Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 6, 2016
`
`THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 277, No. 24, Issue of June 14, pp. 21453–21457, 2002
`Printed in U.S.A.
`
`The Immune Modulator FTY720 Targets Sphingosine
`1-Phosphate Receptors*
`
`Received for publication, March 25, 2002, and in revised form, April 18, 2002
`Published, JBC Papers in Press, April 19, 2002, DOI 10.1074/jbc.C200176200
`
`Volker Brinkmann‡, Michael D. Davis§, Christopher E. Heise¶, Rainer Albert‡, Sylvain Cottens‡,
`Robert Hof‡, Christian Bruns‡, Eva Prieschl储, Thomas Baumruker储, Peter Hiestand**,
`Carolyn A. Foster储, Markus Zollinger‡‡, and Kevin R. Lynch§¶§§
`From the Departments of ‡Transplantation, **Arthritis and Bone Metabolism, ‡‡Preclinical Safety, Novartis Pharma AG,
`Lichtstrasse 35, CH-4002 Basel, Switzerland, the 储Department of Dermatology and Immunopathology, Novartis Research
`Institute, Brunnerstrasse 59, A-1235 Vienna, Austria, and the Departments of §Biochemistry and Molecular Genetics and
`¶Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0735
`
`Immunosuppressant drugs such as cyclosporin have
`allowed widespread organ transplantation, but their
`utility remains limited by toxicities, and they are inef-
`fective in chronic management of autoimmune diseases
`such as multiple sclerosis. In contrast, the immune mod-
`ulating drug FTY720 is efficacious in a variety of trans-
`plant and autoimmune models without inducing a gen-
`eralized immunosuppressed state and is effective in
`human kidney transplantation. FTY720 elicits a lym-
`phopenia resulting from a reversible redistribution of
`lymphocytes from circulation to secondary lymphoid
`tissues by unknown mechanisms. Using FTY720 and sev-
`eral analogs, we show now that FTY720 is phosphoryl-
`ated by sphingosine kinase; the phosphorylated com-
`pound is a potent agonist at
`four
`sphingosine
`1-phosphate receptors and represents the therapeutic
`principle in a rodent model of multiple sclerosis. Our
`results suggest that FTY720, after phosphorylation, acts
`through sphingosine 1-phosphate signaling pathways to
`modulate chemotactic
`responses and lymphocyte
`trafficking.
`
`FTY720 is derived from ISP-1 (myriocin), a fungal metabolite
`that is an eternal youth nostrum in traditional Chinese herbal
`medicine (1). The compound (2-amino-2-[2-(4-octylphenyl)eth-
`yl]propane-1,3-diol) is a novel, high potency immune modulat-
`ing agent that is remarkably effective in a variety of autoim-
`mune and transplant models including islet transplantation (2)
`and has recently proven to be effective in renal transplantation
`in man (3). Unlike the currently used immunosuppressive
`agents (e.g. the calcineurin inhibitors cyclosporin and tacroli-
`mus), FTY720 does not inhibit T cell activation and prolifera-
`tion and in rodent models does not impair immunity to sys-
`temic viral
`infection (4). If confirmed in man, the latter
`property provides a striking advantage over current immuno-
`suppressive therapies. FTY720 apparently sequesters lympho-
`cytes from circulation to secondary lymph tissue compartments
`
`(5) with concomitant reduction of specific effector T cells recir-
`culating from the lymph nodes to inflamed peripheral tissues
`(4) and graft sites (6). FTY720 does not act via the lymphocyte-
`homing chemokine receptor CCR-7 because FTY720 is active
`both in CCR-7-deficient mice and plt (paucity of lymph node T
`cells) mice, which lack CCR-7 ligands (CCL-19 and CCL-21) (7).
`FTY720-induced lymphocyte homing is sensitive to suppres-
`sion by pertussis toxin (6–8), which suggests that the molecu-
`lar target of the drug is a G protein-coupled receptor (GPCR)1
`interacting with heterotrimeric G proteins of the ␣i/o type. The
`affected GPCR(s) is on the lymphocyte since fluorescently la-
`beled lymphocytes treated with pertussis toxin ex vivo and
`transferred to mice are not depleted by FTY720 in vivo (8). The
`structural similarity of FTY720 and sphingosine has prompted
`speculation that the drug might act via the sphingosine 1-phos-
`phate (S1P) receptor S1P4 (formerly Edg-6)2 that is known to
`be expressed by lymphocytes (9).
