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Essential requirement for sphingosine kinase activity in eNOS-dependent NO release and vasorelaxation

Fiorentina Roviezzo^*, Mariarosaria Bucci^*, Chantal Delisle, Vincenzo Brancaleone^*, Annarita Di Lorenzo^*, Inmaculada Posadas Mayo^*, Stefano Fiorucci, Angelo Fontana, Jean-Philippe Gratton and Giuseppe Cirino^*,1

^* Dipartimento di Farmacologia Sperimentale, Università di Napoli Federico II, Naples, Italy;
Laboratory of Endothelial Cell Biology, Institut de Recherches Cliniques de Montreal (IRCM), Montreal, Quebec, Canada;
Dipartimento di Medicina Sperimentale, Perugia, Italy; and
CNR, Istituto Chimico Biomolecolare, Naples, Italy

¹ Correspondence: Departimento di farmacologia Sperimentale, Via Domenico Montesano 49, Napoli 80131, Italy. E-mail: cirino{at}unina.it

SPECIFIC AIMS

Cellular levels of sphigosine-1-phosphate (S1P) are low and tightly regulated by sphingosine kinase (SPK). Recent studies have suggested that eNOS pathway may function as a downstream target for the receptor-mediated biological effects of S1P. Here we have studied the possible interplay between intracellular S1P generation and the eNOS activation pathway. Particularly by using rat aortic rings we have investigated a potential intracellular second messenger role of S1P in eNOS activation and the contribution of this alternate pathway in the control of vascular tone.

PRINCIPAL FINDINGS

1. S1P induces endothelium and receptor-dependent vasorelaxation coupled to an increase in eNOS activity
S1P induces an endothelium-derived NO and concentration-dependent vasorelaxant effect on rat aortic rings (P<0.001). This finding is confirmed by the fact that L-NAME (100µmol/L) significantly inhibits S1P-induced vasodilatation. Among S1P receptors S1P₁ is the most abundantly expressed in rat aorta. S1P₁ receptor signal transduction involves a G_i protein as suggested by pertussis toxin sensitivity. Pertussis toxin abrogates S1P relaxant effect indicating that the effect observed is mediated by a G-protein coupled receptor. To further investigate on the role of eNOS derived NO, we evaluated if S1P (1µmol/L) could increase eNOS phosphorylation on serine 1179. After treatment of aortic rings with S1P, a concentration- and time-dependent increase in eNOS phosphorylation was observed. To gain further insight into the cross-talk between S1P and NO pathway, we evaluated the effect of GA (20 µmol/L), an inhibitor that hinders eNOS activation by blocking its coupling to the eNOS-activating protein hsp90 and LY-294002 (25 µmol/L), an inhibitor of PI-3 kinase activity that prevents downstream Akt-dependent eNOS phosphorylation, against S1P-induced vasorelaxation. GA treatment virtually abolishes S1P-induced vasorelaxation (P<0.001), similar to the effect of endothelium removal or by L-NAME. Conversely, LY-294002 causes a significant (P<0.01) but less marked inhibition of S1P-stimulated vasorelaxation compared with GA or L-NAME.

2.Sphingosine kinase activity is involved in endothelium-dependent vasodilatation
Incubation of aortic rings with Ach but not the vehicle caused a release of 30 ng/mL/wet tissue of S1P as determined by negative ESI LC-MS. After this, we operated a pharmacological modulation through specific inhibitors. Pretreatment of aortic rings with DTD, a specific sphingosine kinase inhibitor, reduces Ach-induced vasodilatation in a concentration-dependent manner (Fig. 1 A). DTD (100 µmol/L) does not interfere with the ability of smooth muscle cells to respond to exogenous NO (Fig. 1B ) and does not modify calcium ionophore A-23187-induced vasorelaxation (Fig. 1C ).

View larger version (13K): [in this window] [in a new window]   Figure 1. A) Pretreatment of aortic rings with DTD, a specific sphingosine kinase inhibitor, reduces in a concentration-dependent manner, Ach-induced vasodilatation at the concentration of 30 µmol/L (n=6; P<0.01) and 100 µmol/L (n=6; P<0.001). B) DTD does not modify SNP-induced vasorelaxation (Fig. 2B , n=6, NS). C) DTD has no effect on the calcium ionophore A-23187-induced vasorelaxation (n=6 NS). Data are expressed as mean ± SE. ***P < 0.001, **P < 0.01 vs. vehicle.

3. Inhibition of sphingolipid biosynthesis reduces Ach-induced vasorelaxation
To assess the involvement of sphingolipid metabolism in endothelium-dependent vasorelaxation, inhibitors of S1P biosynthesis, which interfere at different levels of synthesis, were used. Fumonisin (30 µmol/L), an inhibitor of dihydroceramide, the enzyme that converts sphinganine to dihydroceramide and MAPP (10 µmol/L), a specific inhibitor of the enzyme ceramidase that converts ceramide to sphingosine, were used. Fumonisin and MAPP both inhibit Ach-induced vasorelaxation. These data imply a consistent contribution of sphingolipid pathway, through both sphingosine biosynthesis and its phosphorylation by SPK, in Ach-induced vasorelaxation. To confirm that intracellular S1P is involved in Ach-induced vasorelaxation rat aortic rings were incubated with PTX. Relaxation induced by Ach was unaffected by pretreatment with PTX excluding an extracellular and receptor-mediated role for S1P.

