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Down-regulation of S1P1 Receptor Surface Expression byProtein Kinase C Inhibition*

Received for publication, July 28, 2009, and in revised form, December 18, 2009 Published, JBC Papers in Press, December 23, 2009, DOI 10.1074/jbc.M109.049692

Sven-Christian Sensken and Markus H. Graler1

From the Institute for Immunology, Hannover Medical School, 30625 Hanover, Germany

The sphingosine 1-phosphate receptor type 1 (S1P1) is impor-tant for themaintenance of lymphocyte circulation. S1P1 recep-tor surface expression on lymphocytes is critical for their egressfrom thymus and lymph nodes. Premature activation-inducedinternalization of the S1P1 receptor in lymphoid organs, medi-ated either by pharmacological agonists or by inhibition of theS1P degrading enzyme S1P-lyase, blocks lymphocyte egress andinduces lymphopenia in blood and lymph. Regulation of S1P1receptor surface expression is therefore a promisingway to con-trol adaptive immunity. Hence, we analyzed potential cellulartargets for their ability to alter S1P1 receptor surface expressionwithout stimulation. The initial observation that preincubationof mouse splenocytes with its natural analog sphingosine wassufficient to blockTranswellTM chemotaxis to S1Pdirected sub-sequent investigations to the underlying mechanism. Sphingo-sine is known to inhibit protein kinase C (PKC), and PKC in-hibition with nanomolar concentrations of staurosporine,calphostin C, and GF109203X down-regulated surface expres-sion of S1P1 but not S1P4 in transfected rat hepatoma HTC4cells. The PKC activator phorbol 12-myristate 13-acetate par-tially rescued FTY720-induced down-regulation of the S1P1receptor, linking PKC activation with S1P1 receptor surfaceexpression. FTY720, but not FTY720 phosphate, efficientlyinhibited PKC. Cell-based efficacy was obvious with 10 nM

FTY720, and in vivo treatment of mice with 0.3–3 mg/kg/dayFTY720 showed increasing concentration-dependent effective-ness. PKC inhibition therefore may contribute to lymphopeniaby down-regulating S1P1 receptor cell surface expression inde-pendently from its activation.

Lymphocyte circulation is dependent on cell surface expres-sion of the S1P12 receptor (1). Its endogenous ligand S1P servesas an exit signal in blood and lymph for lymphocytes that areleaving lymph nodes and thymus (2). The lymphocytes arethought to migrate from lymphoid organs with no or low S1Plevels to blood and lymphwith high S1P levels along a proposed

S1P gradient (3). Deletion or down-regulation of the S1P1receptor in lymphocytes prevents their exit from thymus andlymph nodes and renders mice lymphopenic in blood andlymph (1, 4). Premature internalization of the S1P1 receptor isinduced by pharmacological agonists (5–7) and by inhibition ordeletion of the S1P-degrading enzyme S1P-lyase (8, 9). In bothcases the concentration of the endogenous or synthetic S1P1receptor agonist is predominantly increased in lymphoidorgans and prevents re-expression of S1P1 on the surface oflymphocytes (10). The exit signal S1P is consequently not rec-ognized anymore, and lymphocytes are trapped in thymus andlymph nodes (11). Furthermore endothelial cells are stimulatedvia S1P1 and close postulated endothelial cell barriers, againpreventing lymphocytes from passing (12–14). Regulation ofS1P1 receptor surface expression is therefore crucial for main-taining lymphocyte circulation and immune surveillance.The immunomodulator Fingolimod (2-amino-2-(2-[4-octyl-

phenyl]ethyl]-1,3-propanediol, FTY720) is a prodrug in phaseIII clinical trials for treatment ofmultiple sclerosis that serves asan agonist for S1P1 after phosphorylation (6, 15–17). Prematureactivation-induced down-regulation of S1P1 receptor surfaceexpression on lymphocytes is thought to be the main princi-ple for its biological activity in lymphoid organs, emphasizingthe importance of regulation of S1P1 receptor cell surfaceexpression for adaptive immune functions (1, 7, 18). It was alsotested in phase III clinical trials as immunosuppressant afterrenal transplantations but failed because of side effects such asbradycardia (19, 20). FTY720 phosphate (FTY-P) not only acti-vates S1P1 but also three of the four remaining S1P receptorsexcept S1P2 (6, 17). Furthermore FTY720 is cell-permeable andtargets many other intracellular enzymes related to sphingo-lipid metabolism like sphingosine kinases 1 and 2 (SK1/2) (21),phospholipase A2 (PLA2) (22), S1P-lyase (23), and ceramidesynthases (24). Because we originally observed a different spec-ificity for FTY720-induced inhibition of S1P receptors com-pared with FTY-P induced stimulation (7), we aimed to studypotential cellular targets of FTY720 involved in S1P1 receptorsurface expression that are modulated independently from itsphosphorylation to FTY-P.A prominent candidate in this regard is protein kinase C

(PKC), because it is inhibited by the natural analog sphingosine(Sph) (25, 26). Twelve PKC isoforms are known and are classi-fied as conventional, novel, and atypical PKC (27). S1P1 recep-tor phosphorylation and subsequent internalization can be per-formed by PKC after activation (28), and late recovery of S1P1surface expression on lymphocytes after long term exposurewith its endogenous ligand S1P is dependent on PKC� expres-sion (29). We therefore investigated the role of PKC for S1P1

* This work was supported by Deutsche Forschungsgemeinschaft GrantsGR-1943/1-4 of the Emmy Noether Program and GR-1943/2-1 of PriorityProgram 1267 “Sphingolipids: Signal and Disease.”

1 To whom correspondence should be addressed: Institute for Immunology,Hannover Medical School, Bldg. K11, OE 9422, Carl-Neuberg-Str. 1, 30625Hanover, Germany. Tel.: 49-511-532-9779; Fax: 49-511-532-9783; E-mail:[email protected].

