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Am J Physiol Cell Physiol 292: C740-C748, 2007. First published August 30, 2006; doi:10.1152/ajpcell.00117.2006 Free Article
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VASCULAR BIOLOGY

Hydrogen peroxide induces S1P1 receptors and sensitizes vascular endothelial cells to sphingosine 1-phosphate, a platelet-derived lipid mediator

Junsuke Igarashi,1 Megumi Miyoshi,2 Takeshi Hashimoto,1 Yasuo Kubota,2 and Hiroaki Kosaka1

Department of 1Cardiovascular Physiology and 2Dermatology, Kagawa University Faculty of Medicine, Kagawa, Japan

Submitted 14 March 2006 ; accepted in final form 23 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sphingosine 1-phosphate (S1P) is a platelet-derived angiogenic lipid growth factor, modulating G-protein-coupled S1P1 receptors (S1P1-R) to activate endothelial nitric oxide synthase (eNOS), as well as MAPK pathways in endothelial cells. We explored whether and how hydrogen peroxide (H2O2), a representative reactive oxygen species, alters S1P1-R expression and influences S1P signaling in cultured bovine aortic endothelial cells (BAECs). When BAECs are treated with pathophysiologically relevant concentrations of H2O2 (150 µM for 30 min), S1P1-R protein expression levels are acutely augmented by ~30-fold in a dose-dependent fashion. When BAECs have been pretreated with H2O2, subsequent S1P stimulation (100 nM) leads to a higher degree of eNOS enzyme activation (assessed as intracellular cGMP content, 1.7 ± 0.2-fold vs. no H2O2 pretreatment groups, P < 0.05), associated with a higher magnitude of phosphorylation responses of eNOS and MAPK ERK1/2. PP2, an inhibitor of Src-family tyrosine kinase, abolished the effects of H2O2 on both S1P1-R protein upregulation and enhanced BAEC responses to S1P. H2O2 does not augment S1P1 mRNA expression, whereas VEGF under identical cultures leads to increases in S1P1 mRNA signals. Whereas H2O2 attenuates proliferation of BAECs, addition of S1P restores growth responses of these cells. These results demonstrate that extracellularly administered H2O2 increases S1P1-R expression and promotes endothelial responses for subsequent S1P treatment. These results may identify potentially important points of cross-talk between reactive oxygen species and sphingolipid pathways in vascular responses.

sphingolipids; G protein-coupled receptors; reactive oxygen species; signal transduction

SPHINGOSINE 1-PHOSPHATE (S1P) is a platelet-derived lipid mediator that elicits a wide variety of biological actions in numerous mammalian cell types (10). Specifically in vascular endothelium, S1P evokes such important responses as proliferation (21), migration (23), survival (22), vasorelaxation (6), and angiogenic responses (24), among others. These effects of S1P are largely mediated by binding to and activation of a novel family of G protein-coupled receptors termed S1P receptors (13) (previously known as EDG receptors). Stimulation of G protein-coupled S1P receptors in endothelial cells not only mobilizes intracellular calcium (23) but also activates several protein kinase cascades, including MAPK and phosphatidylinositol 3-kinase/Akt cascades (16, 19). Activation of these protein kinase cascades in turn leads to phosphorylation/activation of their cognate substrate proteins, including ERK1/2 and endothelial isoform of nitric oxide synthase (eNOS) (16, 19), ultimately to exhibit aforementioned endothelial responses to S1P. Among five S1P receptor subtypes, S1P1 receptors (S1P1-R) have been considered as the major subtype expressed in endothelial cells (14, 24, 27). The expression levels of S1P1-R are subjected to dynamic regulation by several extracellular stimuli, including phorbol esters such as PMA (14) as well as growth factors such as VEGF (17). Increases in the amounts of S1P1-R mRNA/protein by VEGF are associated with enhanced responses to subsequent S1P treatment (17), indicating that the degrees of S1P1-R expression and magnitudes of S1P actions are functionally coupled in these cells.

