The sphingosine 1-phosphate transporter, SPNS2, functions as a transporter of the phosphorylated form of the immunomodulating agent FTY720

Abstract

FTY720 is a novel immunomodulating drug that can be phosphorylated inside cells; its phosphorylated form, FTY720-P, binds to a sphingosine 1-phosphate (S1P) receptor, S1P(1), and inhibits lymphocyte egress into the circulating blood. Although the importance of its pharmacological action has been well recognized, little is known about how FTY720-P is released from cells after its phosphorylation inside cells. Previously, we showed that zebrafish Spns2 can act as an S1P exporter from cells and is essential for zebrafish heart formation. Here, we demonstrate that human SPNS2 can transport several S1P analogues, including FTY720-P. Moreover, FTY720-P is transported by SPNS2 through the same pathway as S1P. This is the first identification of an FTY720-P transporter in cells; this finding is important for understanding FTY720 metabolism.

FIGURE 1.

hSPNS2-mediated release of endogenous S1P and DH-S1P. Shown are an HPLC diagram (A) and quantitative results (B) of time-dependent secretion of endogenous S1P and DH-S1P from cells. CHO cells expressing SPHK1 and hSPNS2 were incubated with the releasing medium for 2, 6, or 12 h. The releasing medium was collected, and the amounts of secreted S1P and DH-S1P were measured by HPLC. C₁₇-S1P was used as the internal standard. Open and closed circles indicate secreted S1P and DH-S1P, respectively. Experiments were performed more than three times, and error bars indicate the S.D. C, sphingosine kinase activity in culture medium. The activity of SPHK was analyzed by the conversion of [³H]Sph to [³H]S1P. Lipid extracts were separated on a TLC plate, and bands corresponding to [³H]S1P were quantified. The relative amount of [³H]S1P in the medium of CHO/SPHK1 (SPHK1), CHO/SPHK2 (SPHK2), or CHO/SPHK1/hSPNS2 (SPHK1/SPNS2) compared with CHO (mock) cells is indicated at the bottom of the TLC plate image. Experiments were performed more than three times, and values are means with S.D.

FIGURE 2.

DH-S1P, phyto-S1P, and C₁₇-S1P are released from hSPNS2-expressing cells. CHO/SPHK1 (mock) or CHO/SPHK1/hSPNS2 (SPNS2) cells were grown for 2 days and incubated with the releasing medium containing DH-Sph, phyto-Sph, or C₁₇-Sph at 5 μm for 2 h. The cells (A) and releasing medium (B) were collected, and sphingolipid contents were measured by HPLC. C₁₇-S1P was used as the internal standard for measuring DH-S1P and phyto-S1P, whereas phyto-S1P was used as the internal standard for measuring C₁₇-S1P. Experiments were performed more than three times, and error bars indicate the S.D.

FIGURE 3.

Phosphorylation of Sph and FTY720 in SPHK1- or SPHK2-expressing cells. A, chemical structures of S1P and FTY720-P are indicated. B, expression of SPHK1 and SPHK2. Cell lysates prepared from CHO/mock (mock), CHO/SPHK1 (SPHK1::HA), or CHO/SPHK2 (SPHK2::HA) cells were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Western blot analysis was performed using anti-HA and anti-SPHK1 antibodies. Both SPHK1 and SPHK2 stably expressed in CHO cells were tagged at the C terminus with the HA epitope. *, bands not derived from the tagged SPHK1/2 proteins. C, S1P and FTY720-P synthesis in SPHK1- or SPHK2-overexpressing cells. CHO (mock), CHO/SPHK1 (SPHK1), or CHO/SPHK2 (SPHK2) cells were incubated with the releasing medium containing 5 μm Sph or FTY720 for 2 h. The cells were collected, and the S1P and FTY720-P contents were measured by HPLC. C₁₇-S1P was used as the internal standard. Experiments were performed more than three times, and error bars indicate the S.D.

FIGURE 4.