2 The abbreviations used are: S1P1, sphingosine 1-phosphate receptor type 1;PKC, protein kinase C; Sph, sphingosine; PMA, phorbol 12-myristate 13-ac-etate; SK, sphingosine kinase; HA, hemagglutinin; PBS, phosphate-buff-ered saline; ELISA, enzyme-linked immunosorbent assay; LC/MS/MS, liquidchromatography-tandem mass spectrometry; PS, phosphatidylserine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 9, pp. 6298 –6307, February 26, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

6298 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 9 • FEBRUARY 26, 2010

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receptor surface expression and the influence of FTY720 onPKC activity in vitro using N-terminal hemagglutinin (HA)epitope-tagged human S1P1 (S1P1-HA) expressing rat hepa-toma HTC4 cells (7), and in vivo with wild type and SK2-defi-cient mice that are defective for FTY720 phosphorylation(30, 31).

EXPERIMENTAL PROCEDURES

Chemicals and Mice—S1P and the PKC activator phorbol12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich, and the PKC inhibitormyr-�PKC (myristoylated pep-tide: Myr-RFARKGALRQKNV) (32) was from Promega. ThePKC inhibitors calphostin C, GF109203X, and staurosporinewere purchased from Biomol GmbH (Hamburg, Germany), 1mM stock solutions were prepared inMe2SO, and aliquots werestored at �80 °C. Sph was purchased from Otto NordwaldGmbH (Hamburg, Germany). Chemicals and solvents werepurchased fromRoth (Karlsruhe, Germany) if not stated other-wise. AAL(R/S), FTY720, and FTY-P were kindly provided byVolker Brinkmann (Novartis, Basel, Switzerland) (17). FTY-Pwas dissolved at a concentration of 50 mM in Me2SO, 50 mM

HCl and diluted with methanol to 1 mM FTY-P. FTY720,AAL(R/S), and S1Pwere dissolved inmethanol to the final con-centration of 1 mM. C57BL/6 mice were purchased from theHannover Medical School Animal Facility (Hanover, Ger-many). SK2-deficient mice were kindly provided by AndreasBillich (Novartis). The anti-S1P antibody SphingomabTM wasliberally made available by Roger Sabbadini (LPath Inc., SanDiego, CA).Cell Culture—Serum, cell culture media, antibiotics, and

supplements were purchased from PAA Laboratories (Coelbe,Germany) if not otherwise stated. Wild type rat hepatomaHTC4 cells were cultured in Eagle’s minimum essentialmedium with Earle’s salts supplemented with 10% fetal calfserum, 1 mM sodium pyruvate, 2� nonessential amino acids,100 units/ml penicillin G, 100 �g/ml streptomycin, and 2 mM

L-glutamine. HTC4 cells expressing human S1P1-HA orS1P4-HA were cultured in the presence of G418 sulfate (0.4mg/ml). Primary splenocytes were incubated in RPMI 1640supplementedwith 10% fetal calf serum, 100 units/ml penicillinG, 100 �g/ml streptomycin, and 2 mM L-glutamine.Flow Cytometry—Cell staining and analysis were performed

according to standard protocols. Surface expression of S1P1-HA on HTC4 cells was analyzed by fluorescence-activated cellsorting using the FACSCalibur (Becton Dickinson, Heidelberg,Germany). The N-terminal HA epitope (peptide sequence:MGYPYDVPDYAGGP) was detected with the rat anti-HAantibody 3F10, kindly provided by Elisabeth Kremmer (GSF,Munich, Germany), and a goat anti-rat secondary antibodyconjugated with cyanine 2 (Chemicon International, Inc.). Cellstaining was performed with 12.5 �g/ml rat anti-HA and 5�g/ml goat anti-rat-cyanine 2 in phosphate-buffered saline(PBS) at 4 °C for 60 min. Blood was isolated from wild type orSK2-deficient mice by cardiac puncture, and lymphocytes werecounted with Flow Check� flow cytometry particles APCMaxi-Brite (Polysciences Europe GmbH, Eppelheim, Ger-many) after erythrocyte lysis and labeling with anti-CD3�-

fluorescein and anti-CD45R-PE (ImmunoTools, Friesoythe,Germany).PepTag� Fluorescent Protein Kinase C Assay—PKC activity

was tested using the PepTag� fluorescent protein kinase assay(Promega) according to themanufacturer’s protocol. This assayutilizes a brightly colored fluorescent peptide substrate that ishighly specific for PKC. Phosphorylation by PKC changes thenet charge of the substrate from�1 to�1, thereby allowing thephosphorylated and nonphosphorylated derivatives of the sub-strate to be separated by agarose gel electrophoresis. The phos-phorylated species migrates toward the positive electrode,whereas the nonphosphorylated substrate migrates toward thenegative electrode. The gel band containing the phosphoryla-ted peptide can subsequently be visualized under UV light. AllPepTag� PKC assay reaction components were combined onice, and PKC activity was assayed in a final volume of 25 �l ofthe following mixture: 5 �l of 5� PKC reaction buffer (100mM HEPES, pH 7.4, 6.5 mM CaCl2, 5 mM dithiothreitol, 50mM MgCl2, 5 mM ATP), 5 �l of PepTag� C1 peptide(PLSRTLSVAAK, 0.4 �g/�l in water), 5 �l of freshly sonicatedPKC activator solution (1 mg/ml phosphatidylserine in water),1 �l of peptide protection solution, 3.75 �l of water, 1.25 �l ofvehicle (methanol/Me2SO) or compounds, and 4 �l of suppliedPKC (2.5 �g/ml in PKC dilution buffer containing 100 �g/mlbovine serumalbumin and 0.05%TritonX-100�, primarily�,�,and � isotypes with lesser amounts of � and � isotypes, purifiedfrom rat brain (33)). Before adding PKC, themixturewas preincu-bated at 30 °C for 2 min. After the addition of PKC, the entirereaction mixture was incubated at 30 °C for 30 min. The reactionwas stopped by incubation at 95 °C for 10 min. Before loadingsamples on an agarose gel (0.8%agarose in 50mMTris-HCl buffer,pH 8.0), 2 �l of 80% glycerol was added to the sample. Electro-phoresiswas runat 100V for 30min in50mMTris-HClbuffer, pH8.0, andwas imaged immediately underUV lightwithGeneSnap�V6.03 using the Gene Genius transilluminator (Syngene, Cam-bridge, UK) to avoid diffusion of PepTag� peptide. Densitometricquantification of the assaywas performedwithGeneTools�V3.05(Syngene).Preparation of Tissue orCell Samples for PKCAssay—5� 106