Reactive oxygen species (ROS) represent important modulators of vascular endothelial cells under various (patho)physiological conditions. For example, earlier studies established that activated neutrophils release high concentrations of ROS, leading to endothelial injury (reviewed in Ref. 35). Not only being solely toxic, ROS may also function as signaling molecules. More recent studies have been uncovering that ROS at lower concentrations may stimulate vascular endothelial cells to exert angiogenic responses (reviewed in Refs. 4, 26). It has been documented that both extracellularly produced ROS by adjacent cells to endothelium and intracellularly generated ones after receptor stimulation within endothelial cells are capable of inducing various signaling responses within endothelial cells (4, 26). It appeared therefore plausible to us that ROS might also influence sphingolipid signaling in vascular endothelial cells. In the present studies, we show that H2O2, a key ROS, increases amounts of S1P1-R, and modulates responses of vascular endothelial cells to subsequent treatments with S1P.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. FBS was from Hyclone (Logan, UT). All other cell culture reagents and media were from Life Technologies (Rockville, MD). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and VEGF were from Calbiochem (San Diego, CA). S1P was from BioMol (Plymouth Meeting, PA). Anti-S1P1 antibody, raised against NH2-terminal fragment peptide of rat EDG-1 (S1P1) (28), was a gift from Roger A. Sabbadini (San Diego State University). Anti-phospho-eNOS antibody (phosphoserine 1179 in bovine eNOS sequence), anti-phospho-ERK1/2 antibody (Thr202/Tyr204), anti-phospho-JNK antibody (Thr183/Tyr185), anti-phospho-p38 antibody (Thr180/Tyr182), anti-phospho-Akt antibody (Ser473), and anti-Akt polyclonal antibody were from Cell Signaling Technologies (Beverly, MA). Anti-eNOS monoclonal antibody was from Transduction Laboratories (Lexington, KY). Anti-KDR polyclonal antibody (A-3), anti-caveolin polyclonal antibody (sc-894), and protein A/G agarose beads were from Santa Cruz (Santa Cruz, CA). Anti-FLAG monoclonal antibody (M2) was from Sigma (St. Louis, MO). SuperBlock reagents, SuperSignal substrates for chemiluminescence detection, and secondary antibodies conjugated with horseradish peroxidase were from Pierce (Rockford, IL). RNeasy mini columns were from Qiagen (Valencia, CA). SuperScript RNase H– reverse transcriptase (RTase) was from Invitrogen (Carlsbad, CA). Taq DNA polymerase was from Promega (Madison, WI). Specific oligonucleotide primers directed to human, mouse, and rat GAPDH cDNA was from TOYOBO (Osaka, Japan). Other primers were from Hokkaido System Science (Hokkaido, Japan). cGMP assay kit was from Amersham (Piscataway, NJ). Cell counting assay kits using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-8) were from Dojindo (Kumamoto, Japan). Protein determinations were made with the Bio-Rad protein assay kit (Hercules, CA). All other materials were from Sigma.

Cell culture and drug treatments. Bovine aortic endothelial cells (BAECs) were obtained from Cell Systems (Kirkland, WA), maintained in culture as described, and used for experiments between passages 5 and 7 (17). Unless otherwise stated, cells had been incubated with DMEM containing 10% FBS for 4 days; they were then serum starved overnight before being used for experiments to exclude the effects of residual S1P in FBS. PP2 was dissolved into DMSO. All other drug treatments were performed exactly as described previously (17). Final concentrations of the solvents did not exceed 0.1% (wt/vol) in any experiment.

Immunoblot analyses in cultured cells. Immunoblot analyses were performed as described previously (17). Briefly, cells were harvested and lysed into cell lysis buffers, denatured, size-fractionated on SDS-PAGE gels, and transferred to nitrocellulose membranes. Resulting membranes were blocked and incubated with various primary antibodies, followed by incubation with corresponding horseradish peroxidase-conjugated secondary antibodies. Immunoreactive signals were visualized with chemiluminescence substrates with exposure to standard X-ray films (Fuji, Tokyo, Japan).

Semiquantitative RT-PCR analyses. Total RNA was isolated from BAECs using RNeasy mini columns, following supplier's protocol. cDNA was synthesized from 1 µg of cellular RNA using oligo(dT)18 primer and RTase, exactly following supplier's instruction in a total volume of 20 µl. Enzymatic amplification was conducted with a 1-µl aliquot of cDNA mix essentially as described (14). PCR was performed in 10 mM Tris·HCl (pH 9.0 at 25°C), 50 mM KCl, 0.1% Triton X-100, 25 mM MgCl2, 0.2 mM dNTPs, 0.2 µM each of primer pair for S1P1 (or that for GAPDH), and 25 U/ml of Taq DNA polymerase. The reaction mixture was heated at 94°C for 1 min, annealed at 55°C for 2 min, and extended at 72°C for 3 min for 22 repetitive cycles. The primers used were 5'-AAG ACC TGT GAC ATC CTC TTC-3' (sense) and 5'-ATG AAC CCT TTA GGA GCT TGA CAA-3' (antisense) to amplify from nucleotides 1100 to 1702 of human S1P1 (EDG-1) cDNA (14). PCR amplification for GAPDH was performed with commercially available primer pairs: 5'-ACC ACA GTC CAT GCC ATC AC-3' (sense) and 5'-TCC ACC ACC CTG TTG CTG TA-3' (antisense). The reaction mixture was heated at 94°C, annealed at 55°C, and extended at 72°C for 30 s each for 21 repetitive cycles. The PCR product was separated on a 2% agarose gel and visualized with ethidium bromide under UV. Gel image was captured with a charge-coupled device camera system and subjected to densitometric analyses using NIH Image software 1.63. We optimized the assay conditions and verified that increasing amounts of a starting mRNA sample yield increasing amounts of RT-PCR product under these conditions in each primer pair.

Quantitation of intracellular cGMP content. Nitric oxide production of BAECs was assessed as intracellular cGMP content (9). BAECs were treated with IBMX (1 mM) for 30 min before agonist stimulation. Cells were harvested into supplied lysis buffer and subjected to cGMP measurements exactly following supplier's protocol.