`S1P is a pleiotropic lysophospholipid mediator; the promi-
`nent cellular responses to applied S1P are transient calcium
`mobilization, inhibition of adenylyl cyclase, escape from apo-
`ptosis (10), increased cell migration (11, 12), and mitogenesis
`(13). The physiologic role of S1P remains undefined although
`cell culture experiments and the phenotype of a mouse with the
`S1P1 receptor gene ablated suggest a role for S1P in vascular
`maturation (14, 15). Responses to S1P are mediated through a
`set of five cell surface GPCRs (S1P1–5), and the various effects
`of S1P have been attributed to interactions with one or more of
`these receptors (16). S1P is formed by the action of sphingosine
`kinase on sphingosine (17). The activity of this enzyme is
`increased in response to external stimuli (18, 19), and enforced
`expression of sphingosine kinase increases both cell prolifera-
`tion and survival (20). Sphingosine is converted rapidly to S1P
`when added to cells (21), while the route of S1P degradation to
`sphingosine might proceed via an ectophosphatase (22). To
`learn whether FTY720 might participate in the sphingosine-
`S1P signaling cascade, we performed the studies described
`herein.
`
`* Work at the University of Virginia was supported by National
`Institutes of Health Grants R01 GM52722 and R01 CA88994 and a
`research contract grant from Novartis Pharma AG (to K. R. L.) as well
`as National Institutes of Health Predoctoral Fellowship F31 GM64101
`(to M. D. D.). The costs of publication of this article were defrayed in
`part by the payment of page charges. This article must therefore be
`hereby marked “advertisement” in accordance with 18 U.S.C. Section
`1734 solely to indicate this fact.
`§§ To whom correspondence should be addressed: Dept. of Pharma-
`cology, Box 800735, University of Virginia Health System, 1300
`Jefferson Park Ave., Charlottesville, VA 22908-0735. Tel.: 434-924-
`2840; Fax: 434-982-3878; E-mail: krl2z@virginia.edu.
`
`This paper is available on line at http://www.jbc.org
`
`1 The abbreviations used are: GPCR, G protein-coupled receptor; S1P,
`sphingosine 1-phosphate; EAE, experimental autoimmune encephalo-
`myelitis; GTP␥S, guanosine 5⬘-3-O-(thio)triphosphate; HPLC, high
`pressure liquid chromatography.
`2 The International Union of Pharmacology subcommittee on lyso-
`phospholipid receptor nomenclature has recommended that the collo-
`quial “Edg” nomenclature be replaced with S1P (or lysophosphatidic
`acid (LPA)) subscript number, where the number indicates chronology
`of molecular cloning. Thus for S1P receptors, Edg-1 becomes S1P1,
`Edg-5 becomes S1P2, Edg-3 becomes S1P3, Edg-6 becomes S1P4, and
`Edg-8 becomes S1P5.
`21453
`
`Apotex v. Novartis
`IPR2017-00854
`NOVARTIS 2008
`
`

`

`21454
`
`FTY720 and Sphingosine 1-Phosphate Signaling
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 6, 2016
`
`FIG. 1. Structures (not shown are the S-enantiomers of AAL
`and AFD).
`
`of the phosphorylated drug (Fig. 2C). This pattern of active
`tissues best matches the RNA localization of sphingosine ki-
`nase type 1 (27). The concept that phosphorylated FTY720
`might be the active principle is intriguing and suggests that an
`alcohol/phosphate cycling of FTY720/FTY720-P takes place in
`vivo as occurs with sphingosine/S1P. Indeed FTY720 was con-
`verted extensively to FTY720-P in vivo, resulting in up to 4-fold
`higher blood levels of FTY720-P compared with parent FTY720
`(Table I). To learn whether FTY720-P is dephosphorylated in
`vivo, we administered single doses of FTY720-P to mice and
`assayed blood levels of FTY720 after 24 h. FTY720 could be
`detected after the lowest dose (0.1 mg/kg) of FTY720-P and
`increased in a dose-dependent fashion (Table I). We next de-
`termined whether synthetic phosphate derivatives, namely
`FTY720-P, (R)-AFD, and (S)-AFD, which resemble S1P (Fig. 1),
`were agonists at S1P receptors. To interrogate the individual
`S1P receptors, we used a membrane-based [␥-35S]GTP␥S bind-
`ing assay that allows direct comparison of the rank order
`potencies (pEC50) and relative efficacies (Emax) of agonist li-
`gands at isolated receptors (24). All of our compounds were
`agonists at the S1P4 receptor, although FTY720-P and (R)-AFD
`were far more potent than their non-phosphorylated congeners
`(Fig. 3). Both FTY720-P and (R)-AFD were high potency ago-
`nists also at the S1P1, S1P3, and S1P5 receptors (Table II), but
`the corresponding alcohols (FTY720 and AAL) were not effica-
`cious at these three receptors (data not shown). Although
`FTY720-P behaved as a partial agonist in the [␥-35S]GTP␥S
`binding assay (Table II), this compound was a full agonist in
`whole cell assays of inhibition of cAMP accumulation where
`there exists more amplification of signal (data not shown).