4. Sphingosine kinase (SPK) inhibition abrogates receptor-dependent recruitment of hsp90 to eNOS and S1P-induced vasorelaxation
One of the key events in eNOS activation is its association with hsp90, which is necessary for calcium CaM-dependent eNOS activation. To assess the role of SPK in agonist-dependent hsp90 recruitment to eNOS, we performed eNOS immunoprecipitation study in bovine aortic endothelial cells (BAEC). Monitoring of the coprecipitation of the activating protein hsp90, as seen in Fig. 2 A, B), S1P (5 µM), BK (10 µM), and A23187 (5 µM) stimulation of BAEC (10 min) induce an increase in the amount of hsp90 detected in the eNOS immunocomplex (Fig. 2A-C ; compare lanes 1 and 3). Inhibition of SPK by DTD (10 µM) markedly reduces the ability of S1P and BK to promote hsp90 recruitment to eNOS (Fig. 2A, B ; compare lanes 3 and 4). Conversely, DTD fails to inhibit hsp90 recruitment by the receptor independent calcium-mobilizing agent A23187 (Fig. 2C ; compare lanes 3 and 4). Overall, these results suggest that SPK activation is an event upstream to the receptor-induced increase of the intracellular calcium levels, necessary for hsp90 recruitment to eNOS and its activation. In addition, these data are in line with our functional studies showing that SIP-induced vasodilatation of the rat aorta is significantly inhibited by DTD (Fig. 2D ) while A23187-induced effects are unaffected by SPK inhibition.

View larger version (18K): [in this window] [in a new window]   Figure 2. Stimulation of BAEC (10 min) with either BK (10 µmol/L, A) or S1P (5 µmol/L; B), or A23187 (5 µmol/L; C) induces an increase in the amount of hsp90 detected in the eNOS immunocomplex (compare lanes 1 and 3). Inhibition of SPK by DTD (10 µmol/L, 10 min) markedly reduces the ability of S1P (B) or BK (A) to promote hsp90 recruitment to eNOS (compare lanes 3 and 4). IDTD (C) fails to inhibit hsp90 recruitment by the receptor independent calcium mobilizing agent A23187 (compare lanes 3 and 4). SIP-induced vasodilatation of the rat aorta is significantly inhibited by DTD (D). Data are expressed as mean ± SE. ***P < 0.001 vs. vehicle.

5. Intracellular calcium is involved in S1P-induced vasorelaxation
Pretreatment of aortic rings with EGTA, a calcium chelator, significantly inhibits Ach-induced vasodilatation (P<0.01) but has no effect on S1P-induced vasorelaxation, implying a contribution of extracellular calcium only in Ach-induced effect. Conversely, CPA, an inhibitor of Ca²⁺-ATPase in sarcoplasmic reticulum, has a marked effect (70%) both on Ach- (P<0.001) and S1P-induced relaxation (P<0.001), implying a key role for intracellular calcium.

CONCLUSIONS AND SIGNIFICANCE

Regulation of eNOS is a complex process, but it is possible to specifically interfere with eNOS activity at different levels. Among the post-translational modifications that can positively regulate eNOS activity the coupling of the enzyme to hsp90 and/or its phosphorylation on serine 1179 appear to be particularly critical. In our experimental conditions, we were able to show that SIP stimulation of rat aorta induces phosphorylation of eNOS and that the vasorelaxant effects of S1P are mainly dependent on hsp90. This evidence raised the possibility that an interplay may exist between eNOS, hsp90, and S1P. To understand this link, we performed specific experiments using Ach and inhibitors of sphingosine pathway. We first determined by negative ESI LC-MS that after incubation with Ach, S1P was produced, indicating that the sphingolipid pathway may contribute to the agonist-induced vasorelaxation. The contribution of this pathway to signaling appears to be important in vessel function since the blockade of S1P generation reduces by 50% the vasorelaxant effect of Ach. The eNOS/hsp90 co-immunoprecipitation experiments consistently showed increased association of hsp90 with eNOS after exposure of cells to BK or calcium ionophore A-23187. In sharp contrast to A23187, BK effects were significantly inhibited by pretreatment of the cells with the SPK inhibitor DTD. These data imply an involvement of calcium as a trigger for the eNOS/hsp90 coupling. This specificity in the response of BK-induced hsp90 recruitment suggests that sphingosine kinase is a prerequisite for eNOS activation and further corroborates the in vitro data where similar functional effects were clearly evident. Thus, it was necessary to evaluate the role of SPK by using S1P and to evaluate the S1P-induced eNOS/hsp90 interaction. Exogenous S1P induced co-immunoprecipitation of hsp90 with eNOS, abrogated by pretreatment with DTD. DTD abrogated S1P-induced vasorelaxation of rat aortic rings, suggesting that S1P promotes, via S1P₁ activation, its own intracellular synthesis through SPK. Calcium modulation experiments using CPA further suggest that intracellular pool of S1P enhances hsp90/eNOS interaction, possibly through intracellular calcium, and in turn increases NO production. In conclusion, we show that an interplay exists among SPK, S1P, hsp90, and eNOS where intracellularly generated S1P promotes the coupling of eNOS to hsp90 and this contributes to the modulation of vascular tone as demonstrated by our functional and molecular studies.

View larger version (52K): [in this window] [in a new window]   Figure 3. Schematic summary of the interplay among eNOS, S1P/SPK, and hsp90. The dotted arrows depict the classical signal transduction PLC-dependent pathway after acetylcholine stimulation. Black arrows depict the hypothesis of the alternative pathway that leads to release of intracellular S1P through sphingosine kinase. The S1P released intracellularly enhances hsp90/eNOS interaction and in turn increases NO production.

FOOTNOTES

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