FTY720-P is released from hSPNS2-expressing cells. CHO/SPHK1 or CHO/SPHK2 cells were transfected with pEGFP (mock) or pEGFP/hSPNS2 (SPNS2). 24 h after transfection, the cells were incubated with releasing medium containing 5 μm FTY720 (A) or Sph (B) for 2 h. The releasing medium was collected, and the amounts of released FTY720-P (A) and S1P (B) were measured by HPLC as described under “Experimental Procedures.” Experiments were performed more than three times, and error bars indicate the S.D. N.D., not detected.

FIGURE 5.

Tissue distribution of human SPNS2 mRNAs. Quantitative real-time PCR was performed with first strand cDNA synthesized from various human tissue mRNAs. Experiments were performed more than three times, and error bars indicate the S.D.

FIGURE 6.

S1P-releasing assay of cells expressing ABC transporters. A, expression of ABC transporters in the cells transfected with each transporter individually. Membrane (m) and soluble fractions (s) prepared from CHO/SPHK1 cells (mock) or CHO/SPHK1 cells stably expressing mABCA1, hABCB1, mABCC1, or hABCG2 were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Western blot analysis was performed with specific antibodies for each individual ABC transporter as described under “Experimental Procedures.” A ubiquitously expressed membrane protein, Na⁺/K⁺-ATPase, was used as the loading control of each membrane fraction. B and C, S1P or FTY720-P secretion from ABC transporter-expressing cells. CHO/SPHK1 cells (mock) or CHO/SPHK1 cells stably expressing mABCA1, hABCB1, mABCC1, hABCG2, or hSPNS2 were incubated with releasing medium containing 5 μm Sph (B) or FTY720 (C) for 2 h. The releasing medium was collected, and the amounts of released phosphorylated sphingolipids were measured by HPLC. Each diagram is a typical chromatogram of the sphingolipids released from the cells overexpressing an individual ABC transporter. C₁₇-S1P was used as the internal standard. Experiments were performed more than three times.

FIGURE 7.

FTY720 affects the intracellular contents of sphingolipids. CHO/SPHK1 (mock) and CHO/SPHK1-expressing hSPNS2 (SPNS2) cells were incubated with the releasing medium for 2 h in the presence (+) or absence (−) of 5 μm FTY720. The cells were collected, and the amounts of each sphingolipid (S1P (A), DH-S1P (B), Sph (C), and DH-Sph (D)) in the cells were measured by HPLC. Experiments were performed more than three times, and error bars indicate the S.D. *, p < 0.001.

FIGURE 8.

Effect of FTY720-P on the S1P release mediated by hSPNS2. A, inhibition of S1P release from cells with FTY720 treatment. CHO/SPHK1-expressing hSPNS2 cells were incubated with releasing medium containing different concentrations of Sph for 2 h in the presence or absence of 5 μm FTY720. The releasing medium and cells were collected, and the amounts of sphingolipids were measured by HPLC. Closed and open circles indicate S1P released from untreated and FTY720-treated cells, respectively. B–F, the CHO (mock) and CHO/SPHK2 (SPHK2) cells were incubated with releasing medium containing 5 μm Sph for 2 h in the presence (+) or absence (−) of 3 μm FTY720. The contents of intracellular FTY720 (B), intracellular FTY720-P (C), extracellular S1P (D), and intracellular S1P (E) were measured by HPLC. F shows the ratio of the values in D to those in E. Experiments were performed more than three times, and error bars indicate the S.D.

FIGURE 9.

Schematic illustration of the sphingolipid metabolic pathway in CHO cells expressing SPHK1/2 and hSPNS2. S1P and FTY720-P are synthesized through the phosphorylation of Sph and FTY720 by SPHK1 and/or SPHK2, respectively. S1P and FTY720-P are exported through SPNS2. Intracellularly, FTY720 inhibits the conversion of DH-Sph into DH-Cer and S1P into hexadecenal.