Cell proliferation assays. BAECs were seeded on six-well plates and maintained in culture for 24 h. Culture medium was changed to DMEM containing 0.5% FBS, and incubation proceeded for another 24 h. Cells were then stimulated with H2O2 for 30 min, followed by treatment with S1P in DMEM containing 1% FBS for 48 h. They were trypsinized and subjected to cell number counting using a particle analyzer CDA-500 (Kobe, Japan) following supplier's instruction. For experiments that used a cell counting assay kit, BAECs had been seeded on 96-well plates and were subjected to a treatment protocol with H2O2/S1P as above. The degrees of cellular proliferation were assessed as magnitudes of WST-8 reactions [an equivalent of dimethylthiazol-diphenyltetrazoliumbromide assay (29)], using a microplate reader.

Transient transfection, coimmunoprecipitation, and subcellular fractionation. BAECs plated on a 100-mm dish were transfected 48 h after they were split with 12 µg of plasmid DNA encoding FLAG-tagged S1P1 cDNA [FLAG/S1P1 (14, 18)] using Lipofectamine 2000 and OptiMEM (Invitrogen), following supplier's protocol. Six hours after the addition of plasmid DNA, culture medium was switched back to DMEM with 10% FBS. Two days after transfection, cells were serum starved as above and used for experiments.

BAECs that had been transfected with FLAG/S1P1 plasmid DNA were subjected to coimmunoprecipitation analyses as described previously (18), using a monoclonal antibody specific to FLAG peptide and a polyclonal antibody to caveolin-1, followed by immunoblot analyses.

In a separate series of experiments, BAECs were subjected to subcellular fractionation essentially as previously described (31). Briefly, cells were scraped into 1 ml of PBS and spun at 1,000 g for 5 min at 4°C. After the supernatants were discarded, pellets were resuspended into 500 µl of buffer 1-P [50 mM Tris·HCl (pH 7.6 at 25°C), 0.1 mM EDTA, 0.1 mM EGTA, 50 mM sodium fluoride, 2 mM beta-mercaptoethanol, and a cocktail of protease inhibitors]. Cell suspensions were sonicated (three 10-s cycles); 200 µl were saved as homogenate fractions. Remaining samples were spun at 23,500 g for 1 h at 4°C, and the resulting supernatants were saved as soluble fractions. Pellets were washed with buffer 1-P and spun for another 30 min. After the supernatants were discarded, pellets were resuspended into 300 µl of buffer 1-P and saved as particulate fractions. Protein sample buffer for S1P1-R immunoblot analyses containing 1.7% SDS and 100 mM DTT (600 µl; see Ref. 17 for detailed composition) was added to each fraction. Fractionated cellular lysates were passed through a 21-gauge needle, denatured by sonication (17, 20), separated on an acrylamide gel, and subjected to immunoblot analyses as described above.

Other methods. All experiments were performed at least three times. Mean values for individual experiments are expressed as means ± SE. Statistical differences were analyzed by ANOVA followed by Scheffé's F-test using StatView II (Abacus Concepts). P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We first studied the effects of H2O2 on the expression levels of S1P1-R protein in BAECs. As shown in Fig. 1A, when BAECs are treated with H2O2 (150 µM), the expression of S1P1-R protein increases within 15 min of H2O2 addition. H2O2 does not alter the protein abundance of kinase Akt. H2O2 within these time periods also did not modulate expression levels of several other signaling proteins, including those of eNOS, caveolin-1, and the principal VEGF receptor subtype KDR (data not shown). A dose response for H2O2-mediated S1P1-R induction is shown in Fig. 1B. In these dose-response experiments, BAECs were treated with various concentrations of H2O2 for 30 min, and the lysates derived from these cells were analyzed in immunoblots probed with the S1P1-R antibody. As shown in Fig. 1B, H2O2-mediated induction of S1P1-R protein occurred in a dose-dependent fashion between 100 and 200 µM, values within the pathophysiological range for many other endothelial responses of this ROS (35).


Figure 1
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  		Fig. 1. Effects of H2O2 on expression of S1P1-R protein. Shown are the results of a protein immunoblot assay analyzed in cell lysates derived from bovine aortic endothelial cells (BAECs) treated with H2O2. A, top: results of time course experiments. BAECs were treated with H2O2 (150 µM) for the times indicated, and equal quantities of cellular protein (20 µg/lane) were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to immunoblot analyses probed with antibodies directed against S1P1 or Akt as indicated. Experiment is representative of 4 independent experiments that produced similar results. A, bottom: results of densitometric analyses from pooled data, plotting the fold increase of the degree of expression levels of S1P1-R protein at the time after H2O2 addition as indicated, relative to the signals obtained in the absence of H2O2. Data points are means ± SE derived from 4 independent experiments. B, top: results of dose-response experiments. BAECs were treated with H2O2 for 30 min at the indicated concentrations. Equal quantities of cellular protein (20 µg/lane) were analyzed in immunoblots probed with an antibody specific to S1P1; immunoblots were reprobed for Akt as above to confirm equal loading (data not shown). Data are representative of 4 independent experiments that yielded similar results. B, bottom: results of densitometric analyses from pooled data, plotting the fold increase of the degree of expression levels of S1P1-R at the H2O2 concentration indicated, relative to the signals obtained in the absence of H2O2. Data points are means ± SE derived from 4 independent experiments. *P < 0.05 vs. vehicle treatment.

