Subcellular distribution of FTY720 and FTY720-phosphate in immune cells – another aspect of Fingolimod action relevant for therapeutic application
-
Matthias Schröder
, Olga Arlt , Helmut Schmidt , Andrea Huwiler , Carlo Angioni , Josef M. Pfeilschifter , Anja Schwiebsund Heinfried H. Radeke
Abstract
FTY720 (Fingolimod; Gilenya®) is an immune-modulatory prodrug which, after intracellular phosphorylation by sphingosine kinase 2 (SphK2) and export, mimics effects of the endogenous lipid mediator sphingosine-1-phosphate. Fingolimod has been introduced to treat relapsing-remitting multiple sclerosis. However, little has been published about the immune cell membrane penetration and subcellular distribution of FTY720 and FTY720-P. Thus, we applied a newly established LC-MS/MS method to analyze the subcellular distribution of FTY720 and FTY720-P in subcellular compartments of spleen cells of wild type, SphK1- and SphK2-deficient mice. These studies demonstrated that, when normalized to the original cell volume and calculated on molar basis, FTY720 and FTY720-P dramatically accumulated several hundredfold within immune cells reaching micromolar concentrations. The amount and distribution of FTY720 was differentially affected by SphK1- and SphK2-deficiency. On the background of recently described relevant intracellular FTY720 effects in the nanomolar range and the prolonged application in multiple sclerosis, this data showing a substantial intracellular accumulation of FTY720, has to be considered for benefit/risk ratio estimates.
Acknowledgments
We would like to thank J. Textor, University of Lübeck, for his helpful advice regarding species-specific details of lymphocyte sizes and volumes. This work was supported by grants from the Sonderforschungsbereich 1039, Deutsche Forschungsgemeinschaft (Graduate school GRK1172 and FOG784), Merck KGaA, LOEWE Lipid Signaling Center LiFF and the Swiss National Science Foundation.
References
Alemany, R., van Koppen, C.J., Danneberg, K., Ter Braak, M., and Meyer Zu Heringdorf, D. (2007). Regulation and functional roles of sphingosine kinases. Naunyn Schmiedebergs Arch. Pharmacol. 374, 413–428.Suche in Google Scholar
Anada, Y., Igarashi, Y., and Kihara, A. (2007). The immunomodulator FTY720 is phosphorylated and released from platelets. Eur. J. Pharmacol. 568, 106–111.10.1016/j.ejphar.2007.04.053Suche in Google Scholar PubMed
Arlt, O., Schwiebs, A., Japtok, L., Ruger, K., Katzy, E., Kleuser, B., and Radeke, H.H. (2014). Sphingosine-1-phosphate modulates dendritic cell function: focus on non-migratory effects in vitro and in vivo. Cell. Physiol. Biochem. 34, 27–44.10.1159/000362982Suche in Google Scholar PubMed
Bandhuvula, P., Tam, Y.Y., Oskouian, B., and Saba, J.D. (2005). The immune modulator FTY720 inhibits sphingosine-1-phosphate lyase activity. J. Biol. Chem. 280, 33697–33700.10.1074/jbc.C500294200Suche in Google Scholar PubMed
Blaho, V.A. and Hla, T. (2014). An update on the biology of sphingosine 1-phosphate receptors. J. Lipid Res. 55, 1596–1608.10.1194/jlr.R046300Suche in Google Scholar PubMed PubMed Central
Blom, T., Back, N., Mutka, A.L., Bittman, R., Li, Z., de Lera, A., Kovanen, P.T., Diczfalusy, U., and Ikonen, E. (2010). FTY720 stimulates 27-hydroxycholesterol production and confers atheroprotective effects in human primary macrophages. Circ. Res. 106, 720–729.10.1161/CIRCRESAHA.109.204396Suche in Google Scholar PubMed PubMed Central
Brinkmann, V., Billich, A., Baumruker, T., Heining, P., Schmouder, R., Francis, G., Aradhye, S., and Burtin, P. (2010). Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discov. 9, 883–897.10.1038/nrd3248Suche in Google Scholar PubMed
Bryan, L., Kordula, T., Spiegel, S., and Milstien, S. (2008). Regulation and functions of sphingosine kinases in the brain. Biochim. Biophys. Acta 1781, 459–466.10.1016/j.bbalip.2008.04.008Suche in Google Scholar PubMed PubMed Central
Cartwright, T.A., Campos, C.R., Cannon, R.E., and Miller, D.S. (2013). Mrp1 is essential for sphingolipid signaling to p-glycoprotein in mouse blood-brain and blood-spinal cord barriers. J. Cereb. Blood Flow Metab. 33, 381–388.10.1038/jcbfm.2012.174Suche in Google Scholar PubMed PubMed Central