`None of our compounds were active at the S1P2 receptor in our
`assays at concentrations up to 10 ␮M. The receptor activation
`data are consistent with ligand binding measurements, which
`demonstrate a high affinity interaction between FTY720-P and
`(R)-AFD but not (S)-AFD and the S1P receptors (data not
`shown). Finally the compounds were not active at the three
`receptors for a structurally related lysophospholipid mediator,
`lysophosphatidic acid (data not shown).
`To learn whether this pattern of activity is recapitulated in
`vivo, we determined the potency of our compounds in reducing
`numbers of circulating T lymphocytes. FTY720 and (R)-AAL
`
`EXPERIMENTAL PROCEDURES
`Sphingosine Kinase Assay—The assays were performed as described
`previously (23) using mouse recombinant sphingosine kinase 1a ex-
`pressed in Escherichia coli. The reaction buffer contained 50 mM Hepes,
`pH 7.4, 15 mM MgCl2, 10% glycerol, and 0.05% Triton X-100. Substrates
`(sphingosine, FTY720, or AAL) were incubated at various concentra-
`tions with 10 nM sphingosine kinase, 10 ␮M ATP/0.5 ␮l of [␥-32P]ATP
`(3000 Ci/mmol, Amersham Biosciences) at 30 °C for 1 h. Lipids were
`extracted with 2 volumes of CHCl3/methanol (1:2), the organic extrac-
`tion product was dried, and the pellet was redissolved. The lipids were
`separated on a thin layer chromatography plate using a 1-butanol/
`acetic acid/water (6:2:2) solvent system after which the plate was ex-
`posed to x-ray film to detect phosphorus-32-labeled lipids.
`Organ Culture—Mouse organs (as indicated) were prepared and kept
`for 24 h in medium containing tritiated FTY720. Single cells were
`prepared from those organs by cell strainer, cells were lysed, and a lipid
`extraction was performed from 106 cells of each organ or from the
`corresponding culture supernatant. Extraction and analysis by thin
`layer chromatography was done as described above.
`[␥-35S]GTP␥S Binding Assays—Membranes were prepared from ei-
`ther insect Sf9 cells that were infected with recombinant baculoviruses
`encoding receptor and G proteins (for the S1P1 receptor), HEK293T
`cells transfected with DNAs encoding S1P receptors as well as G pro-
`teins (S1P4 and S1P5 receptors), or rat hepatoma RH7777 cells that
`were transfected with receptor DNA alone (S1P3 receptor). After 48 h,
`cells were collected, and crude microsomal membranes were prepared.
`Ligand stimulation of [␥-35S]GTP␥S binding was performed as de-
`scribed previously (24). In membranes from all three cell types, agonist
`stimulation of [␥-35S]GTP␥S binding was entirely dependent on exoge-
`nous receptor.
`Measurement of Circulating Lymphocytes—FTY720 and AAL were
`dissolved in water and administered by gavage to Lewis rats at various
`doses. FTY720-P and AFD were dissolved in water:Me2SO (5:1, v/v) and
`injected intraperitoneally into C3H mice (1 mg/kg). Blood was collected
`from the tail vein of mice or the sublingual vein of rats 6 h after drug
`administration and subjected to hematology using an automated Tech-
`nicon H1-E analyzer (Bayer Diagnostics, Zu¨ rich, Switzerland).
`Apoptosis Assay—Human CD4⫹ T cells were negatively selected from
`Ficoll-isolated peripheral blood mononuclear cells by magnetic cell sort-
`ing according to standard procedures using anti-CD8-, anti-CD20-, and
`anti-CD14-coated Miltenyi Biotec (Gladbach, Germany) magnetic
`beads. CD4⫹ T cells (106/ml) were incubated with increasing concentra-
`tions of compounds for 4 h. Cells were stained for expression of phos-
`phatidylserine in the outer leaflet of the membranes using the Annexin-
`V-FLUOS staining kit (Roche Molecular Biochemicals), and positive
`cells were detected by fluorescence-activated cell sorting.
`Experimental Autoimmune Encephalomyelitis (EAE) Model—Wistar
`rats were immunized with an emulsion of bovine spinal cord in com-
`plete Freund’s adjuvant as described previously (25). Two-week oral
`treatment with FTY720 (aqueous solution) or the enantiomers (dis-
`solved in water:Me2SO, 10:1, v/v) was started on day 0 using a dose of
`0.3 mg/kg/day. Positive controls received vehicle alone. Animals (10 per
`group) were monitored daily and graded according to disease symp-
`toms: 1, flaccid tail; 2, hind limb weakness or ataxia; 3, full paralysis of
`hind limbs.