 
We then sought to explore the mechanisms whereby H2O2 modulates S1P1-R protein expression levels. Because several earlier studies implicated Src-family tyrosine kinases (SFK) in H2O2-mediated endothelial signaling pathways (1, 3, 33), we employed PP2, an agent that attenuates activity of SFK with relatively high specificity. As shown in Fig. 2, pretreatment with PP2 (1 µM for 30 min) abolishes H2O2-mediated upregulation of S1P1-R protein expression levels, suggesting that SFK pathways may play a major role in this response. A distinct pharmacological inhibitor of SFK pathways, herbimycin A (1 µM for 30 min), also attenuated H2O2-promoted induction of S1P1-R protein (data not shown).


Figure 2
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  		Fig. 2. Effects of 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) on H2O2-mediated upregulation of S1P1-R protein abundance. Top: effects of PP2, an inhibitor of Src-family tyrosine kinases, on the H2O2-induced increase in S1P1-R protein abundance. BAECs were treated with PP2 (1 µM for 30 min) or vehicle and then incubated with H2O2 (150 µM for 30 min) or vehicle, as indicated. Equal quantities of cellular protein (20 µg/lane) were analyzed in immunoblots probed with an antibody specific to S1P1; immunoblots were reprobed for Akt to confirm equal loading (data not shown). Data are representative of 4 independent experiments, which yielded identical results. Bottom: results of densitometric analyses from pooled data, plotting the fold increase of the degree of expression levels of S1P1-R relative to the signals obtained in the absence of H2O2. Data points are means ± SE derived from 4 independent experiments. *P < 0.05 vs. vehicle treatment. {dagger}P < 0.05 vs. cells treated with H2O2 but without PP2.

 
To explore whether H2O2 upregulates S1P1-R protein expression by modulating S1P1 transcripts, we next performed RT-PCR analyses of RNA isolated from H2O2-treated endothelial cells, using specific oligonucleotide primers for S1P1 (14). As shown in Fig. 3, the abundance of S1P1 transcripts does not increase within 60 min of H2O2 addition to BAEC at the time points tested. In contrast, VEGF increased abundance of S1P1 mRNA at 15 and 60 min after drug addition under identical conditions. Together, these data suggest that H2O2-mediated upregulation of S1P1-R protein, which appears to depend on endothelial SFK pathway activity, may proceed without altering expression levels of S1P1 transcripts.


Figure 3
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  		Fig. 3. Effects of H2O2 and VEGF on S1P1 transcript abundance. Shown are the results of RT-PCR analyses directed for S1P1 transcripts in BAECs treated with H2O2 or VEGF. Top: results of RT-PCR analyses with total RNA derived from BAECs. Cells were treated with H2O2 (150 µM) or VEGF (10 ng/ml) for the times indicated. Total RNA was isolated, reverse transcribed, and amplified with specific oligonucleotide primers directed to S1P1 or GAPDH as described in detail in the text. Data are representative of 5 independent experiments, which yielded similar results. Bottom: results of densitometric analyses from pooled data, plotting the fold increase of the degree of expression levels of S1P1 transcripts relative to those of GAPDH at the treatment condition as indicated, relative to the signals obtained in the absence of drug treatments. Data points are means ± SE derived from 5 independent experiments. *P < 0.05 vs. vehicle treatment.

 
The functional consequences of the induction of S1P1-R in H2O2-treated BAECs were explored in a series of experiments. S1P has been previously shown to promote the phosphorylation of eNOS and of protein kinases ERK1/2 (16). We performed immunoblot analyses in lysates prepared from H2O2-pretreated BAECs that had been subsequently exposed to S1P, using phospho-specific antibodies as probes. Figure 4A shows the results of Western blot analysis with antibodies specific to phosphorylated forms of ERK1/2 and eNOS in these cells. BAECs were first incubated with H2O2 (150 µM for 30 min) or its vehicle and then treated with S1P (100 nM for up to 60 min). S1P induced phosphorylation of these proteins in BAECs that had not been pretreated with H2O2 (Fig. 4, A and B; also see Ref. 16). When BAECs were first preincubated with H2O2 for 30 min, the degrees of subsequent phosphorylation of ERK1/2 and eNOS-Ser1179 induced by S1P were augmented (Fig. 4, A and B). Pretreatment with H2O2 also promoted S1P-elicited phoshorylation of kinase Akt (Ser473; data not shown), which acts as an upstream regulator of eNOS-Ser1179 phosphorylation (16). In Fig. 4, C and D, we performed dose-response studies of S1P in BAECs pretreated with H2O2. In H2O2-pretreated BAECs, the increases in ERK1/2 phosphorylation in response to S1P became significant at lower concentrations compared with those not pretreated with H2O2. H2O2 augmented the degrees of eNOS phosphorylation at higher S1P concentrations (Fig. 4, C and D). Whereas H2O2 induces phosphorylation of JNK and p38, S1P does not induce phosphorylation responses of these MAPKs and H2O2 pretreatment does not augment them (data not shown).