Cinamon, G., Zachariah, M.A., Lam, O.M., Foss, F.W., and Cyster, J.G. (2008). Follicular shuttling of marginal zone B cells facilitates antigen transport. Nat. Immunol. 9, 54–62.10.1038/ni1542Suche in Google Scholar PubMed PubMed Central
Ferreirós, N., Labocha, S., Schröder, M., Radeke, H.H., and Geisslinger, G. (2012). LC-MS/MS determination of FTY720 and FTY720-phosphate in murine intracellular compartments and human plasma. J. Chromatogr. B Analyt. Technol. Biomed. Life. Sci. 887–888, 122–127.10.1016/j.jchromb.2012.01.023Suche in Google Scholar PubMed
Fukuhara, S., Simmons, S., Kawamura, S., Inoue, A., Orba, Y., Tokudome, T., Sunden, Y., Arai, Y., Moriwaki, K., Ishida, J., et al. (2012). The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice. J. Clin. Invest. 122, 1416–1426.10.1172/JCI60746Suche in Google Scholar PubMed PubMed Central
Hait, N.C., Allegood, J., Maceyka, M., Strub, G.M., Harikumar, K.B., Singh, S.K., Luo, C., Marmorstein, R., Kordula, T., Milstien, S. et al. (2009). Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate. Science 325, 1254–1257.10.1126/science.1176709Suche in Google Scholar PubMed PubMed Central
Hisano, Y., Kobayashi, N., Kawahara, A., Yamaguchi, A., and Nishi, T. (2011). The sphingosine 1-phosphate transporter, SPNS2, functions as a transporter of the phosphorylated form of the immunomodulating agent FTY720. J. Biol. Chem. 286, 1758–1766.10.1074/jbc.M110.171116Suche in Google Scholar PubMed PubMed Central
Honig, S.M., Fu, S., Mao, X., Yopp, A., Gunn, M.D., Randolph, G.J., and Bromberg, J.S. (2003). FTY720 stimulates multidrug transporter- and cysteinyl leukotriene-dependent T cell chemotaxis to lymph nodes. J. Clin. Invest. 111, 627–637.10.1172/JCI200316200Suche in Google Scholar
Huwiler, A. and Pfeilschifter, J. (2008). New players on the center stage: sphingosine 1-phosphate and its receptors as drug targets. Biochem. Pharmacol. 75, 1893–1900.10.1016/j.bcp.2007.12.018Suche in Google Scholar PubMed
Igarashi, N., Okada, T., Hayashi, S., Fujita, T., Jahangeer, S., and Nakamura, S. (2003). Sphingosine kinase 2 is a nuclear protein and inhibits DNA synthesis. J. Biol. Chem. 278, 46832–46839.10.1074/jbc.M306577200Suche in Google Scholar PubMed
Kappos, L., Radue, E.W., O’Connor, P., Polman, C., Hohlfeld, R., Calabresi, P., Selmaj, K., Agoropoulou, C., Leyk, M., Zhang-Auberson, L., et al. (2010). A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N. Engl. J. Med. 362, 387–401.10.1056/NEJMoa0909494Suche in Google Scholar PubMed
Lim, K.G., Sun, C., Bittman, R., Pyne, N.J., and Pyne, S. (2011). (R)-FTY720 methyl ether is a specific sphingosine kinase 2 inhibitor: effect on sphingosine kinase 2 expression in HEK 293 cells and actin rearrangement and survival of MCF-7 breast cancer cells. Cell. Signal. 23, 1590–1595.10.1016/j.cellsig.2011.05.010Suche in Google Scholar PubMed PubMed Central
Limaye, V. (2008). The role of sphingosine kinase and sphingosine-1-phosphate in the regulation of endothelial cell biology. Endothelium 15, 101–112.10.1080/10623320802125342Suche in Google Scholar PubMed
Liu, M., Seo, J., Allegood, J., Bi, X., Zhu, X., Boudyguina, E., Gebre, A.K., Avni, D., Shah, D., Sorci-Thomas, M.G. et al. (2014). Hepatic apolipoprotein M (ApoM) overexpression stimulates formation of larger ApoM/sphingosine 1-phosphate-enriched plasma high density lipoprotein. J. Biol. Chem. 289, 2801–2814.10.1074/jbc.M113.499913Suche in Google Scholar PubMed PubMed Central
Mandala, S., Hajdu, R., Bergstrom, J., Quackenbush, E., Xie, J., Milligan, J., Thornton, R., Shei, G.J., Card, D., Keohane, C., et al. (2002). Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 296, 346–349.10.1126/science.1070238Suche in Google Scholar
Matloubian, M., Lo, C.G., Cinamon, G., Lesneski, M.J., Xu, Y., Brinkmann, V., Allende, M.L., Proia, R.L., and Cyster, J.G. (2004). Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427, 355–360.10.1038/nature02284Suche in Google Scholar
Moriue, T., Igarashi, J., Yoneda, K., Nakai, K., Kosaka, H. and Kubota, Y. (2008). Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in endothelial cells. Biochem. Biophys. Res. Commun. 368, 852–857.10.1016/j.bbrc.2008.01.155Suche in Google Scholar