`
`RESULTS
`We used a set of FTY720-like compounds to determine sphin-
`gosine kinase and S1P receptor activity and correlate these
`with assays of lymphocyte function. In addition to FTY720,
`we tested both enantiomers (AAL) of an analog described by
`Kiuchi et al. (26) wherein a hydroxymethylene substituent of
`FTY720 was replaced by a methyl group (Fig. 1). These enan-
`tiomers have very different activities; the ID50 values for de-
`creasing circulating T lymphocytes in rats were reported to be
`0.009 and ⬎1 mg/kg for the R- and S-enantiomers, respectively,
`while the ID50 value for FTY720 in the same system was 0.024
`mg/kg (26).
`We first asked whether these compounds were substrates for
`sphingosine kinase. Recombinant mouse sphingosine kinase 1a
`catalyzed the phosphorylation of FTY720 and (R)-AAL but not
`(S)-AAL (Fig. 2, A and B). Moreover lymphoid tissue including
`Peyer’s patches, spleen, and lymph nodes effectively phospho-
`rylated FTY720, while heart, liver, and kidney contained little
`
`

`

`FTY720 and Sphingosine 1-Phosphate Signaling
`
`21455
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 6, 2016
`
`FIG. 2. A and B, in vitro kinase assay with recombinant mouse sphingosine kinase 1a using FTY720, (R)-AAL, (S)-AAL, and sphingosine as
`substrates. The phosphorylated compounds, which were labeled with phosphorus-32, were detected by autoradiography. The substrate alcohols
`were detected by spraying the TLC plate with Fluram威 (a fluorescent dye that binds to compounds with a primary amine) followed by photography
`with UV illumination. Thus the image shown in A or B is a composite of two images. The compound used as a substrate as well as the concentration
`(in ␮M) is indicated underneath the panels, 10 ␮M sphingosine (Sph) was used as a reference point. No phosphorylated form(s) was detected in this
`in vitro assay when omitting either the enzyme or the substrates. N gives a normalization control for equal loading/extraction after “staining” the
`thin layer chromatography plate with Fluram. Conversion rates of sphingosine to S1P ranged, depending on the enzyme preparation and the assay
`conditions, between 0.5 and 10% in this assay. The autoradiogram in B represents an ⬃50-fold longer exposure time than that presented in A. C,
`the fate of [3H]FTY720 in various mouse tissues (normalized on a cell basis of 106 cells). Shown are autoradiograms of thin layer chromatography
`plates whereby FTY720 and its phosphorylated form were resolved; the positions of FTY720 and FTY720-P are indicated at the right. Further
`details are provided under “Experimental Procedures.”
`
`TABLE I
`Metabolism of FTY720 and FTY720-P in rodents
`Compound detecteda
`
`Compound
`administered
`
`Dose
`
`FTY720
`
`FTY720-P
`
`3 h
`
`8 h
`
`24 h
`
`72 h
`
`3 h
`
`8 h
`
`24 h
`
`72 h
`
`ng/ml
`mg/kg
`109
`437
`24
`179 383
`133 73
`85
`7.5
`FTY720b
`FTY720c
`NDd ND
`1.15 ND ND ND ND ND
`0.1
`FTY720c
`ND ND
`6.20 ND ND ND ND ND
`0.3
`FTY720-Pc
`ND ND
`0.15 ND ND ND ND ND
`0.1
`FTY720-Pc
`ND ND
`1.06 ND ND ND ND ND
`0.3
`FTY720-Pc
`ND ND
`4.91 ND ND ND ND ND
`1.0
`a Compound was detected by HPLC separation after extraction from
`blood pools from three animals.
`b FTY720 administered to Wistar rats (oral).
`c FTY720 or FTY720-P administered to C3H mice (intraperitoneal).
`d ND, not determined.
`
`potently reduced circulating T lymphocyte levels in rats in a
`dose-dependent manner, whereas (S)-AAL and sphingosine
`were completely inactive at doses up to 1 mg/kg (Fig. 4A). We
`obtained the analogous result with the respective phosphoryl-
`ated compounds; a 1 mg/kg bolus injection of FTY720-P or
`(R)-AFD reduced circulating T cells by about 70%, whereas
`(S)-AFD and S1P were inactive with this dosing regimen (Fig.
`4B). The lymphopenic activity of FTY720 and (R)-AAL in vivo
`(Fig. 4 and Ref. 25) thus can be explained by their metabolism
`to the phosphorylated forms (FTY720-P and (R)-AFD), which
`are potent agonists at multiple S1P receptors. The lack of
`efficacy of (S)-AAL in decreasing circulating lymphocytes can-
`not be credited only to its failure as a substrate for sphingosine
`kinase because even when phosphorylated (synthetically) to
`form (S)AFD, it lacks affinity for S1P receptors (Table II).