HTC4 cells were incubated for 15 h with 10–1000 nM AAL(R),AAL(S), or FTY720. HTC4 cells were detached from cultureflasks with trypsin, pelleted by centrifugation at 400 � g for 5min at 4 °C, washed once with PBS, and centrifuged again asabove. The pellet was suspended in 250 �l of ice-cold PBS,including a protease inhibitormixture (RocheApplied Science)and homogenized on ice with a precooled Dounce homoge-nizer by 50 strokes. The lysate was centrifuged for 1 min at 4 °Cand 10,000 � g in a microcentrifuge, and the supernatant wascollected for PKC activity screening. For in vivo PKC activitystudies, the mice were treated orally with 20 mg/kg/day DOPand 3 mg/kg/day AAL(S) or FTY720. After isolation of lymphnodes, spleen, and thymus, the organs were homogenized asdescribed above. The crude extracts were freshly prepared foreach experiment and assayed the same day they were preparedto obtain maximal activity. A total volume of 14 �l of extractwas applied in each PepTag� PKC assay reaction.Protein Kinase C Isotype Activity ELISA—The influence of

FTY720 and AAL(S) on PKC activity of PKC isotypes �, �I, �II,

S1P1 Down-regulation by PKC Inhibition

FEBRUARY 26, 2010 • VOLUME 285 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 6299


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�, �, �, �, �, , and (Calbiochem/Merck KGaA, Darmstadt,Germany) was analyzed with the PKC kinase activity assay kit(Assay Designs, Ann Arbor, MI) according to the manufactur-er’s protocol. Briefly, 20 units of each PKC isotype were mixedwith FTY720 or AAL(S) (final concentration, 10 �M) in kinaseassay dilution buffer and adjusted to 30�l of total volume. Afterthe addition of 10 �l of ATP solution, the mixture was trans-ferred to a pre-equilibrated 96-well ELISA plate coated withPKC substrate, which was incubated for 90 min at 30 °C withgentle shaking every 20 min. After subsequent incubations ofphospho-specific substrate antibody, anti-rabbit IgG-horserad-ish peroxidase conjugate, and tetramethylbenzidine substratewith intermittent washing, the absorbance at 450 nm wasmeasured with the microplate reader Tecan Infinite� 200(Crailsheim, Germany). The results are expressed as relativePKC activity in percentages normalized to vehicle control reac-tions of each PKC isotype, which were set to 100%.FTY720 PKC Substrate Test—Phosphorylation of FTY720 by

PKC was analyzed by incubation of 1.15 �l of 30 �M FTY720with amixture of PKC isotypes�,�I,�II, �, �, �, �,�, , and (20units each) for 15 h at 30 °C. The total assay volume was 25 �l,and buffer conditions were similar as described under PepTag�fluorescent protein kinase C assay procedure. FTY720 andFTY-P concentrations were analyzed by LC/MS/MS asdescribed previously (10).Splenocyte Isolation—Spleens frommice were minced on ice

and pushed through a 70-�mmesh. Red blood cells were lysedin 138 mMNH4Cl, 1 mM KHCO3, and 0.1 mM EDTA for 5 min,and the remaining splenocytes were washed three times withPBS supplemented with 10% fetal calf serum. Some splenocytesuspensions were stimulated by incubation for 15 h in 10-cmcell culture dishes on 30 �g each of adherent anti-CD3 andanti-CD28 monoclonal antibodies (BD Biosciences, Heidel-berg, Germany). For selected experiments, the nuclei were pel-leted by centrifugation at 300 � g and 4 °C for 5 min afterhomogenization with a Dounce homogenizer by 20 strokes onice and washed twice with ice-cold PBS.TranswellTM in Vitro Chemotaxis Assay—Migration of pri-

mary mouse splenocytes was analyzed in 24-well TranswellTMchambers (Costar, Cambridge,MA)with 6.5-mmdiameter and5-�m pore polycarbonate filters, which were coated on thelower side for 15 h at 4 °Cwith 600�l of a 100�g/ml solution ofhuman collagen type IV (Sigma-Aldrich) in 0.5 M acetic acid,washed three times with 600 �l of PBS, and air-dried. Thesplenocytes were prepared as described above, and 2� 106 cellsin 100 �l of RPMI 1640 supplemented with 0.1% fatty acid-freebovine serum albumin (U.S. Biological, Swampscott, MA), 100units/ml penicillin G, 100 �g/ml streptomycin, 2 mM L-gluta-mine, and 25 mM HEPES buffer were placed on the top of theTranswellTM inserts. 600 �l of medium supplemented with 20nM S1P or 38 nM (300 ng/ml) mouse recombinant SDF1�(CXCL-12) (ImmunoTools) as the chemotactic stimulus wereadded to the lower chamber. Migration was performed for 4 hat 37 °C in a humidified 5.0% CO2 atmosphere incubator. Theinserts were removed, and the number of migrated cells wasassessed by flow cytometry using Flow Check� flow cytometryparticles APC Maxi-Brite (Polysciences Europe GmbH) as aninternal standard. To establish the number of cells that

migrated nonspecifically, migration assays were performed inparallel in the absence of chemoattractants. The results areexpressed as fold increases of specificmigration over unspecificmigration without chemoattractant.Immunoprecipitation—The tissues were homogenized in

250 �l of PBS on ice with a precooled Dounce homogenizer by50 strokes. The cells and tissue homogenates were lysed in 20mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1%Triton X-100, 20 mM NaF, 0.1 mM Na3VO4, and complete pro-tease inhibitor mixture (Roche Applied Science) for 120 min at4 °C. The concentration of protein was quantified using theBCA method (Pierce). For immunoprecipitation 10–50 mg oflysates were incubated with 3 �g of mouse anti-human-S1P1(clone H-60; Santa Cruz Biotechnology, Heidelberg, Germany)or rat anti-HA (clone 3F10) antibodies for 4 h. Subsequently,the antibody-protein conjugates were bound to a 1:1mixture ofprotein A- and G-agarose beads (GE Healthcare) overnight at4 °C. The beads were washed twice, dissolved in 50�l of samplebuffer (Invitrogen), and taken for Western blot analysis ofbound proteins.Protein Detection by Western Blot—Western blots were per-

formed according to standard protocols. The tissues werehomogenized in 250 �l of PBS on ice with a precooled Douncehomogenizer by 50 strokes. The cells and tissue homogenateswere lysed in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM

