Sphingosine 1-Phosphate Activates Akt, Nitric Oxide Production, and Chemotaxis through a Gi Protein/Phosphoinositide 3-Kinase Pathway in Endothelial Cells*

Manuel Morales-RuizDagger , Menq-Jer Lee§, Stefan ZöllnerDagger , Jean-Philippe GrattonDagger , Ramona ScotlandDagger , Ichiro Shiojima||, Kenneth Walsh||, Timothy Hla§**, and William C. SessaDagger DaggerDagger

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>−</SUP></UP>, the stable breakdown product of NO, from BLMVECs treated with or without SPP (10-500 nM), LY294002 (10 µM), L-N-mono-methyl-arginine (L-NMMA) (1 mM), or PTx (200 ng/ml). Cells, plated on 60-mm2 dishes were equilibrated for 30 min at 37 °C in Dulbecco's modified Eagle's medium without fetal bovine serum. To stimulate NO release, test reagents were added for 30 min, and the supernatant was collected for analysis of NO by chemiluminescence. Samples (10 µl) containing NO<UP><SUB>2</SUB><SUP>−</SUP></UP> were refluxed in glacial acetic acid containing sodium iodide. Under these conditions, NO<UP><SUB>2</SUB><SUP>−</SUP></UP> was quantitatively reduced to NO, which was quantified by a chemiluminescence detector after reaction with ozone in a NO analyzer (Sievers). Net NO<UP><SUB>2</SUB><SUP>−</SUP></UP> per µg of protein was calculated after subtracting background levels of NO<UP><SUB>2</SUB><SUP>−</SUP></UP> found in the media. In experiments confirming the involvement of Gi protein or PI 3-kinase, we pre-incubated the cells with LY294002 (10 µM for 1 h) or PTx (200 ng/ml for 6 h) in Dulbecco's modified Eagle's medium without fetal bovine serum. In some experiments, BLMVECs were infected with an adenovirus that expresses dominant negative Akt, which was hemagglutinin (HA)-tagged, and contained mutations at K179A, T308A, and S473A (AAA-Akt) or a green fluorescent protein (GFP)-expressing virus. Endothelial cells were infected with adenovirus (multiplicity of infection of 25) containing GFP or AAA-Akt for 4 h. The virus was removed, and cells were left to recover for 12 h in complete medium. These conditions resulted in uniform expression of the transgenes in close to 100% of the cells (determined by GFP expression in living cells).

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (69K):
[in this window]
[in a new window]
 
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).

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.


View larger version (11K):
[in this window]
[in a new window]
 
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.

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.


View larger version (23K):
[in this window]
[in a new window]
 
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.

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).


View larger version (26K):
[in this window]
[in a new window]
 
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<UP><SUB>2</SUB><SUP>−</SUP></UP> per µg of protein after subtracting background levels of NO<UP><SUB>2</SUB><SUP>−</SUP></UP> found in the media. Similar results were obtained in three additional experiments. B, serum-starved BLMVECs were pre-treated without or with 10 µM LY294002 for 1 h or 200 ng/ml PTx for 6 h and then stimulated with 500 nM SPP at different times. Cell lysates were analyzed by Western blotting (80 µg) with antibodies to phospho-eNOS (upper panel) or total eNOS (lower panel). Representative results from three separate experiments are shown. D.units reflects relative densitometric units of p-eNOS to total eNOS.

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.


View larger version (32K):
[in this window]
[in a new window]
 
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.

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.


View larger version (16K):
[in this window]
[in a new window]
 
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

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 p110gamma , 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 alpha vbeta 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.

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.

    ACKNOWLEDGEMENT

We thank Genentech for the generous supply of VEGF.

    FOOTNOTES

* 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.