`FTY720 evokes apoptosis in lymphocytes at micromolar con-
`centrations, prompting the idea that the drug acts by killing
`lymphocytes (28). We consider this mechanism highly unlikely
`
`FIG. 3. Concentration-response curves of compounds at the
`recombinant human S1P4 receptor. A membrane-based [␥-35S]
`GTP␥S binding assay was used to determine the relative potencies and
`efficacies of the compounds. Each point represents triplicate measure-
`ments, and these data are representative of those used to generate the
`values presented in Table II. E, (R)-AFD; ‚, FTY720-P; f, S1P; Œ,
`FTY720; ⽧, (R)-AAL; , (S)-AAL; 䡺, (S)-AFD.
`
`in view of the low nanomolar levels (Cmax ⬍ 50 nM) of FTY720
`realized in the blood of rats treated with the high dose of 1
`mg/kg (29). Nevertheless we determined whether the apoptotic
`potential of our compounds correlated with activities in vitro or
`in vivo. We observed apoptotic responses only in T cells treated
`with micromolar concentrations of non-phosphorylated com-
`pounds (Table III). This pattern is reminiscent of reports of the
`activity of sphingosine and S1P where sphingosine is associated
`with apoptosis and S1P is associated with protection from apo-
`ptosis (10, 30). The apoptotic responses elicited by the non-phos-
`phorylated compounds were neither stereoselective nor inhibited
`
`

`

`21456
`
`FTY720 and Sphingosine 1-Phosphate Signaling
`
`TABLE II
`Potency (pEC50) and efficacy (Emax) values of phosphorylated compounds at human S1P receptors
`Drug
`
`Parameter
`
`Receptor
`
`S1P1
`
`S1P3
`
`S1P4
`
`S1P5
`
`a
`
`(S)-AFD
`⬍5
`
`⬍5
`
`⬍5
`
`⬍5
`
`(R)-AFD
`FTY720-P
`S1P
`8.6 (8.4–8.8)
`8.2 (8.1–8.2)
`7.1 (7.0–7.3)b
`pEC50
`0.92 (0.84–1.03)
`0.92 (0.84–1.03)
`1.00
`Emax
`8.4 (7.8–9.4)
`8.4 (7.8–9.4)
`8.7 (8.4–8.9)
`pEC50
`0.34 (0.27–0.38)
`0.34 (0.27–0.38)
`1.00
`Emax
`8.4 (8.3–8.4)
`7.2 (6.6–7.9)
`6.1 (5.9–6.1)
`pEC50
`1.01 (0.92–1.09)
`0.70 (0.62–0.75)
`1.00
`Emax
`8.9 (8.0–9.7)
`8.2 (7.2–9.1)
`7.7 (7.4–8.0)
`pEC50
`0.64 (0.57–0.68)
`0.61 (0.56–0.66)
`1.00
`Emax
`a pEC50, ⫺log molar concentration of compound resulting in 50% of maximal GTP␥S binding; Emax, maximal [␥-35S]GTP␥S binding as a fraction
`of S1P signal (set at 100% ⫽ 1.00).
`b Average value of three to six experiments (range of values).
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 6, 2016
`
`FIG. 4. Dose-dependent depletion of circulating peripheral
`blood lymphocytes in rats. Phosphorylated compounds (B) were
`administered at 1 mg/kg. In all cases, blood was drawn 6 h after drug
`administration. ⽧, FTY720; Œ, (R)-AAL; ●, (S)-AAL; E, sphingosine.
`
`TABLE III
`Induction of apoptosis in human CD4⫹ T cells
`
`FIG. 5. Prophylactic effect of FTY720, (R)-AAL, and (S)-AAL in
`EAE in rats. Compounds were administered orally to rats at 0.3
`mg/kg/day. Disease symptom grades are as follows: 1, flaccid tail; 2,
`hind limb weakness or ataxia; 3, full paralysis of hind limbs. E, vehicle;
`●, FTY720; f, (R)-AAL; , (S)-AAL. p.o., per os.
`
`Compound
`
`30 ␮M
`
`Apoptosis in the presence of compound
`a
`
`10 ␮M
`
`3 ␮M
`
`1 ␮M
`
`0.3 ␮M
`
`%
`0.9
`12.7
`24.0
`FTY720
`0.7
`0.8
`0.4
`FTY720-P
`2.6
`10.6
`33.7
`(R)-AAL
`(R)-AFD, phos.b
`0
`1.0
`2.6
`2.9
`13.1
`34.9
`(S)-AAL
`1.6
`0.9
`1.3
`(S)-AFD, phos.
`a Data are representative of three experiments.
`b phos. ⫽ phosphorylated compound.
`
`1.4
`0.7
`0.6
`1.2
`0.4
`0.6
`
`0
`1.8
`0.5
`1.3
`2.4
`0.2
`
`by prior pertussis toxin treatment (data not shown); both prop-
`erties contrast the behavior of these compounds in mice or rats
`regarding the depletion of circulating lymphocytes (7, 26).
