FTY720 inhibits ceramide synthases and up-regulates dihydrosphingosine 1-phosphate formation in human lung endothelial cells

Abstract

Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent sphingosine 1-phosphate (S1P)-like action through S1P(1,3-5) receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with l-[U-(13)C, (15)N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, sphingosine, and S1P but increased levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent K(i) of 2.15 microm. FTY720-induced up-regulation of DHS1P level was mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells.

FIGURE 1.

Development of LC/MS/MS methods for quantitation of FTY720 and FTY720-P. A, positive product ion spectrum of FTY720. B, chromatographic separation of FTY720 and sphingoid bases. 1, FTY720; 2, C₁₇-Sph; 3, Sph; 4, DHSph. C and D, standard curves for FTY720 with C₁₇-Sph as the internal standard without interfering lipid background (C) and in the presence of total lipid extract (D). E, negative product ion spectrum of FTY720-P. F, chromatographic separation of FTY720-P and sphingoid base 1-phosphates. 1, FTY720-P; 2, C₁₇-S1P; 3, S1P; 4, DHS1P. G and H, standard curves for FTY720-P with C₁₇-S1P as the internal standard without interfering lipid background (G) and in the presence of total lipid extract (H). Refer to text for further details. C.P.S., counts/s.

FIGURE 2.

The effect of FTY720 on intracellular level of signaling sphingolipids in HPAEC. Cells were treated with increasing concentrations of FTY720 for 2 h, and S1P, DHS1P, and FTY720-P (A) and Sph, DHSph, ceramides, and FTY720 (B) were quantified by LC/MS/MS. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control. Error bars represent S.E.

FIGURE 3.

The effects of FTY720 and FB1 on the incorporation of l-[U-¹³C, ¹⁵N]serine into signaling sphingolipids. HPAECs were pretreated with the indicated concentrations of FTY720 or FB1 for 3 h, and then the cells were labeled with l-[U-¹³C, ¹⁵N]serine for 2 h. Label-incorporated sphingoid bases, sphingoid base 1-phosphates, ceramides, and dihydroceramides were quantified by LC/MS/MS. The point of interference for both compounds with sphingolipidde novo biosynthesis is indicated in the scheme (star indicates labeled molecules). **, p < 0.01; ***, p < 0.001 versus control. Palm., palmitoyl; GlyCer, glycosylceramide; SM, sphingomyelin. Error bars represent S.E.

FIGURE 4.

The effects of FTY720 and FB1 on the de novo generation of individual ceramide molecular species. HPAECs were pretreated with FTY720 (5 μm) or FB1 (10 μm) for 3 h at 37 °C, and then the cells were labeled with l-[U-¹³C, ¹⁵N]serine for 2 h. Label-incorporated 16:0-, 24:1-, and 24:0-ceramides were quantified by LC/MS/MS. Data are presented as percentage of control label incorporation into the same ceramide molecular species in vehicle-treated cells. Error bars represent S.E.

FIGURE 5.

FTY720 is a competitive inhibitor of ceramide synthase 2. A, concentration range of FTY720 was tested in an in vitro ceramide synthase assay using 5 μm 22:0-coenzyme A, 100 nm DHSph, and 50 μg of total cell lysate protein/assay as described under “Experimental Procedures.” Data are plotted as percentage of the activity in the presence of vehicle (0.1% BSA). 22:0-Dihydroceramide was quantified by LC/MS/MS. B, total cell lysate proteins were incubated with 10 μm FTY720 for 2 min. Then part of the protein preparation was diluted 10- or 20-fold with 0.1% BSA in the reaction buffer, and the ceramide synthase reaction was initiated by the addition of 5 μm 22:0-CoA and 100 nm DHSph. 22:0-DHCer was quantified by LC/MS/MS. Data are expressed as the percentage of the activity in the presence of vehicle for each corresponding protein concentration and demonstrate the reversibility of enzyme inhibition on FTY720 dilution. C, left, inhibition of ceramide synthase 2 activity by FTY720 (2.5 μm) with increasing concentrations of DHSph, 5 μm 22:0-CoA, and 50 μg of total cell lysate protein/reaction. 22:0-DHCer was quantified by LC/MS/MS. Right, the same experimental data are presented as a Lineweaver-Burk plot; the results typify a competitive inhibition of DHSph binding to ceramide synthase by FTY720. Error bars represent S.E.

FIGURE 6.

Effect of FTY720 on inhibition of CerS isozymes in the cell lysate assay. FTY720 (5 and 10 μm) was tested in the in vitro ceramide synthase assay with 100 nm DHSph, 50 μg of total cell lysate protein, and 5 μm 16:0-, 18:0-, 20:0-, 22:0-, or 24:1-CoA as described under “Experimental Procedures.” Dihydroceramides were quantified by LC/MS/MS. Data are expressed as a percentage of the activity in the presence of vehicle (0.1% BSA in the reaction buffer). Error bars represent S.E.

FIGURE 7.

FB1 is more potent than FTY720 in inhibiting ceramide synthases in the in vitro assay. CerS activity was determined using 5 μm 16:0- or 24:1-CoA, 100 nm DHSph, and 50 μg of total cell lysate protein with the indicated concentrations of FTY720 or FB1 as described under “Experimental Procedures.” 16:0- and 24:1-DHCer were quantified by LC/MS/MS. Error bars represent S.E.

FIGURE 8.

SphK1 but not SphK2 is involved in FTY720-mediated DHS1P generation in HPAECs. A, SphK1 or SphK2 were silenced by siRNA, and then HPAECs were treated with 5 μm FTY720 for 2 h. The lipids were extracted and analyzed for the presence of S1P, DHS1P, and FTY720-P. Values above bars represent -fold increase versus corresponding controls. Statistical significance is not shown for better presentation of the data. B, silencing of SphK1 and SphK2 by siRNA for 72 h decreases protein (top) and mRNA (bottom) levels of SphK1 and SphK2 in HPAECs. Protein and real time PCR data are matched with lipid data presented in A. Scr (or sc), scrambled. Error bars represent S.E.

FIGURE 9.

Multiple points of FTY720 interference with sphingolipid metabolism and signaling. FTY720 is phosphorylated by SphK2, and the formed FTY720-P activates S1P_1,3–5 receptors. Also FTY720 can inhibit the cannabinoid receptor 1 (CB1). Intracellularly FTY720 inhibits S1P lyase, cPLA₂, and ceramide synthases. Overall FTY720 action favors anti-inflammatory responses. Cer1P, ceramide 1-phosphate.