Neumann, S. and van Meer, G. (2008). Sphingolipid management by an orchestra of lipid transfer proteins. Biol. Chem. 389, 1349–1360.10.1515/BC.2008.154Suche in Google Scholar
Nishi, T., Kobayashi, N., Hisano, Y., Kawahara, A. and Yamaguchi, A. (2014). Molecular and physiological functions of sphingosine 1-phosphate transporters. Biochim. Biophys. Acta 1841, 759–765.10.1016/j.bbalip.2013.07.012Suche in Google Scholar
Oo, M.L., Chang, S.H., Thangada, S., Wu, M.T., Rezaul, K., Blaho, V., Hwang, S.I., Han, D.K., and Hla, T. (2011). Engagement of S1P(1)-degradative mechanisms leads to vascular leak in mice. J. Clin. Invest. 121, 2290–2300.10.1172/JCI45403Suche in Google Scholar
Oskeritzian, C.A., Alvarez, S.E., Hait, N.C., Price, M.M., Milstien, S., and Spiegel, S. (2008). Distinct roles of sphingosine kinases 1 and 2 in human mast-cell functions. Blood 111, 4193–4200.10.1182/blood-2007-09-115451Suche in Google Scholar
Paugh, S.W., Payne, S.G., Barbour, S.E., Milstien, S., and Spiegel, S. (2003). The immunosuppressant FTY720 is phosphorylated by sphingosine kinase type 2. FEBS Lett. 554, 189–193.10.1016/S0014-5793(03)01168-2Suche in Google Scholar
Payne, S.G., Oskeritzian, C.A., Griffiths, R., Subramanian, P., Barbour, S.E., Chalfant, C.E., Milstien, S., and Spiegel, S. (2007). The immunosuppressant drug FTY720 inhibits cytosolic phospholipase A2 independently of sphingosine-1-phosphate receptors. Blood 109, 1077–1085.10.1182/blood-2006-03-011437Suche in Google Scholar PubMed PubMed Central
Price, M.M., Oskeritzian, C.A., Falanga, Y.T., Harikumar, K.B., Allegood, J.C., Alvarez, S.E., Conrad, D., Ryan, J.J., Milstien, S., and Spiegel, S. (2013). A specific sphingosine kinase 1 inhibitor attenuates airway hyperresponsiveness and inflammation in a mast cell-dependent murine model of allergic asthma. J. Allergy Clin. Immunol. 131, 501–511. e501.10.1016/j.jaci.2012.07.014Suche in Google Scholar PubMed PubMed Central
Qin, J., E. Berdyshev, Goya, J., Natarajan, V., and Dawson, G. (2010). Neurons and oligodendrocytes recycle sphingosine 1-phosphate to ceramide: significance for apoptosis and multiple sclerosis. J. Biol. Chem. 285, 14134–14143.10.1074/jbc.M109.076810Suche in Google Scholar PubMed PubMed Central
Rathmell, J.C., Farkash, E.A., Gao, W., and Thompson, C.B. (2001). IL-7 enhances the survival and maintains the size of naive T cells. J Immunol 167, 6869–6876.10.4049/jimmunol.167.12.6869Suche in Google Scholar PubMed
Saddoughi, S.A., Song, P., and Ogretmen, B. (2008). Roles of bioactive sphingolipids in cancer biology and therapeutics. Subcell. Biochem. 49, 413–440.10.1007/978-1-4020-8831-5_16Suche in Google Scholar PubMed PubMed Central
Saddoughi, S.A., Gencer, S., Peterson, Y.K., Ward, K.E., Mukhopadhyay, A., Oaks, J., Bielawski, J., Szulc, Z.M., Thomas, R.J., Selvam, S.P., et al. (2013). Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis. EMBO Mol. Med. 5, 105–121.10.1002/emmm.201201283Suche in Google Scholar PubMed PubMed Central
Schwab, S.R., Pereira, J.P., Matloubian, M., Xu, Y., Huang, Y., and Cyster, J.G. (2005). Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients. Science 309, 1735–1739.10.1126/science.1113640Suche in Google Scholar PubMed
Sensken, S.C., Bode, C., and Graler, M.H. (2009). Accumulation of fingolimod (FTY720) in lymphoid tissues contributes to prolonged efficacy. J. Pharmacol. Exp. Ther. 328, 963–969.10.1124/jpet.108.148163Suche in Google Scholar PubMed
Simons, K. and van Meer, G. (1988). Lipid sorting in epithelial cells. Biochemistry 27, 6197–6202.10.1021/bi00417a001Suche in Google Scholar PubMed
Skoura, A. and Hla, T. (2009). Lysophospholipid receptors in vertebrate development, physiology, and pathology. J. Lipid Res. 50 (Suppl.), S293–298.10.1194/jlr.R800047-JLR200Suche in Google Scholar PubMed PubMed Central
Spiegel, S. and Milstien, S. (2011). The outs and the ins of sphingosine-1-phosphate in immunity. Nat. Rev. Immunol. 11, 403–415.10.1038/nri2974Suche in Google Scholar PubMed PubMed Central