Figure 4
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  		Fig. 4. Effects of H2O2 on S1P-mediated phosphorylation of endothelial nitric oxide synthase (eNOS) and ERK1/2. Shown are the results of a protein immunoblot assay probed with antibodies directed against phosphorylated forms of eNOS and ERK1/2. A: results of time course studies in which BAECs were incubated with H2O2 (150 µM for 30 min) or vehicle and then treated with S1P (100 nM). After addition of S1P, cells were harvested at the times indicated, and equal quantities of cell lysate (20 µg/lane) were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies directed against phospho (p)-eNOS and phospho-ERK1/2. Equal loading of samples was confirmed by reprobing the immunoblots with an antibody against (total) eNOS. Results are representative of an experiment that was independently repeated 5 times with similar results. B: results of densitometric analyses from pooled data, plotting the fold increase of the degree of phosphorylation levels of eNOS or ERK1/2 at the S1P treatment conditions as indicated, relative to the signals obtained in the absence of H2O2 and S1P. Data points are means ± SE derived from 5 independent experiments. C: results of dose-response studies in which BAECs were incubated with H2O2 (150 µM for 30 min) or vehicle and then treated with S1P for 5 min at the concentrations indicated. After addition of S1P, cell lysates were prepared and subjected to phospho-Western analyses. Results are representative of an experiment that was independently repeated 4 times with similar results. D: results of densitometric analyses from pooled data, plotting the fold increase of the degree of phosphorylation levels of eNOS or ERK1/2 at the S1P treatment conditions indicated, relative to the signals obtained in the absence of H2O2 and S1P. Data points are means ± SE derived from 4 independent experiments. *P < 0.05 vs. cells without H2O2 pretreatment at the identical S1P treatment protocol.

 
We then sought to explore how pretreatment with H2O2 influences S1P-mediated eNOS activation. We measured intracellular cGMP content of BAECs as an index of eNOS activity. S1P increased cGMP content even in BAECs that had not been pretreated with H2O2, reflecting S1P-induced eNOS activation. When BAECs were first preincubated with H2O2 and then treated with S1P, the magnitudes of S1P-induced increases in cGMP were augmented (Fig. 5). PP2, an inhibitor of SFK pathways that abolishes H2O2-mediated induction of S1P1-R protein (Fig. 2), completely blocked the ability of H2O2 to enhance S1P-mediated phosphorylation of eNOS and of ERK1/2 but had no effect on the S1P-elicited phosphorylation response in control BAECs not treated with H2O2 (Fig. 6).


Figure 5
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  		Fig. 5. Effects of H2O2 on S1P-mediated augmentation of cGMP content in BAEC. Shown are the results of cGMP measurements in the cell lysates derived from BAECs. BAECs were incubated with H2O2 (150 µM for 30 min) or vehicle and then treated with S1P for 10 min at the indicated concentrations. Cells were harvested, and resulting lysates were subjected to cGMP assay as described in the text. Data points are means ± SE of the pooled data derived from 6 independent experiments. *P < 0.05 vs. vehicle treatment. {dagger}P < 0.05 vs. cells without H2O2 pretreatment at the identical S1P treatment protocol.

 

Figure 6
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  		Fig. 6. Effects of PP2 on H2O2-induced enhancement of S1P-mediated phosphorylation of eNOS and ERK1/2. Shown are the results of immunoblots probed with antibodies directed against phosphorylated eNOS or phosphorylated ERK1/2. BAECs were pretreated with PP2 (1 nM for 30 min) or vehicle, followed by H2O2 (150 µM for 30 min) or vehicle, and then treated with S1P (100 nM for 5 min). Cell lysates (20 µg/lane) were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies as indicated. Equal loading of samples was confirmed by reprobing the immunoblots with antibodies against (total) eNOS (not shown). Results are representative of an experiment that was independently repeated 4 times with equivalent results.

 
It is well established that S1P promotes proliferation responses in many cell types. We therefore explored whether S1P promotes endothelial proliferation in H2O2-treated BAECs. BAECs were first stimulated with H2O2 for 30 min and then treated with S1P for 2 days. Cells were then collected and subjected to cell counting assay as described above. Incubation of BAECs for 48 h after stimulation with H2O2 decreased cell numbers; however, when cells were incubated with S1P for 48 h after H2O2 treatment for 30 min, BAECs exhibited cell counts at a level comparable to those incubated without H2O2 and S1P (Fig. 7A). In endothelial cells, both eNOS and ERK1/2 have been proposed to mediate S1P-induced proliferative responses (23, 30). Figure 7B demonstrates that inhibitors of both an ERK pathway (PD-98059) and nitric oxide synthase [NG-nitro-L-arginine methyl ester (L-NAME)] are capable of counteracting S1P-induced recovery of cell growth responses in H2O2-treated BAECs. These results suggest that activation of ERK1/2 and that of eNOS contribute to S1P's ability to restore cell growth retardation of H2O2-treated endothelial cells. An independent approach was taken to confirm that S1P counteracts delay of BAEC proliferation induced by H2O2. In an equivalent protocol, the degrees of cellular proliferation were studied with WST-8 reagent, which is an alternative of MTT assay (29). Figure 7C demonstrates that S1P reverses the decreases in WST-8 reactions of H2O2-treated BAECs.