EDTA, pH 8.0, 1% Triton X-100, 20 mM NaF, 0.1 mM Na3VO4,and complete protease inhibitor mixture (Roche Applied Sci-ence). The lysates (30–50�g)were subjected toNuPage 4–12%Bis-Tris gels (Invitrogen) according to the manufacturer’s pro-tocol, and the proteins were transferred to Hybond-P polyvi-nylidene difluoride membranes (GE Healthcare) by wet blot-ting. The membranes were subsequently blocked with 5%SlimFast chocolate powder (Allpharm-Vertriebs-GmbH, Mes-sel, Germany) in Tris-buffered saline and probed with mouseanti-phosphothreonine (clone H-2, 2 �g/ml; Santa Cruz Bio-technology), rabbit anti-human-S1P1 (cloneH-60, 2�g/ml), ratanti-HA (clone 3F10, 2 �g/ml), or rabbit anti-human histonedeacetylase 1 (clone H-51, 2 �g/ml; Santa Cruz Biotechnology)antibodies overnight. After incubation with specific horserad-ish peroxidase-labeled secondary antibodies against mouse(400 ng/ml; Abnova, Heidelberg, Germany), rabbit (160 ng/ml;Santa Cruz Biotechnology), and rat (160 ng/ml; Santa Cruz Bio-technology), the signals were visualized with the ECL detectionsystem according to the manufacturer’s instructions (GEHealthcare).Confocal Microscopy—HTC4 cells were seeded on glass cov-

erslips in 6-well plates. The next day the cells were fixedwith 5%paraformaldehyde in PBS for 15 min and permeabilized with0.5% Triton X-100 for 5 min on ice. After two washes, the cellswere stained with fluorescein isothiocyanate-labeled anti-HAFab fragments (500 ng/ml, clone 3F10; Roche Applied Science)and 4�,6-diamidino-2�-phenylindole dihydrochloride (1 �g/ml;Roche Applied Science) for 2 h on ice. Subsequently the cellswere washed an mounted on microscope slides with Mowiol4-88. Confocalmicroscopic analysis was performed using aDMIRB with a TCS SP2 AOBS scan head (Leica Microsystems,Wetzlar, Germany).




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LC/MS/MS Analysis—Quantification of S1P, Sph, FTY720,and FTY-P was performed as described (34).

RESULTS

Sph Blocks TranswellTM Chemotaxis to S1P—We haverecently shown that Sph efficiently incorporates and accumu-lates in primarymouse splenocytes (35). To test whether or notSph accumulation affects lymphocyte migration, freshly iso-lated primarymouse splenocytes were preincubated for 15minwith 300 nM and 1 �M Sph and, after several washes, subjectedto TranswellTM chemotaxis assays. In contrast to controlsplenocytes, preincubation with Sph greatly inhibited chemo-taxis to S1P, but not to the chemokine SDF1� (CXCL-12), the

ligand for the chemokine receptor CXCR4 (Fig. 1). Similar Sphincubations with splenocytes did not result in detectable phos-phorylation to S1P (35). To exclude the possibility that smallamounts of S1P formed by the cells were responsible for desen-sitization of S1P receptors and the observed inhibition ofsplenocyte migration to S1P, equimolar concentrations of theanti-S1P antibody SphingomabTM were added to the cells dur-ing preincubation with Sph. 100 nM SphingomabTM was addi-tionally added to the cells in the TranswellTM chamber duringchemotaxis to prevent autocrine stimulation of S1P receptorsby secreted S1P. SphingomabTM was previously shown to bindand inactivate S1P (36). Indeed, 40 nM SphingomabTM added to20 nMS1P as stimulus completely blocked splenocytemigrationin TranswellTM migration experiments (Fig. 1). Despite thisS1P blocking activity of SphingomabTM, its presence did notprevent Sph-induced inhibition of splenocytemigration. Theseexperiments therefore suggested that Sph may influencesplenocyte migration to S1P in its nonphosphorylated state.PKC Inhibitors Down-regulate the S1P1 Receptor—A critical

requirement for splenocyte migration to S1P is surface expres-sion of the S1P1 receptor. The latter is modulated by PKCamong others, and PKC was shown to be inhibited by Sph (37).To test the relevance of PKC activation for S1P1 receptor sur-face expression, rat hepatoma HTC4 cells expressing thehuman N-terminal hemagglutinin epitope-tagged S1P1 recep-tor (S1P1-HA) were treated with the specific PKC inhibitormyr-�PKC. This PKC pseudosubstrate nonapeptide is a selec-tive PKC inhibitor without cross-reactivity for other proteinkinases (32). S1P1-HA surface expression was determined byflow cytometry. Compared with vehicle-treated S1P1-HAHTC4 cells, treatment with 50 �M of the PKC inhibitor myr-�PKC resulted in down-regulation of receptor surface expres-sion (Fig. 2A). The effect was weaker compared with treatmentwith 300 nM FTY720 but clearly distinguishable from vehicle-treated control cells. Down-regulation of receptor surface

expression through PKC inhibitionwas specific for S1P1 and not seen inthe same cell system with a compa-rable construct bearing the S1P4receptor (Fig. 2A). The fluorescencesignal was specific for receptorsurface expression and did not shiftin nonexpressing control cells upontreatment with the PKC inhibitormyr-�PKC (Fig. 2A). To furtherdemonstrate the influence of PKCinhibition on S1P1 receptor down-regulation, surface expression ofS1P1 was analyzed after treatmentwith nanomolar concentrations ofthe additional PKC inhibitorsstaurosporine, calphostin C, andGF109203X. All of the PKC inhibi-tors induced severe down-regula-tion of S1P1 receptor surface ex-pression, with 100 nM calphostin Cbeing even more efficient than 300nM FTY720, 50 nM staurosporine,

FIGURE 1. Inhibition of TranswellTM chemotaxis to S1P by Sph. C57BL/6mouse splenocytes were preincubated for 15 min at 37 °C with 0.3 or 1.0 �M Sphin the presence and absence of equimolar concentrations of the anti-S1P anti-body (Ab) SphingomabTM as indicated and washed twice with PBS, before theywere added to the upper wells of 5-�m pore polycarbonate tissue culture insertswith and without 100 nM SphingomabTM added. Migration toward 20 nM S1P, 20nM S1P in the presence of 40 nM SphingomabTM, or 38 nM SDF1� was performedat 37 °C for 4 h, and migrated cells were counted by flow cytometry. Each columnrepresents the mean � S.E. of three to four independent experiments.