Dagger Dagger 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Spiegel, S., and Merrill, A. H., Jr. (1996) FASEB J. 10, 1388-1397[Abstract/Free Full Text]
2. Hla, T., and Maciag, T. (1990) J. Biol. Chem. 265, 9308-9313[Abstract/Free Full Text]
3. MacLennan, A. J., Browe, C. S., Gaskin, A. A., Lado, D. C., and Shaw, G. (1994) Mol. Cell. Neurosci. 5, 201-209[CrossRef][Medline] [Order article via Infotrieve]
4. Graler, M. H., Bernhardt, G., and Lipp, M. (1999) Curr. Top. Microbiol. Immunol. 246, 131-136[Medline] [Order article via Infotrieve]
5. Im, D. S., Heise, C. E., Ancellin, N., O'Dowd, B. F., Shei, G. J., Heavens, R. P., Rigby, M. R., Hla, T., Mandala, S., McAllister, G., George, S. R., and Lynch, K. R. (2000) J. Biol. Chem. 275, 14281-14286[Abstract/Free Full Text]
6. Pyne, S., and Pyne, N. J. (2000) Biochem. J. 349, 385-402[CrossRef][Medline] [Order article via Infotrieve]
7. Okamoto, H., Takuwa, N., Gonda, K., Okazaki, H., Chang, K., Yatomi, Y., Shigematsu, H., and Takuwa, Y. (1998) J. Biol. Chem. 273, 27104-27110[Abstract/Free Full Text]
8. Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167[Abstract]
9. Lee, O. H., Kim, Y. M., Lee, Y. M., Moon, E. J., Lee, D. J., Kim, J. H., Kim, K. W., and Kwon, Y. G. (1999) Biochem. Biophys. Res. Commun. 264, 743-750[CrossRef][Medline] [Order article via Infotrieve]
10. Boguslawski, G., Lyons, D., Harvey, K. A., Kovala, A. T., and English, D. (2000) Biochem. Biophys. Res. Commun. 272, 603-609[CrossRef][Medline] [Order article via Infotrieve]
11. Wang, F., Van Brocklyn, J. R., Hobson, J. P., Movafagh, S., Zukowska-Grojec, Z., Milstien, S., and Spiegel, S. (1999) J. Biol. Chem. 274, 35343-35350[Abstract/Free Full Text]
12. Lee, M. J., Thangada, S., Claffey, K. P., Ancellin, N., Liu, C. H., Kluk, M., Volpi, M., Sha'afi, R. I., and Hla, T. (1999) Cell 99, 301-312[Medline] [Order article via Infotrieve]
13. Liu, Y., Wada, R., Yamashita, T., Mi, Y., Deng, C. X., Hobson, J. P., Rosenfeldt, H. M., Nava, V. E., Chae, S. S., Lee, M. J., Liu, C. H., Hla, T., Spiegel, S., and Proia, R. L. (2000) J. Clin. Invest. 106, 951-961[Abstract/Free Full Text]
14. Rakhit, S., Conway, A. M., Tate, R., Bower, T., Pyne, N. J., and Pyne, S. (1999) Biochem. J. 338, 643-649[CrossRef][Medline] [Order article via Infotrieve]
15. Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997) Cell 88, 435-437[Medline] [Order article via Infotrieve]
16. Downward, J. (1998) Curr. Opin. Cell Biol. 10, 262-267[CrossRef][Medline] [Order article via Infotrieve]
17. Fulton, D., Gratton, J. P., McCabe, T. J., Fontana, J., Fujio, Y., Walsh, K., Franke, T. F., Papapetropoulos, A., and Sessa, W. C. (1999) Nature 399, 597-601[CrossRef][Medline] [Order article via Infotrieve]
18. Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., and Zeiher, A. M. (1999) Nature 399, 601-605[CrossRef][Medline] [Order article via Infotrieve]
19. Michell, B. J., Griffiths, J. E., Mitchelhill, K. I., Rodriguez-Crespo, I., Tiganis, T., Bozinovski, S., de Montellano, P. R., Kemp, B. E., and Pearson, R. B. (1999) Curr. Biol. 9, 845-848[CrossRef][Medline] [Order article via Infotrieve]
20. Papapetropoulos, A., Fulton, D., Mahboubi, K., Kalb, R. G., O'Connor, D. S., Li, F., Altieri, D. C., and Sessa, W. C. (2000) J. Biol. Chem. 275, 9102-9105[Abstract/Free Full Text]
21. Fujio, Y., and Walsh, K. (1999) J. Biol. Chem. 274, 16349-16354[Abstract/Free Full Text]
22. Gerber, H. P., McMurtrey, A., Kowalski, J., Yan, M., Keyt, B. A., Dixit, V., and Ferrara, N. (1998) J. Biol. Chem. 273, 30336-30343[Abstract/Free Full Text]
23. Rossig, L., Fichtlscherer, B., Breitschopf, K., Haendeler, J., Zeiher, A. M., Mulsch, A., and Dimmeler, S. (1999) J. Biol. Chem. 274, 6823-6826[Abstract/Free Full Text]
24. Morales-Ruiz, M., Fulton, D., Sowa, G., Languino, L. R., Fujio, Y., Walsh, K., and Sessa, W. C. (2000) Circ. Res. 86, 892-896[Abstract/Free Full Text]