Figure 7
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  		Fig. 7. Effects of H2O2 and S1P on BAEC proliferation. A and B: results of cell counting assays. In A, BAECs were treated with H2O2 (150 µM for 30 min) or vehicle and then treated with S1P or vehicle (indicated concentrations for 48 h). In B, some cells were also cotreated with S1P (100 nM) and with PD-98059 (PD; 10 µM) or NG-nitro-L-arginine methyl ester (L-NAME; 5 mM), as indicated. Cells were then trypsinized and subjected to cell counting assays as described in MATERIALS AND METHODS. C: results of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-8) assays. BAECs seeded on 96-well plates were treated as above and subjected to measurement of WST-8 reactions, an alternative of dimethylthiazol-diphenyltetrazoliumbromide (MTT) assays. Data points are means ± SE of the pooled data derived from 5 independent experiments. *P < 0.05 vs. untreated cells. {dagger}P < 0.05 vs. cells treated with H2O2 but without S1P. {ddagger}P < 0.05 vs. cells treated with both H2O2 and S1P.

 
We have previously reported that S1P/S1P1-R signaling is associated with caveolae, which are flask-shaped specialized signaling compartments of plasma membrane, using a heterologous expression system of COS-7 cells (18). In seeking to explore the involvement of caveolae in S1P/S1P1-R signaling in native BAECs treated with H2O2, we found that endogenous S1P1-R protein becomes undetectable after subcellular fractionation with currently available antibodies in immunoblot analyses (data not shown). To overcome this experimental limitation, we exploited several approaches. We examined whether transfected S1P1-R protein coimmunoprecipitates with native caveolin-1, a resident protein of caveolae, in BAECs. Figure 8A indicates that an antibody specific to caveolin-1 leads to coimmunoprecipitation of transfected FLAG/S1P1-R and vice versa. Note that these combinations of coimmunoprecipitation do not take place with nonimmune IgG (18). Figure 8A also demonstrates that treatment with H2O2 does not change the recovery of coimmunoprecipitated proteins. We then performed subcellular fractionation experiments in which BAEC homogenates were sonicated in hypotonic buffer and centrifuged to separate them into soluble and particulate fractions. In this preparation, kinase Akt and caveolin-1 were exclusively recovered in soluble and particulate fractions, respectively (Fig. 8B). We found that, under these conditions, transfected FLAG/S1P1-R is recovered in particulate fractions along with caveolin-1; H2O2 treatment does not alter its subcellular distribution. Methyl beta-cyclodextrin (MBCD) is an agent that depletes cholesterol from cellular plasma membrane, thereby abrogating caveolae-associated signal transduction (15). When BAECs were pretreated with MBCD, S1P-induced phosphorylation responses of eNOS and ERK1/2 are abolished (Fig. 8C). MBCD also abolished eNOS/ERK1/2 responses to S1P in H2O2-pretreated BAECs. Collectively, these data suggest that S1P1-R are targeted to plasmalemmal caveolae in native BAECs, in association with caveolin-1, a scaffolding protein of caveolae. These data also suggest that S1P1-R stays associated with caveolae/caveolin-1 even after stimulation with H2O2, rather than undergoing subcellular redistribution.


Figure 8
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  		Fig. 8. Involvement of caveolae/caveolin-1 (Cav-1) in S1P/S1P1-R signaling of BAEC. A: results of coimmunoprecipitation analyses of FLAG-tagged S1P1-R (FLAG/S1P1-R) and caveolin-1. BAECs were transfected with plasmid DNA encoding FLAG/S1P1-R and treated with H2O2 (150 µM for 30 min) or vehicle. Cell lysates were prepared from these cells, and an aliquot (2%) was saved. Sample remainders were immunoprecipitated (IP) with an antibody specific to FLAG peptide or to caveolin-1 as indicated. Lysates and IP proteins were subjected to immunoblot (IB) analyses, using antibodies as shown. B: results of subcellular fractionation experiments. BAECs that had been transfected with plasmid DNA encoding FLAG/S1P1-R were treated with H2O2 (150 µM for 30 min) or vehicle. Cell homogenates were prepared with a hypotonic buffer, and an aliquot was removed (homogenates). The remaining was subjected to subcellular fractionation. Total cell homogenates and subcellular fractions (soluble and particulate) were subjected to immunoblot analyses, using antibodies as indicated. C: effects of methyl-beta-cyclodextrin (MBCD) on phosphorylation responses of eNOS and ERK1/2. BAECs were subjected to analyses without transfection. Cells were treated with H2O2 (150 µM for 30 min) or vehicle and then treated with S1P or vehicle (100 nM for 5 min). Some cells had been incubated with MBCD (10 mM for 50 min) before addition of S1P. These cells were lysed and subjected to phospho-Western analyses as above. Blots were reprobed with an antibody specific to (total) eNOS to confirm equal loading (not shown). Experiments shown in each panel were repeated 3 times with equivalent results; representative data are shown.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that H2O2, a representative ROS, exerts distinct alterations in the expression levels of S1P1-R and exhibits remarkable influences on endothelial responses for subsequent stimulation with S1P, a platelet-derived lipid mediator. Exogenously administered H2O2 upregulates expression levels of S1P1-R protein (Fig. 1). The induction of S1P1-R protein by H2O2 is very acute and reaches a maximum at 30 min after drug addition (Fig. 1A). The concentrations of H2O2 that increase S1P1-R protein (100–200 µM; Fig. 1B) have been shown to occur in vivo in various situations and are considered pathophysiologically relevant to endothelial cells in the context of vascular regulation, rather than being merely toxic (4, 26). Thus, expression levels of S1P1-R might be subjected to dynamic regulation by ROS in vivo as well. To our surprise, exogenously added H2O2 does not increase S1P1 mRNA steady-state abundance, whereas VEGF clearly does so under identical conditions (Fig. 3). This may suggest that increases in S1P1-R protein after H2O2 addition are mediated by mechanisms distinct from those mediating actions of VEGF, which increases the expression levels of S1P1 at levels of both protein and mRNA (Fig. 3; see also Ref. 17). Because PP2 reverses H2O2-modulated increases in S1P1-R protein (Fig. 2) and enhancement of endothelial responses to S1P (Fig. 6), SFK pathways appear to be involved in these responses. Previous studies documented that H2O2 activates c-Src in BAECs (1, 3, 33). However, detailed molecular mechanisms regarding how endothelial SFK pathways, following H2O2, increase S1P1-R protein abundance in BAEC by promoting the synthesis of (or possibly by attenuating the degradation of) S1P1-R protein remain to be elucidated. It is of note that PP2 per se does not affect S1P-induced phosphorylation responses (Fig. 6); this is consistent with earlier findings from our group (17, 19), which established that S1P-mediated signaling responses via G-protein-coupled S1P receptors by themselves are largely independent of tyrosine kinase pathways.