FIGURE 2. Down-regulation of the S1P1 receptor by PKC inhibition. A, S1P1-HA expressing HTC4 cells (leftpanel) were incubated for 15 h at 37 °C with 50 �M PKC inhibitor myr-�PKC (blue line) or 300 nM FTY720 (redline). HTC4 wild type cells are shown as negative control (green line), and vehicle-treated control cells arerepresented by a black line. Similar treatments are shown for S1P4-HA (middle panel) and untransfected HTC4control cells (right panel). B, expression of S1P1-HA (left panel) analyzed after treatment with 300 nM FTY720 (redline) and PKC inhibitors staurosporine (50 nM, blue line), calphostin C (100 nM, purple line), or GF109203X (100 nM,brown line) for 15 h. Vehicle-treated control cells are shown as a black line, and HTC4 wild type cells are repre-sented by a green line. S1P4-HA-expressing (middle panel) and untransfected HTC4 control cells (right panel) areshown after similar incubations. One representative histogram of three similar experiments is shown in eachfigure.




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and 100 nM GF109203X (Fig. 2B). Consistent with the PKCinhibitor myr-�PKC, none of the tested PKC inhibitorsinduced down-regulation of S1P4 receptor surface expressionor shifted the fluorescence signal in nonexpressing control cells(Fig. 2B).PMA Partially Rescues S1P1 Receptor Down-regulation by

FTY720—Because S1P1-HA surface expression was decreasedafter treatment with several different PKC inhibitors, the nextstep was to investigate the effect of the PKC activator PMA onFTY720-induced down-regulation of S1P1-HA surface expres-sion. Compared with control cells, treatment with 10 nMFTY720 already resulted in significant down-regulation ofS1P1-HAsurface expression (Fig. 3). Simultaneous treatment ofcells with 10 �M PMA partially prevented from FTY720-in-duced down-regulation of S1P1-HA surface expression (Fig. 3).PMA treatment had no effect on S1P4 receptor surface expres-sion, and no differences in fluorescence intensitywere observed

in nontransfected vehicle- and com-pound-treated HTC4 cells (Fig. 3).Altered S1P1 Receptor Phosphor-

ylation—To test whether or notPKC activation or inhibition altersthe phosphorylation state of theS1P1 receptor, S1P1-HA-expressingHTC4 cells were treated for 15 hwith 10 �M PMA, 10 nM FTY720,and both stimuli together, similar toFig. 3, and with 50 �M myr-�PKC,300 nM FTY720, 50 nM staurospo-

rine, 100 nM calphostin C, and 100 nM GF109203X, similar toFig. 2. After immunoprecipitation of the S1P1-HA receptorwith an anti-HA antibody, receptor phosphorylation wasdetected byWestern blot using an anti-phosphothreonine anti-body. The observed threonine phosphorylation of the S1P1-HAreceptor reflected the observed surface expression (Figs. 2 and3). Treatment with the PKC activator PMA resulted in slightlyincreased receptor phosphorylation, whereas single andco-treatment with 10 nM FTY720 attenuated S1P1 receptorphosphorylation comparedwith untreated control cells (Fig. 4).A higher concentration of FTY720 (300 nM) did not fur-ther enhance inhibition of receptor phosphorylation. ThePKC inhibitors myr-�PKC, staurosporine, calphostin C, andGF109203X were all effective (Fig. 4).FTY720, but Not FTY-P, Inhibits PKC—The influence of the

PKC activator PMA on FTY720-induced down-regulation ofS1P1-HA surface expression pointed at a potential direct cellu-lar interaction of FTY720 with PKC. Detection of PKC activitywith the PepTag� fluorescent protein kinase assay revealed apotent inhibition of a mixture of predominantly conventionalisoforms of PKC (33) by FTY720 (Fig. 5A). Comparable inhibi-tion was not observed with FTY-P. The effective amount ofFTY720 was highly dependent on the amount of added PKCactivator phosphatidylserine (PS). PS added at concentrationsof 50 and 500�Mmaximally activated PKC, whereas lower con-centrations were less effective (Fig. 5B). PKC inhibition of 500�M FTY720 in the presence of 500 �M PSwas similarly efficientas 50 �M FTY720 with 50 �M PS added (Fig. 5C). Therefore theratio of PS to FTY720 was the critical determinant for efficientPKC inhibition rather than absolute FTY720 concentrations. Aratio of 1:1 PS:FTY720 proved to be maximally effective withnoticeable PKC inhibition starting at a ratio of 10:1 (Fig. 5C).Ten PKC isoforms including the conventional PKC �, �1, �2,and �, the novel PKC �, �, �, and , and the atypical PKC � and were tested with regard to FTY720-induced inhibition usingan ELISA-based PKC activity assay kit. Therewas no significantisotype specificity for FTY720 among these PKC isoforms,demonstrating that FTY720 serves as a pan-PKC inhibitor (Fig.5D). AAL(S), an inactive stereospecific isoform of FTY720 thatis neither phosphorylated by sphingosine kinases nor inducinglymphopenia in rodents (17), showed amuch lower potency forPKC inhibition thanFTY720 (Fig. 5E). The activity ofmost PKCisoforms was less affected by AAL(S) than FTY720; inhibition,however, was obvious for conventional PKC �2, novel PKC ,and atypical PKC . PKC itself had no phosphorylating activity

FIGURE 3. FTY720-induced S1P1 receptor down-regulation is partially rescued by PMA. S1P1-HA express-ing HTC4 cells (left panel) were incubated for 15 h at 37 °C with 10 �M PKC activator PMA (blue line), 10 nM FTY720(red line), or PMA combined with FTY720 (purple line). A black line represents vehicle-treated control cells, andHTC4 wild type cells are shown as negative control (green line). Similar incubations using S1P4-HA (middle panel)or wild type HTC4 cells (right panel) are illustrated as control experiments. One representative histogram ofthree similar experiments is shown in each figure.

