalytic subunit
P110
can directly respond to the G protein
subunit (45).
Interestingly, lysophosphatidic acid, another ligand for EDG receptors,
induces PI3-K/Akt activation in COS-7 cells (42). Taken together with
our observations, these studies suggest that endothelial cells have
signal transduction machinery that connects EDG receptor stimulation
with activation of the PI3-K/Akt pathway.
We were surprised to find that the intracellular calcium chelator BAPTA abrogated Akt activation in BAEC (Fig. 3). It is unlikely that this effect of BAPTA reflects nonspecific or toxic effects of the compound because identical treatments with BAPTA did not block S1P-mediated ERK1/2 activation. To our knowledge, there has been no prior report documenting that the activation of PI3-K/Akt by G protein-coupled receptors is calcium-dependent. However, the fact that BAPTA abrogates Akt activation (and Ser1179 eNOS phosphorylation) suggests that elevation of intracellular calcium concentration may be required for S1P-mediated activation of the PI3-K/Akt pathway in endothelial cells. Interestingly, BAPTA did not abolish ERK1/2 phosphorylation but instead reproducibly induced hyperphosphorylation of these kinases (Fig. 3). The differential effects of BAPTA on ERK1/2 versus Akt pathways may indicate the divergence of signaling pathways downstream from S1P-mediated EDG receptor activation in endothelial cells. However, it must be also noted that different EDG receptor subtypes may have significantly different pharmacological properties in response to the same lipid ligands (38, 46, 47). Although we have shown that the EDG-1 receptor subtype is fully able to activate Akt and eNOS in transfected COS-7 cells (Fig. 5), it is quite plausible that BAEC express more than one EDG receptor isoform (9, 10). Thus, other EDG receptors such as EDG-3 may also be able to activate Akt and/or eNOS in endothelial cells.
The PI3-K inhibitor wortmannin inhibited S1P-mediated Akt activation and Ser1179 eNOS phosphorylation while having no substantive effect on S1P-mediated ERK1/2 activation (Fig. 3). By contrast, the MEK inhibitor PD98059 blocked ERK1/2 activation while having no substantive effect on S1P-mediated Akt activation, Ser1179 eNOS phosphorylation (Fig. 3), or eNOS enzyme activity (Fig. 4). Thus in these experimental settings, S1P-induced MAP kinase activation does not seem to play a major role in eNOS regulation. Nonetheless, MAP kinase activation by S1P plays many other important roles, for example, the formation of cadherin-based cell-cell junctions (5, 17) or cell migration (9, 10) in vascular endothelial cells.
The present study provides the first evidence that G protein-coupled receptor stimulation can lead to Akt-dependent eNOS phosphorylation. However, not all of S1P's effect on eNOS activation is the result of Ser1179 eNOS phosphorylation: as shown in Fig. 5, the S1179A eNOS mutant still undergoes S1P-dependent activation (albeit less than the wild-type enzyme), and the PI3-K inhibitor wortmannin only partially blocks S1P-induced eNOS activation (Fig. 4), whereas this inhibitor completely blocks S1P-induced Akt activation (Fig. 3). These results also point up key differences between two different G protein-coupled receptors, the EDG receptor pathway activated by S1P, and the B2 receptor pathway activated by bradykinin. In contrast to S1P, bradykinin does not activate Akt kinase nor promote Ser1179 eNOS phosphorylation (Fig. 6). However, bradykinin still promotes robust activation of eNOS to levels comparable to S1P without appearing to involve the PI3-K pathway.
S1P and EDG receptors are implicated in signal transduction pathways leading to angiogenesis (17). Interestingly, eNOS and PI3-K/Akt are also implicated independently in angiogenesis. For example, angiogenic polypeptide growth factors such as vascular endothelial growth factor lead to eNOS enzyme activation (48, 49), and overexpression of a dominant-negative PI3-K construct markedly attenuates angiogenesis (27). Moreover, these signaling molecules share common features of their subcellular localization: plasmalemmal caveolae serve as the sites for localization of eNOS (19), EDG-1 receptor (18), as well as members of the PI3-K/Akt pathway (50). The present study establishes that S1P activates eNOS as well as the PI3-K/Akt pathway and may implicate this signaling system in regulation of angiogenic responses in vascular endothelial cells.
Remarkable differences between S1P and bradykinin-mediated regulation
of PI3-K/Akt, MAP kinase, and eNOS may identify additional points for
control of nitric oxide-dependent signal transduction pathways in vascular endothelial cells. Furthermore, it is notable that
S1P, which had not been previously identified as an activator of NO
synthesis in endothelial cells, has been shown in these studies to
activate eNOS to a level comparable to that elicited by bradykinin.
These cellular responses to S1P occur at concentrations well within the
physiological range of this sphingolipid. Taken together, our
observations suggest that S1P may have an important role in the
modulation of NO-dependent signaling in the vasculature.
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ACKNOWLEDGEMENT |
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We thank Dr. Timothy Hla for providing cDNA encoding FLAG/EDG-1.
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FOOTNOTES |
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* This work was supported in part by awards and grants (to T. M.) from the National Institutes of Health and the Burroughs Wellcome Fund.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.
§ Recipient of a Thomas W. Smith postdoctoral research fellowship award (Brigham and Women's Hospital, 1999-2000).
¶ Supported by a fellowship award from the Heart and Stroke Foundation of Canada. Present address: Research Center of Hospital Saint Justine, University of Montreal, Quebec, Canada H3T 1C5.
** To whom correspondence should be addressed: Cardiovascular Division, Brigham and Women's Hospital, Thorn Bldg., Rm. 1210A, 75 Francis St., Boston, MA 02115. Tel.: 617-732-7376; Fax: 617-732-5132; E-mail: michel@calvin.bwh.harvard.edu.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M008375200
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ABBREVIATIONS |
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The abbreviations used are: S1P, sphingosine 1-phosphate; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAP/ERK kinase; eNOS, endothelial nitric-oxide synthase; PI3-K, phosphoinositide 3-kinase; FBS, fetal bovine serum; dihydro-S1P, sphinganine 1-phosphate; BAPTA, 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; FLAG/EDG-1, EDG-1 receptor epitope-tagged with FLAG peptide; PAGE, polyacrylamide gel electrophoresis.
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