These studies also indicate that exogenously added H2O2 (150 µM) is capable of sensitizing BAECs to subsequently added S1P, demonstrating that increases in S1P1-R protein expression levels are functionally coupled with the enhancement of S1P actions on endothelial cells. H2O2 sensitization to S1P takes place at a level of the activation of protein kinase cascades, MAPK and phosphatidylinositol 3-kinase/Akt, which are assessed as degrees of phosphorylation of ERK1/2 (Thr202/Tyr204) and eNOS (Ser1179), representative substrate proteins of each protein kinase cascade modulated by S1P (16). The degrees of maximum phosphorylation elicited by S1P are remarkably higher in ERK1/2, eNOS, and Akt of H2O2-pretreated cells than in nonpretreated cells (Fig. 4, A and B, and data not shown). In dose-response experiments, H2O2-pretreated BAECs exhibited higher degrees of ERK1/2 and eNOS phosphorylation at the identical S1P concentration than those not pretreated with H2O2 (Fig. 4, C and D). To examine whether pretreatment with H2O2 enhances nitric oxide production elicited by S1P, we measured intracellular cGMP contents, which have been exploited as an index of nitric oxide production of these cell types (9). Consistent with the results of phospho-Western analyses, pretreatment with H2O2 clearly augmented S1P-mediated increases in cGMP (Fig. 5). Because both eNOS and ERK1/2 mediated several important endothelial responses to S1P, including vasorelaxation, survival, migration, and angiogenesis (8, 12), H2O2 may ultimately lead these cells to exhibit enhanced downstream reactions modulated by increased signaling responses to S1P. It is also interesting to speculate that ROS may modulate S1P contents in endothelial cells by altering activities of various S1P-metabolizing enzymes. Abolishment of H2O2-induced upregulation of S1P1-R protein by PP2 (Fig. 2) completely reversed augmented endothelial reactions for S1P in H2O2-pretreated BAECs (Fig. 6), consistent with the hypothesis that increases in S1P1-R protein play a central role in mediating H2O2-induced enhancement of S1P signaling in these cells.

In the time window in which we treated BAECs with H2O2, the expression levels of several other signaling proteins, including Akt and eNOS, were not altered (Figs. 1 and 4 and data not shown). However, longer treatment with H2O2 of cultured endothelial cells increased expression of eNOS at the levels of mRNA, protein, and enzyme activity; H2O2 induction of eNOS occurs at concentrations similar to those used in the present studies but starts several hours after the drug addition (7). Thus our results that H2O2 sensitizes endothelial cells to activate eNOS in response to S1P at ~ 30 min (Figs. 1, 4, and 5) may identify a mechanism of much shorter endothelial response to this ROS. ROS derived from various cardiovascular origins acutely react with nitric oxide and thereby decrease its effective concentrations (5). Our studies demonstrate that BAECs, which have been preexposed to H2O2, produce higher amounts of nitric oxide in response to S1P (Fig. 5). eNOS-derived nitric oxide plays central roles in regulating interaction of endothelial cells with adjacent cells (25), for example, by attenuating expression levels of endothelial adhesion molecules, leading to lower degrees of leukocyte/platelet adhesion with vascular endothelium (2). Because S1P stands for a bioactive lipid mediator derived from activated platelets (36), it is tempting to speculate that enhancement of eNOS activity by S1P in H2O2-pretreated endothelial cells may represent a novel "negative-feedback" mechanism against excessive ROS production, which might occur at stimulated vasculature (5). It is interesting that platelets themselves express functional S1P receptors (37), as well as eNOS (32), whereas regulation of S1P1/eNOS by ROS remains to be elucidated in nonendothelial cell types. These considerations add another level of complexity in our understanding of endothelial interaction with other cardiovascular cell types, modulated by nitric oxide/ROS and by sphingolipid mediators.