`The unique mechanism that apparently underlies the im-
`mune modulating effects of FTY720, i.e. increased homing of
`T cells toward the lymphatic system and away from inflam-
`matory tissues (5), provides an opportunity for therapy of
`autoimmune disorders that does not exist with current im-
`munosuppressive agents. Therefore, we tested FTY720 and
`both enantiomers of AAL in EAE, which is a primary model of
`human multiple sclerosis (31). Treatment of Wistar rats with
`FTY720 or (R)-AAL (0.3 mg/kg/day) completely prevented the
`development of EAE, whereas (S)-AAL was entirely inactive
`(Fig. 5). Thus the prophylactic activities of the compounds in
`this model are entirely consistent with their activity on S1P
`receptors
`and
`their
`potential
`to
`reduce
`circulating
`lymphocytes.
`
`DISCUSSION
`Administration of FTY720 somehow resets a rheostat that
`apportions lymphocytes between the circulatory and secondary
`
`lymphoid tissue compartments. We have now documented that
`FTY720 and an analog ((R)-AAL), after phosphorylation to
`FTY720-P and (R)-AFD, are high affinity agonists at four (of
`five) S1P receptors. The correlation of substrate activity (Fig.
`2), agonism at S1P receptors (Fig. 3 and Table II), induction of
`lymphopenia (Fig. 4), and activity in the EAE model (Fig. 5)
`suggests strongly that FTY720 and an analog (i.e. (R)-AAL)
`function ultimately as S1P mimetics that increase the lympho-
`cyte homing response.
`The failure of sphingosine and S1P to evoke lymphopenia
`when administered to rats (Fig. 4B) might relate to an approx-
`imately 1 log order higher potency of FTY720-P and (R)-AFD
`compared with S1P at the S1P1 and S1P4 receptors (Table I)
`but could also be due to a different metabolic rate for the
`naturally occurring compound. For example, the rate of de-
`phosphorylation of S1P, FTY720-P, and (R)-AFD, presumably
`proceeding either through the non-selective lipid phosphate
`phosphohydrolases (22) or the S1P phosphatase (30), might be
`different with resultant differences in accumulation. Alter-
`nately FTY720 and (R)-AAL might be more effective substrates
`of the sphingosine kinase in vivo.
`Our discovery that FTY720 can be phosphorylated to yield a
`potent S1P mimetic has substantial implications for S1P biol-
`ogy. This pleiotropic lipid mediator is most often characterized
`as promoting angiogenesis, cell proliferation, and escape from
`apoptosis. To this list must now be added immune system
`modulation via changes in lymphocyte trafficking. In addition
`to increasing knowledge of lysophospholipid medicinal chemis-
`try, our results reinforce the notion that sphingosine partici-
`pates in a cycle of phosphorylation/dephosphorylation that gov-
`erns the levels of the alcohol (sphingosine) and phosphate (S1P)
`forms and thus establishes the S1P tone of tissues. The ability of
`FTY720 to participate in this cycle to establish an exaggerated
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 6, 2016
`
`FTY720 and Sphingosine 1-Phosphate Signaling
`
`21457
`
`S1P tone probably underlies the high potency and efficiency of
`this drug. The existence of this cycle and its effect on lymphocyte
`trafficking might confound development of drugs that are sphin-
`gosine kinase inhibitors or S1P receptor antagonists.
`FTY720-P and its active analog, (R)AFD, are potent agonists
`at four S1P receptors, three of which (S1P1, S1P4, and S1P5)
`are expressed by lymphocytes. Although pertussis toxin treat-
`ment of lymphocytes ex vivo demonstrates that a G protein
`signaling pathway in the lymphocyte is essential for the
`FTY720-promoted homing response (7, 8), S1P receptors on
`endothelial cells (including S1P1 and S1P3) might participate
`in the process also. Thus only the S1P2 receptor, at which
`FTY720-P and (R)-AFD are inactive, is eliminated from con-
`tention. Likewise we cannot know from present data what
`sphingosine kinase isoform or what phosphatase isoform is
`relevant to the metabolism of FTY720. Additional chemical
`entities and genetically modified mice lacking one or more S1P
`receptor genes or sphingosine kinase genes are needed to de-
`fine the FTY720 target(s) more precisely.
`An interesting finding reported recently by Hla and col-
`leagues (21) is that sphingosine kinase 1a can be released from
`cultured cells. Although their experimental system was neces-
`sarily artificial and its prediction requires confirmation, taken
`at face value it suggests the intriguing notion that the phos-
`phorylation of sphingosine might be extracellular. Since the
`lipid phosphate phosphohydrolases are clearly ectophosphata-
`ses (22), the entire cycle might proceed in the extracellular
`compartment. Such a system would provide a route whereby
`lysophospholipid receptors could be accessed using orally avail-
`able lipid alcohol compounds as exemplified by FTY720.