25. Radisavljevic, Z., Avraham, H., and Avraham, S. (2000) J. Biol. Chem. 275, 20770-20774[Abstract/Free Full Text]
26. Dimmeler, S., Dernbach, E., and Zeiher, A. M. (2000) FEBS Lett. 477, 258-262[CrossRef][Medline] [Order article via Infotrieve]
27. Noiri, E., Lee, E., Testa, J., Quigley, J., Colflesh, D., Keese, C. R., Giaever, I., and Goligorsky, M. S. (1998) Am. J. Physiol. 274, C236-C244[Medline] [Order article via Infotrieve]
28. Murohara, T., Asahara, T., Silver, M., Bauters, C., Masuda, H., Kalka, C., Kearney, M., Chen, D., Symes, J. F., Fishman, M. C., Huang, P. L., and Isner, J. M. (1998) J. Clin. Invest. 101, 2567-2578[Abstract/Free Full Text]
29. Kureishi, Y., Luo, Z., Shiojima, I., Bialik, A., Fulton, D., Lefer, D. J., Sessa, W. C., and Walsh, K. (2000) Nat. Med. 6, 1004-1010[CrossRef][Medline] [Order article via Infotrieve]
30. Sessa, W. C., Garcia-Cardena, G., Liu, J., Keh, A., Pollock, J. S., Bradley, J., Thiru, S., Braverman, I. M., and Desai, K. M. (1995) J. Biol. Chem. 270, 17641-17644[Abstract/Free Full Text]
31. Garcia-Cardena, G., Fan, R., Stern, D. F., Liu, J., and Sessa, W. C. (1996) J. Biol. Chem. 271, 27237-27240[Abstract/Free Full Text]
32. Sowa, G., Liu, J., Papapetropoulos, A., Rex-Haffner, M., Hughes, T. E., and Sessa, W. C. (1999) J. Biol. Chem. 274, 22524-22531[Abstract/Free Full Text]
33. Vanhaesebroeck, B., Leevers, S. J., Panayotou, G., and Waterfield, M. D. (1997) Trends Biochem. Sci. 22, 267-272[CrossRef][Medline] [Order article via Infotrieve]
34. Yamamura, S., Yatomi, Y., Ruan, F., Sweeney, E. A., Hakomori, S., and Igarashi, Y. (1997) Biochemistry 36, 10751-10759[CrossRef][Medline] [Order article via Infotrieve]
35. Wang, F., Nohara, K., Olivera, A., Thompson, E. W., and Spiegel, S. (1999) Exp. Cell Res. 247, 17-28[CrossRef][Medline] [Order article via Infotrieve]
36. Kimura, T., Watanabe, T., Sato, K., Kon, J., Tomura, H., Tamama, K., Kuwabara, A., Kanda, T., Kobayashi, I., Ohta, H., Ui, M., and Okajima, F. (2000) Biochem. J. 348, 71-76[CrossRef][Medline] [Order article via Infotrieve]
37. Kon, J., Sato, K., Watanabe, T., Tomura, H., Kuwabara, A., Kimura, T., Tamama, K., Ishizuka, T., Murata, N., Kanda, T., Kobayashi, I., Ohta, H., Ui, M., and Okajima, F. (1999) J. Biol. Chem. 274, 23940-23947[Abstract/Free Full Text]
38. Li, Z., Jiang, H., Xie, W., Zhang, Z., Smrcka, A. V., and Wu, D. (2000) Science 287, 1046-1049[Abstract/Free Full Text]
39. Sasaki, T., Irie-Sasaki, J., Jones, R. G., Oliveira-dos-Santos, A. J., Stanford, W. L., Bolon, B., Wakeham, A., Itie, A., Bouchard, D., Kozieradzki, I., Joza, N., Mak, T. W., Ohashi, P. S., Suzuki, A., and Penninger, J. M. (2000) Science 287, 1040-1046[Abstract/Free Full Text]
40. Hirsch, E., Katanaev, V. L., Garlanda, C., Azzolino, O., Pirola, L., Silengo, L., Sozzani, S., Mantovani, A., Altruda, F., and Wymann, M. P. (2000) Science 287, 1049-1053[Abstract/Free Full Text]
41. Zheng, D. Q., Woodard, A. S., Tallini, G., and Languino, L. R. (2000) J. Biol. Chem. 275, 24565-24574[Abstract/Free Full Text]
42. Igarashi, J., and Michel, T. (2000) J. Biol. Chem. 275, 32363-32370[Abstract/Free Full Text]
43. Fisslthaler, B., Dimmeler, S., Hermann, C., Busse, R., and Fleming, I. (2000) Acta Physiol. Scand. 168, 81-88[CrossRef][Medline] [Order article via Infotrieve]
44. Haynes, M. P., Sinha, D., Russell, K. S., Collinge, M., Fulton, D., Morales-Ruiz, M., Sessa, W. C., and Bender, J. R. (2000) Circ. Res. 87, 677-682[Abstract/Free Full Text]
45. McCabe, T. J., Fulton, D., Roman, L. J., and Sessa, W. C. (2000) J. Biol. Chem. 275, 6123-6128[Abstract/Free Full Text]
46. Forstermann, U., Gorsky, L. D., Pollock, J. S., Ishii, K., Schmidt, H. H., Heller, M., and Murad, F. (1990) Mol. Pharmacol. 38, 7-13[Abstract]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.