van Meer, G. and de Kroon, A.I. (2011). Lipid map of the mammalian cell. J. Cell Sci. 124, 5–8.10.1242/jcs.071233Suche in Google Scholar PubMed
von Andrian, U.H. and Mackay, C.R. (2000). T-cell function and migration. Two sides of the same coin. N. Engl. J. Med 343, 1020–1034.10.1056/NEJM200010053431407Suche in Google Scholar PubMed
©2015 by De Gruyter
Artikel in diesem Heft
- Frontmatter
- Guest Editorial
- Highlight: Molecular Medicine of Sphingolipids
- HIGHLIGHT: MOLECULAR MEDICINE OF SPHINGOLIPIDS
- The role of serum amyloid A and sphingosine-1-phosphate on high-density lipoprotein functionality
- Sphingolipids in viral infection
- Tackling the biophysical properties of sphingolipids to decipher their biological roles
- Ceramide and sphingosine in pulmonary infections
- Molecular mechanisms of erythrocyte aging
- Sphingolipids in liver injury, repair and regeneration
- Ultrasound-stimulated microbubble enhancement of radiation response
- Innate immune responses in the brain of sphingolipid lysosomal storage diseases
- Novel mechanisms of action of classical chemotherapeutic agents on sphingolipid pathways
- The role of sphingolipids in endothelial barrier function
- The effect of altered sphingolipid acyl chain length on various disease models
- Secretory sphingomyelinase in health and disease
- Preclinical development of a C6-ceramide NanoLiposome, a novel sphingolipid therapeutic
- Sphingomyelin breakdown in T cells: role in activation, effector functions and immunoregulation
- The molecular medicine of acid ceramidase
- Caenorhabditis elegans as a model to study sphingolipid signaling
- S1PR4 is required for plasmacytoid dendritic cell differentiation
- Antinociceptive effects of FTY720 during trauma-induced neuropathic pain are mediated by spinal S1P receptors
- Subcellular distribution of FTY720 and FTY720-phosphate in immune cells – another aspect of Fingolimod action relevant for therapeutic application
- Downregulation of sphingosine 1-phosphate (S1P) receptor 1 by dexamethasone inhibits S1P-induced mesangial cell migration
- Sphingosine kinase 2 deficiency increases proliferation and migration of renal mouse mesangial cells and fibroblasts
- Obituary
- The life and work of Dr. Robert Bittman (1942–2014)
Artikel in diesem Heft
- Frontmatter
- Guest Editorial
- Highlight: Molecular Medicine of Sphingolipids
- HIGHLIGHT: MOLECULAR MEDICINE OF SPHINGOLIPIDS
- The role of serum amyloid A and sphingosine-1-phosphate on high-density lipoprotein functionality
- Sphingolipids in viral infection
- Tackling the biophysical properties of sphingolipids to decipher their biological roles
- Ceramide and sphingosine in pulmonary infections
- Molecular mechanisms of erythrocyte aging
- Sphingolipids in liver injury, repair and regeneration
- Ultrasound-stimulated microbubble enhancement of radiation response
- Innate immune responses in the brain of sphingolipid lysosomal storage diseases
- Novel mechanisms of action of classical chemotherapeutic agents on sphingolipid pathways
- The role of sphingolipids in endothelial barrier function
- The effect of altered sphingolipid acyl chain length on various disease models
- Secretory sphingomyelinase in health and disease
- Preclinical development of a C6-ceramide NanoLiposome, a novel sphingolipid therapeutic
- Sphingomyelin breakdown in T cells: role in activation, effector functions and immunoregulation
- The molecular medicine of acid ceramidase
- Caenorhabditis elegans as a model to study sphingolipid signaling
- S1PR4 is required for plasmacytoid dendritic cell differentiation
- Antinociceptive effects of FTY720 during trauma-induced neuropathic pain are mediated by spinal S1P receptors
- Subcellular distribution of FTY720 and FTY720-phosphate in immune cells – another aspect of Fingolimod action relevant for therapeutic application
- Downregulation of sphingosine 1-phosphate (S1P) receptor 1 by dexamethasone inhibits S1P-induced mesangial cell migration
- Sphingosine kinase 2 deficiency increases proliferation and migration of renal mouse mesangial cells and fibroblasts
- Obituary
- The life and work of Dr. Robert Bittman (1942–2014)