`FTY720 is the first in a class of new immune system modu-
`lators that may allow both better management of allograft
`recipients and more effective treatment of patients with auto-
`immune disorders, which is a substantially unmet medical
`need. The drug is apparently less toxic than existing regimens,
`and in striking contrast to classical
`immunosuppressants,
`FTY720 did not impair immunity to systemic viral infection (4),
`suggesting that treatment with FTY720 could reduce the inci-
`dence of opportunistic infections in transplant patients. The
`drug may even be used to treat inflammatory processes asso-
`ciated with chronic viral infection since in a model of viral
`myocarditis, FTY720 (but not cyclosporin) reduced inflamma-
`tory processes and pathology without accelerating virus repli-
`cation (32). Very encouraging is the activity of FTY720 in
`models of autoimmune disorders such as EAE (Fig. 5), a model
`of human multiple sclerosis. Calcineurin inhibitors (cyclos-
`porin and tacrolimus), which block T cell activation, are of
`limited use in the chronic management of such diseases. Per-
`haps the effects of FTY720 in EAE may relate to a direct effect
`on neuronal cells and/or oligodendrocytes expressing S1P re-
`ceptors (33). Activation of S1P receptors can antagonize apo-
`ptotic processes (21), which are associated with early stages of
`progressive neurodegenerative and demyelinating diseases
`(34, 35).
`
`Acknowledgments—We thank C. Wilt, C. Kristofic, C. Pally, C.
`Simeon, H. Wiegand, and J.-P. Baldeck of Novartis and R. Jarosz of the
`University of Virginia for excellent technical assistance.
`
`Addendum—While this paper was in review, a report (36) was pub-
`lished describing the metabolism of FTY720 in rodents and the agonist
`activity of FTY720-P at S1P receptors.
`
`REFERENCES
`1. Fujita, T., Inoue, K., Yamamoto, S., Ikumoto, T., Sasaki, S., Toyama, R., Chiba,
`K., Hoshino, Y., and Okumoto, T. (1994) J. Antibiot. 47, 208–215
`2. Brinkmann, V., Pinschewer, D. D., Feng, L., and Chen, S. (2001) Transplan-
`tation 72, 764–769
`3. Tedesco, T., Kahan, B., Mourad, G., Vanrenterghem, Y., Grinyo, J., Weimar,
`W., Pellet, P., Chodoff, L., and Sablinski, T. (2001) Am. J. Transplant. 1,
`S243
`4. Pinschewer, D. D., Ochsenbein, A. F., Odermatt, B., Brinkmann, V.,
`Hengartner, H., and Zinkernagel, R. M. (2000) J. Immunol. 164, 5761–5770
`5. Chiba, K. Yanagawa, Y., Yasubuchi, Y., Kataoka, H., Kawaguchi, T., Ohtsuki,
`M., and Hoshino, Y. (1998) J. Immunol. 160, 5037–5044
`6. Yanagawa, Y., Sugahara, K., Kataoka, H., Kawaguchi, T., Masubuchi, Y., and
`Chiba, K. (1998) J. Immunol. 160, 5493–5499
`7. Henning, G., Ohl, L., Junt, T., Reiterer, P., Brinkmann, V., Nakano, H.,
`Hohenberger, W., Lipp, M., and Fo¨rster, R. (2001) J. Exp. Med. 194,
`1875–1881
`8. Brinkmann, V., Pinschewer, D. D., Chiba, K., and Feng, L. (2000) Trends
`Pharmacol. Sci. 21, 49–52
`9. Gra¨ler, M. H., Bernhardt, G., and Lipp, M. (1998) Genomics 53, 164–169
`10. Cuvillier, O., Pirianov, G., Kleusar, B., Vanek, P. G., Coso, O. A., Gurkind,
`J. S., and Spiegel, S. (1996) Nature 381, 800–803
`11. Palik, J. H., Chae, S.-S., Lee, M.-J., Thangada, S., and Hla, T. (2001) J. Biol.
`Chem. 276, 11830–11837
`12. Hobson, J. P., Rosenfeldt, H. M., Barak, L. S., Olivera, A., Poulton, S., Caron,
`M. G., Milstein, S., and Spiegel, S. (2001) Nature 291, 1800–1803
`13. Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S.
`(1991) J. Cell Biol. 114, 155–167
`14. 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
`15. 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. Investig. 106, 951–961
`16. Hla, T., Lee, M. J., Ancellin, N., Paik, J. K., and Kluk, M. J. (2001) Science 294,
`1875–1878
`17. Kohama, T. A., Olivera, A., Edsall, L., Nagiec, M. M., Dickson, R., and Spiegel,
`S. (1998) J. Biol. Chem. 273, 23722–23728
`18. Xia, P., Gamble, J. R., Rye, K. A., Wang, L., Hii, C. S. T., Cockerill, P.,
`Khew-Goodall, Y., Bert, A. G., Barter, P. J., and Vadas, M. A. (1998) Proc.