In an attempt to explore pathophysiological consequences of S1P1 induction by H2O2, we studied the effects of S1P on endothelial proliferation in H2O2-treated BAECs. H2O2 decreased cell numbers 48 h after drug addition (Fig. 7). We interpret these data that H2O2 delayed cellular proliferation rather than caused cell death because these concentrations (100–150 µM) of H2O2 have been established not to cause endothelial injury of aortic origin (4). Indeed, we did not observe significant endothelial injury under these treatment protocols (assessed by morphological examination and release of lactate dehydrogenase into culture media; data not shown). Thus, our data that addition of S1P to H2O2-pretreated cells leads to restoration of cell numbers (Fig. 7) demonstrate that S1P is able to counteract H2O2-mediated retardation of endothelial cell growth. In many pathological states, defects of endothelial layer in injured vasculature are associated with deleterious outcomes in various cardiovascular diseases (11). Thus, our data suggest that S1P can counteract deleterious effects of ROS on vascular endothelium. Both eNOS and ERK1/2 participate in endothelial proliferative responses promoted by S1P (23, 30); pharmacological inhibitors of these pathways (L-NAME and PD-98059) attenuated S1P-elicited counteraction of cell number decreases in H2O2-treated BAECs (Fig. 7B). Our results with L-NAME (Fig. 7B) are also consistent with the hypothesis that promotion of nitric oxide production after H2O2/S1P, assessed as cGMP content (Fig. 5), is indeed biologically active. Thus elevated degrees of activation of these endothelial effector molecules in H2O2-pretreated BAECs, which express higher amounts of S1P1-R protein (Fig. 1), may play major roles in mediating S1P-evoked counteraction against growth retardation in H2O2-treated endothelial cells. It is potentially interesting to explore the roles of H2O2 on other S1P-related endothelial reactions, including migration and apoptosis.

Caveolae are flask-shaped specialized signaling compartments of endothelial plasma membrane (reviewed in Ref. 34). We have previously documented that S1P1-R are targeted to caveolae and associated with caveolin-1, a scaffolding protein of caveolae, and that targeting of S1P1-R to caveolae/caveolin-1 modulates S1P signaling in COS-7 cells heterologously expressing S1P1-R (18). We found that native S1P1-R protein is no longer detectable in immunoblot analyses after subcellular fractionation (data not shown). This likely reflects lower abundance of native S1P1-R protein in BAECs and/or lack of sensitivity in presently available anti-S1P1 antibodies. To overcome this experimental limitation, we chose to transiently transfect FLAG-tagged S1P1-R construct (14, 18) to BAECs. Our results indicate that FLAG/S1P1-R and caveolin-1 coimmunoprecipitates with each other (Fig. 8A) and that FLAG/S1P1-R proteins are recovered in particulate fractions along with caveolin-1 following subcellular fractionation (Fig. 8B). Importantly, treatment of transfected BAECs with H2O2 does not alter the degree of coimmunoprecipitation or recovery in particulate fractions of S1P1-R protein (Fig. 8, A and B). Although the expression level of exogenously transfected FLAG/S1P1-R protein is not promoted by H2O2, unlike native S1P1-R, these results suggest that S1P1-Rs remain associated with caveolae/caveolin-1 even when stimulated with H2O2, rather than undergoing subcellular redistribution. MBCD is an agent that depletes cholesterol from cellular plasma membrane, thereby inhibiting caveolae-associated receptor signaling. Our experiments demonstrate that MBCD abrogates phosphorylation responses of eNOS and ERK1/2 elicited by S1P, both with and without pretreatment with H2O2 (Fig. 8C). Together, these results are consistent with the hypothesis that S1P1-R protein in native vascular endothelial cells is associated with caveolae and caveolin-1. They also suggest that H2O2 is not likely to elicit subcellular redistribution of S1P1-R protein. At this stage, however, detailed subcellular distribution of native S1P1-R protein in vascular endothelial cells remains to be determined, awaiting innovation of more sensitive antibodies specific to S1P1-R.

In conclusion, the present study identified that H2O2 upregulates expression levels of S1P1-R of cultured vascular endothelial cells. Extracellularly added H2O2 increases S1P1-R protein abundance via SFK pathways. H2O2-dependent upregulation of S1P1-R is associated with enhanced responses to S1P of eNOS, as well as those of MAPKs ERK1/2, and with S1P-promoted restoration of H2O2-induced delay of endothelial cell proliferation. Thus our study may identify another point of control at which ROS and sphingolipids exert cross-talk of signaling pathways, which may modulate responses of vascular endothelial cells to platelet-derived bioactive molecules.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was in part supported by Grants-in-Aid to J. Igarashi (15790119) and to H. Kosaka (15590186) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a Grant-in-Aid to J. Igarashi by Ono Medical Research Foundation (Osaka, Japan).


   ACKNOWLEDGMENTS
 
The authors thank Drs. Roger A. Sabbadini and Timothy Hla for providing anti-EDG-1 (S1P1) antibody and FLAG/S1P1 cDNA, respectively. We are also grateful to Dr. Shigemoto Fujii (Kagawa University) for helpful discussions in performing RT-PCR analysis and to Kayoko Osumi for help in preliminary experiments of this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Igarashi, Dept. of Cardiovascular Physiology, Kagawa Univ. Faculty of Medicine, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793 Japan (e-mail: igarashi{at}med.kagawa-u.ac.jp)

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.


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