FIGURE 4. Attenuation of S1P1 receptor phosphorylation by PKC inhibi-tion. S1P1-HA expressing HTC4 cells were incubated for 15 h at 37 °C with 10�M PKC activator PMA, 10 nM FTY720, PMA, and FTY720 together, 50 �M PKCinhibitor myr-�PKC, 300 nM FTY720, 50 nM PKC inhibitor staurosporine, 100nM PKC inhibitor calphostin C, and 100 nM PKC inhibitor GF109203X. Afterimmunoprecipitation with anti-HA antibody, phosphorylation of the S1P1-HAreceptor was detected by Western blot using anti-phosphothreonine anti-body. The total amount of S1P1-HA was determined with the anti-HA anti-body. Signals derived from two independent experiments were quantifiedwith ImageJ 1.42q (National Institutes of Health). Shown are the means � S.E.




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on FTY720, and incubation of FTY720 with PKC did not resultin FTY-P production (Fig. 5F).Similar Receptor Internalization Induced by S1P1 Activation

and PKC Inhibition—Cyclical alteration of S1P1 receptor sur-face expression on T and B cells was suggested to be critical forregulation of lymphocyte circulation (3). S1P1 receptor inter-nalization was therefore recognized as an important step in thisprocess. Besides agonist-induced activation and internalizationof the S1P1 receptor by S1P andFTY-P,which directs the recep-tor via the endocytic pathway back to the plasmamembrane forreceptor recycling or into the Golgi complex for its degrada-tion, T cell activation with anti-CD3 and anti-CD28 antibodiesled to S1P1 receptor translocation to the nuclear envelope (38).To investigate the S1P1 receptor internalization pathway afterPKC inhibition, S1P1-HA expressing HTC4 cells were stimu-lated for 15 hwith 300 nM FTY720 and 100 nM calphostin C andfor 1 h with 1 �M S1P. The cells were fixed, permeabilized, and

FIGURE 5. PKC is blocked by FTY720, but not FTY-P. PKC activity wasaddressed with the PepTag� fluorescent PKC assay as described under“Experimental Procedures.” A, representative UV-illuminated agarose gelwith fluorescently labeled C1 peptide phosphates. FTY720 and FTY-P wereadded at a concentration of 500 �M. B, PS dependence of PepTag� fluores-cent PKC assay. C, inhibition of PKC by FTY720 in dependence of PS (500 �M,left panel; 50 �M, right panel). The reactions were performed with varyingconcentrations of FTY720 or vehicle as indicated. Relative enzyme activitywas quantified by densitometric analysis of the fluorescence intensity ofphosphorylated PKC substrate and is expressed as a percentage of PKC activ-ity. D and E, influence of 10 �M FTY720 (D) or AAL(S) (E) on enzyme activity ofdifferent PKC-isotypes as indicated. PKC activity was quantified by ELISA as

illustrated under “Experimental Procedures.” The results represent normal-ized PKC activity in percent (vehicle control was set to 100%). F, FTY720 andFTY-P concentrations after incubation with PKC, analyzed by LC/MS/MS. Theresults represent the means � S.E. of three separate experiments in eachfigure.

FIGURE 6. Internalization of S1P1-HA by PKC inhibition. A, S1P1-HA-trans-fected HTC4 cells were stimulated for 1 h with 1 �M S1P and for 15 h with 300nM FTY720 and 100 nM calphostin C as indicated. The cells were fixed, perme-abilized, and stained with anti-HA antibody for expression and localization ofthe S1P1-HA receptor (green), and with 4�,6-diamidino-2�-phenylindole dihy-drochloride (DAPI) for visualization of nuclei (blue). B, mouse splenocyteswere stimulated for 15 h with 100 nM calphostin C (Cal C) and immobilizedantibodies against CD3 and CD28 (30 �g each) and for 1 h with 300 nM FTY720and 1 �M S1P as indicated. Endogenous S1P1 receptor was detected in iso-lated nuclear cell fractions by Western blot using anti-S1P1 antibody. Expres-sion of the nuclear protein histone deacetylase 1 (HDAC1) is shown as control(Cont.). Signals derived from two independent experiments were quantifiedwith ImageJ 1.42q (National Institutes of Health). Shown are the means � S.E.




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stained for S1P1 receptor expression and nuclear DNA. Confo-cal microscopy revealed that treatment with calphostin C,FTY720, and S1P resulted in efficient S1P1 receptor internal-ization (Fig. 6A). No co-staining of the S1P1 receptor and thenuclei was detected. To confirm this result in primary lympho-

cytes, mouse splenocytes were treated for 15 h with 100 nMcalphostin C and cross-linked antibodies against CD3 andCD28 and for 1 h with 1 �M S1P and 300 nM FTY720. Westernblots revealed similar amounts of S1P1 receptor in nuclearsplenocyte fractions after stimulation with anti-CD3 and anti-




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CD28 antibodies, FTY720, or S1P, and stimulation with cal-phostin C in tendency resulted in lower nuclear S1P1 signal,although these differences were not statistically significant (Fig.6B). PKC inhibition thus internalized the S1P1 receptor via theendocytic pathway similar to S1P and FTY-P without detecta-ble translocation to the nuclear envelope.Cell-based and in Vivo Inhibition of PKC by FTY720—To

demonstrate the relevance of FTY720-induced PKC inhibitionfor S1P1 receptor down-regulation and induction of lymphope-nia, PKCactivitywas determined in S1P1-HA-expressingHTC4cells treated with different concentrations of FTY720 and itsstereoselective analogs AAL(R/S). Compared with vehicle-treated control cells, treatment with 10 nM FTY720 alreadyresulted in considerable PKC inhibition (Fig. 7A). AAL(R) andparticularly AAL(S) were noticeably less efficient PKC inhibi-tors than FTY720 in this cell-based assay, requiring concentra-tions of 100 nM to 1 �M for similar effectiveness (Fig. 7A). ThePKC inhibitors staurosporine, calphostin C, and GF109203Xwere similarly or even less effective than FTY720 (Fig. 7A). PKCinhibition by FTY720 was also tested in vivo. C57BL/6 micewere treated orally for 3 days with 0.3, 1, and 3 mg/kg/dayFTY720, and after their lymphoid organs, lymph nodes, spleen,and thymus were harvested, they were tested for PKC activity.Compared with untreated control mice, all lymphoid organsfrom FTY720-treated mice revealed decreased PKC activity(Fig. 7B). The loss of PKC activity was most dramatic in spleen,followed by lymph nodes and thymus. The onset of PKC inhi-bition was already seen with the lowest concentration of 0.3mg/kg/day FTY720 (Fig. 7B). Treatment of mice with either 20mg/kg/dayDOP,which increased endogenous levels of S1P andSph in lymphoid organs, or 3 mg/kg/day AAL(S) did not resultin PKC inhibition (Fig. 7B).In Vivo Inhibition of S1P1 Receptor Phosphorylation—Similar