`Natl. Acad. Sci. U. S. A. 95, 14196–14201
`19. Melendez, A., Floto, R. A., Gillooly, D. J., Harnett, M. M., and Allen, J. M.
`(1998) J. Biol. Chem. 273, 9393–9402
`20. Olivera, A., Kohama, T., Edsall, L., Nava, V., Cuvillier, O., Poulton, S., and
`Spiegel, S. (1999) J. Cell Biol. 147, 545–557
`21. Ancellin, N., Colmont, C., Su, J., Li, Q., Mittereder, N., Chae, S.-S., Stefansson,
`S., Liau, G., and Hla, T. (2002) J. Biol. Chem. 277, 6667–6675
`22. Kai, M., Wada, I., Imai, S., Sakane, F., and Kanoh, H. (1997) J. Biol. Chem.
`272, 24572–24578
`23. Prieschl, E. E., Csonga, R., Novotny, V., Kikuchi, G., and Baumruker, T. (1999)
`J. Exp. Med. 190, 1–8
`24. Im, D.-S., Clemens, J., Macdonald, T. L., and Lynch, K. R. (2001) Biochemistry
`40, 14053–14060
`25. Bolton, C., Borel, J. F., and Cuzner, M. L. (1982) J. Neurol. Sci. 56, 147–153
`26. Kiuchi, M., Adachi, K., Kohara, T., Minoguchi, M., Hanano, T., Aoki, Y.,
`Mishina, T., Arita, M., Nakao, N., Ohtsuki, M., Hoshino, Y., Teshima, K.,
`Chiba, K., Sasaki, S., and Fujita, T. (2000) J. Med. Chem. 43, 2946–2961
`27. Liu, H., Sugiura, M., Nava, V. E., Edsall, L. C., Kono, K., Poulton, S., Milstein,
`S., Kohama, T., and Spiegel, S. (2000) J. Biol. Chem. 275, 19513–19520
`28. Matsuda, S., Minowa, A., Suzuki, S., and Koyasu, S. (1999) J. Immunol. 162,
`3321–3326
`29. Nikolova, Z., Hof, A., Baumlin, Y., and Hof, R. P. (2001) Transplantation 72,
`168–171
`30. Mandala, S. M., Thornton, R., Galve-Roperh, I., Poulton, S., Peterson, C.,
`Olivera, A., Bergstrom, J., Kurtz, M. B., and Spiegel S. (2000) Proc. Natl.
`Acad. Sci. U. S. A. 97, 7859–7864
`31. Steinman, L. (1999) Neuron 24, 511–514
`32. Miyamoto, T., Matsumori, A., Hwang, M. W., Nishio, R., Ito, H., and
`Sasayama, S. (2001) J. Am. Coll. Cardiol. 37, 1713–1718
`33. Beer, M. S., Stanton, J. A., Salim, K., Rigby, M., Heavens, R. P., Smith, D., and
`McAllister, G. (2000) Ann. N. Y. Acad. Sci. 905, 118–131
`34. Kanazawa, I. (2000) Trends Mol. Med. 7, 339–344
`35. Mattson, M. P. (2000) Nat. Rev. Mol. Cell. Biol. 1, 120–129
`36. Mandala, S., Hajdu, R., Bergstrom, J., Quackenbush, E., Xie, J., Milligan, J.,
`Thornton, R., Shei, G. J., Card, D., Keohane, C., Rosenbach, M., Hale, J.,
`Lynch, C. L., Rupprecht, K., Parsons, W., and Rosen, H. (2002) Science 296,
`346–349
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 6, 2016
`
`The Immune Modulator FTY720 Targets Sphingosine 1-Phosphate Receptors
`Volker Brinkmann, Michael D. Davis, Christopher E. Heise, Rainer Albert, Sylvain
`Cottens, Robert Hof, Christian Bruns, Eva Prieschl, Thomas Baumruker, Peter Hiestand,
`Carolyn A. Foster, Markus Zollinger and Kevin R. Lynch
`2002, 277:21453-21457.J. Biol. Chem.
`
`doi: 10.1074/jbc.C200176200 originally published online April 19, 2002
`
`
`
`10.1074/jbc.C200176200Access the most updated version of this article at doi:
`
`Alerts:
`•
`•
`
`Click here
`
`
`When this article is cited
`When a correction for this article is posted
`
` to choose from all of JBC's e-mail alerts
`
`This article cites 34 references, 18 of which can be accessed free at
`http://www.jbc.org/content/277/24/21453.full.html#ref-list-1
`
`
`