to S1P1-HA receptor phosphorylation inHTC4 cells (Fig. 4), wealso tested the phosphorylation state of the endogenous S1P1receptor in lymph nodes, spleen, and thymus frommice treatedfor 3 days with 1 or 3mg/kg/day FTY720, 3 mg/kg/day AAL(S),and 20 mg/kg/day DOP. Treatment of mice with the highestconcentration of FTY720 demonstrated much lower S1P1receptor phosphorylation in all examined lymphoid tissuescompared with untreated control mice (Fig. 7C). Phosphoryla-tion was not altered with the lower dose of 1 mg/kg/dayFTY720. In line with the PKC activity measurements (Fig. 7B),treatment of mice with AAL(S) and DOP did not change thephosphorylation state of the S1P1 receptor in lymphnodes, thy-mus, and spleen (Fig. 7D). PKC inhibition by FTY720 therefore

resulted in reduced S1P1 receptor phosphorylation in vivo,whereas AAL(S) and DOP were ineffective.Contribution of FTY720-induced PKC Inhibition to Lym-

phopenia Induction—FTY720-induced lymphopenia may arisefrom its phosphorylation by SK2 to FTY-P and consequentS1P1 receptor stimulation (30, 31) or by the herein describedphosphorylation- and agonist-independent S1P1 receptordown-regulation via PKC inhibition. To test the contribution ofboth mechanisms, SK2-deficient mice were treated with 3mg/kg/day FTY720 and analyzed after 5 days for the onset oflymphopenia. PKC inhibition in lymphoid organs was evidentunder these conditions (Fig. 7B), and phosphorylation ofFTY720 to FTY-P was less than 1% compared with wild typemice because of the lack of SK2 expression (Fig. 7E). Sph andS1P levels in plasma and lymphoid tissues were not consider-ably altered during FTY720 treatment (Fig. 7E). In comparisonwith wild type control mice, which developed a severe B and Tcell lymphopenia, SK2-deficient mice only responded with acomparably mild phenotype (Fig. 7F). The share of FTY720-induced PKC inhibition and down-regulation in the onset oflymphopenia was therefore limited, and S1P1 receptor agonismof FTY-P proved to be the principal mechanism of FTY720-induced lymphopenia. Attempts to use PKC inhibitors in vivofor the investigation of lymphopenia failed because of severeside effects and circulation-independent alterations of lympho-cyte populations in blood and lymphoid tissues.

DISCUSSION

Two different mechanisms are currently used to explain theblock of lymphocyte egress by FTY720: Down-regulation of theexit signal-sensing S1P1 receptor on lymphocytes (1) and acti-vation of S1P1 on sinus-lining endothelial cells, resulting in theclosure of suggested portals (14). Whereas the first mechanismbasically relies on S1P1 receptor inhibition, the latter proposesS1P1 receptor activation as the critical event for lymphopeniainduction. Evidence exists for both hypotheses: lymphocyte-specific S1P1 receptor-deficient mouse mutants manifest asevere block in thymocyte and lymphocyte egress from thymusand lymph nodes, respectively, demonstrating the importanceof S1P1 receptor expression on thymocytes and patrolling lym-phocytes for their exit from thymus and secondary lymphoidorgans (1, 4). On the other hand S1P1 receptor agonists preventlymphocytes from crossing the lymphatic endothelium, whichwas blocked by otherwise inactive S1P1 receptor antagonists(14, 39). Furthermore B cells seem to exit lymph nodes inde-pendently from S1P-mediated chemotaxis (40). The contribu-

FIGURE 7. FTY720 blocks PKC in vivo and in vitro. A, 5 � 106 HTC4 cells were incubated for 15 h with indicated concentrations of AAL(R), AAL(S), FTY720,staurosporine, calphostin C, and GF109203X. After homogenization of cells, the lysates were screened for PKC activity as described under “ExperimentalProcedures.” B, C57BL/6 mice were treated orally with 20 mg/kg/day DOP and 3 mg/kg/day AAL(S) or 0.1, 0.3, and 3 mg/kg/day FTY720. After isolation oflymphoid organs, the lysates were tested for PKC activity. One representative UV-illuminated agarose gel is shown in each figure. Relative enzyme activitieswere quantified by densitometric analysis of fluorescence intensity of phosphorylated PKC substrate and are expressed as the percentages of PKC activity. Theresults represent the means � S.E. of three separate experiments. C and D, mice were treated as mentioned in B, and the endogenous S1P1 receptor wasimmunoprecipitated in lysed tissue homogenates from peripheral lymph nodes (pLN), thymus, and spleen. Phosphorylation of the S1P1 receptor was detectedby Western blot using anti-phosphothreonine antibody. Total amount of S1P1 was determined with anti-S1P1 antibody. The signals derived from two separateblots were quantified with ImageJ 1.42q (National Institutes of Health). Shown are the means � S.E. E, plasma, spleen, thymus, and peripheral lymph nodesfrom Balb/c wild type and SK2-deficient mice untreated and treated orally with 3 mg/kg/day FTY720 for 5 days were analyzed by LC/MS/MS regarding their Sph, S1P,FTY720, and FTY-P concentration. Shown are the means�S.E. (n �3). F, wild type or SK2-deficient Balb/c mice were treated orally with 3 mg/kg/day FTY720 for 5 days,and the lymphocyte counts were analyzed in blood as depicted the “Experimental Procedures.” The results represent the means � S.E. of three separate experimentsin each figure. Statistical analysis was done by Student’s t test. *, p � 0.05; **, p � 0.01; ***, p � 0.001. n.s., not significant; Cont., control.




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tion of both proposed mechanisms to FTY720-induced lym-phopenia is therefore unclear. The presented data show thatS1P1 receptor surface expression can be abolished by PKC inhi-bition without S1P1 receptor activation. In vivo inhibition ofPKC, however, only induced a very mild lymphopenia (Fig. 7F),indicating that S1P1 receptor stimulation mostly accounts forFTY720-induced egress inhibition, and S1P1 receptor down-regulation is a secondary, less important event, at least in case ofFTY720 treatment. This is in line with recent reports showingthe requirement of FTY720 phosphorylation by SK2 for effi-cient lymphopenia at low FTY720 concentrations (30, 31) andthe independence of B cell exit from S1P-mediated chemotaxis(40). It also explains the observation that S1P1 receptor agonistsand not antagonists were able to induce lymphopenia in vivo,although antagonists could reverse lymphopenia induced byagonists (39). It should be noted here that the preferential stim-ulating activity of FTY-P over FTY720-induced S1P1 receptordown-regulation does not exclude the imperative role of S1P1receptor cell surface expression for lymphocyte exit. But it isobvious from the presented studies that S1P1 receptor down-regulation has to be rather complete to significantly affect lym-phocyte egress. In contrast to S1P1 receptor antagonists, ago-nists may be more potent either by establishing endothelial cellbarriers (14) or by neutralizing a postulated gradient of theendogenous ligand S1P in lymphoid tissues (3).The presented data demonstrate that modulation of S1P1

receptor surface expression is possible without extracellularstimuli. Another agonist-independent mechanism for modula-tion of S1P1 receptor surface expression is the observed directinteraction of S1P1 with CD69, which prevents cell surfaceexpression of both molecules when co-expressed (41). Trans-genic expression of CD69 in T cells led to accumulation ofmature T cells in thymus (42), resembling a similar phenotypeto S1P1 receptor deficiency (1, 4) and FTY720 treatment(43). PKC inhibition also leads to S1P1 receptor down-regu-lation, and FTY720 itself is a potent PKC inhibitor (Figs. 2and 5C). Although the major immunomodulatory activity atlow nanomolar FTY720 concentrations derives mainly fromits phosphorylated derivative and S1P1 receptor agonistFTY-P (30, 31), mild lymphopenia in FTY720 phosphoryla-tion-defective SK2-deficient animals after treatment with 3mg/kg/day FTY720 can be attributed to FTY720-inducedPKC inhibition and consequent down-regulation of the S1P1receptor on lymphocytes (Fig. 7, B and F). Watterson et al.(28) reported that PKC stimulation with PMA leads to S1P1receptor phosphorylation and subsequent internalization.These studies, however, focused on the immediate effects ofPKC stimulation within minutes, whereas onset of S1P1receptor down-regulation by PKC inhibitors typicallyrequired several hours (Fig. 2). It is likely that PKC directlyphosphorylates S1P1 and induces immediate and transientreceptor down-regulation and additionally alters receptoravailability by targeting S1P1 cell surface expression indi-rectly via phosphorylation of other signaling molecules withthe observed delayed onset and prolonged efficacy for recep-tor internalization after PKC inhibition. The data fromWatterson et al. (28) are therefore not contradictory to thisstudy but highlight a different regulatory aspect.

Sph is able to accumulate in lymphocytes just like FTY720(35). Preincubation of primary mouse splenocytes with Sphspecifically inhibited chemotaxis to S1P (Fig. 1). Significantincreases in S1P levels were not observed, and combined pre-and co-incubation of mouse splenocytes with the S1P-blockinganti-S1P antibody SphingomabTM did not prevent inhibition ofchemotaxis to S1P by Sph (Fig. 1). An alternative explanationfor agonist-induced receptor down-regulation and inhibition ofchemotaxis to S1P is the repression of PKC by Sph (25). ThePKC-specific inhibitor myr-�PKC also induced partial down-regulation of the S1P1 receptor (Fig. 2). But Sph levels typicallydo not reach the concentrations necessary for PKC inhibition,and treatment ofmice with the S1P-lyase inhibitor DOP, whichalso induced Sph accumulation, did not inhibit PKC activity(Fig. 7B). PKC inhibition by Sph therefore does not seem tooccur even after DOP treatment, possibly because of misplacedsubcellular localization, whereas PKC inhibition by FTY720was obvious after treatment (Fig. 7B).Although no direct PKC phosphorylation sites are currently

known for the S1P1 receptor, computational analysis revealedThr236 and Thr257 as putative PKC phosphorylation sites forboth human and mouse S1P1 receptors. In line with previousstudies (28, 29), PKC inhibition resulted in attenuated S1P1receptor phosphorylation in S1P1-HA-transfected HTC4 cells(Fig. 4). Although S1P1 receptor phosphorylation was only par-tially reduced in these overexpressing cells after addition ofvarious PKC inhibitors, a pronounced defect in phosphoryla-tion of the endogenous mouse S1P1 receptor was observed inlymphoid tissues from mice treated with 3 mg/kg/day FTY720for 3 days (Fig. 7C). High dosage of FTY720 therefore inhibitsPKC and impairs S1P1 receptor phosphorylation in vivo.FTY720was shown to directly affect several different cellular

enzymes including PLA2 (22), S1P-lyase (23), SK1/2 (21), andceramide synthases (24). But S1P-lyase inhibition was not evi-dent in the in vivo setting (23), and possible contributions ofPLA2, SK1/2, and ceramide synthase for the in vivo efficacy ofFTY720 were not even tested (21, 22, 24). This study clearlyidentifies FTY720 as a pan-PKC inhibitor not only in vitro butalso in vivo at physiologically relevant concentrations (Figs. 5,A–D, and 7, A and B). PKC inhibition resulted in S1P1 receptordown-regulation (Fig. 2). Although the contribution of PKCinhibition to the onset of lymphopenia was minor comparedwith S1P1 receptor agonism (Fig. 7F), it may explain observa-tions linking FTY720 treatment to increased apoptosis (44),tumor suppression (45), and clearance of chronic viral infection(46). Vice versa, PKC inhibitors may affect lymphocyte circula-tion by altering S1P1 receptor cell surface expression.

Acknowledgments—We thank Anika Munk for excellent technicalassistance and Constantin Bode for performing LC/MS/MSmeasurements.We are grateful to Elisabeth Kremmer for the anti-HAantibody and to Roger Sabbadini for the anti-S1P antibodySphingomabTM.

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Sven-Christian Sensken and Markus H. GrälerInhibition

Receptor Surface Expression by Protein Kinase C1Down-regulation of S1P

doi: 10.1074/jbc.M109.049692 originally published online December 23, 20092010, 285:6298-6307.J. Biol. Chem.

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