Sphingosine Kinase 2 Is Required for Modulation of Lymphocyte Traffic by FTY720

Abstract

Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.Sphingosine 1-phosphate (S1P) 2 receptor agonists are likely to be the next generation of pharmacologic agents used to modulate immune system function. The prototype drug of this class is FTY720, which is highly efficacious in prolonging allograft survival and in ameliorating autoimmune disease in a variety of animal models (1-4). FTY720 is being tested in human trials for the indications renal transplantation and multiple sclerosis (5). Further, there is mounting evidence that S1P agonists are efficacious in animal models of atherosclerosis (6), renal ischemia-reperfusion injury (7), and acute lung injury (8).FTY720 is a sphingosine analog that, after activation by phosphorylation (to FTY720-P), disrupts lymphocyte trafficking by decreasing lymphocyte egress from lymph nodes and the thymus (9, 10). Although the precise mechanisms that underlie this phenomenon are uncertain, the profound lymphopenia that is the index of FTY720 action is dependent on agonist action at lymphocyte S1P 1 receptors. Since FTY720-P is also a potent agonist at the S1P 3 , S1P 4 , and S1P 5 receptors (11, 12), it remains unknown whether the multiple therapeutic benefits of the drug correlate with agonist activity at the S1P 1 receptor. The propensity for S1P 1 receptor responses to desensitize (13) and the similar behaviors of S1P 1 receptor null thymocytes and FTY720-treated mouse lymphocytes have led to the suggestion that FTY720-P is a functional antagonist (14). In this scenario, the drug exaggerates S1P tone to the extent that the lymphocyte S1P 1 receptor signaling is chronically down-regulated.The kinase(s) responsible for FTY720 activation is the gateway whereby S1P signaling can be accessed readily with a therapeutic agent. Knowledge of this enzyme is important specifically to guide S1P prodrug design and generally to gain insight into the normal role of S1P in immune function. The identity of the kinase is not known currently; two candidates are sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2). These enzymes, which are expressed widely, catalyz...

Sphingosine‐1‐phosphate signaling in physiology and diseases

“…The myosin light chain kinase activation by PLC-Ca 2+ , together with myosin phosphatase inhibition by Rho-Rho kinase, efficiently increases myosin light chain phosphorylation and, thereby, vascular contraction. S1P 2 is also suggested to contribute to vascular tone through a mechanism involving the action on the 20 endothelium although the precise mechanism remains to be defined [48]. S1P contributes to vascular barrier integrity.…”

Asymmetric Synthesis of Conformationally Constrained Fingolimod Analogues—Discovery of an Orally Active Sphingosine 1-Phosphate Receptor Type-1 Agonist and Receptor Type-3 Antagonist

“…22 Mouse Sphk2 was expressed by the introduction of plasmid DNA into HEK293T cells. Sphk2 activity was measured in a solution that consisted of 20 mM Tris-Cl (pH 7.4), 1 mM 2-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM sodium orthovanadate, 40 mM β-glycerophosphate, 15 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 mM MgCl 2 , 0.5 mM 4-deoxypyridoxine, 10% glycerol, and 0.01 mg/mL each of leupeptin, aprotinin, and soybean trypsin inhibitor.…”

FTY720 Ameliorates Th1-Mediated Colitis in Mice by Directly Affecting the Functional Activity of CD4+CD25+ Regulatory T Cells

“…Currently, the discussion about its mode of action is mainly focused on its property to cause lymph node homing or sequestration of T cells. In this scenario, FTY720 activates sphingosine-1-phosphate (S1P) 4 receptors and modulates migration after being effectively phosphorylated in vivo by sphingosine kinase 2 (SphK2) (5,6). FTY720-phosphate (FTY720-P) exhibits a potency comparable to S1P itself as an agonist at four of the five known G-protein-coupled S1PRs (S1P 1,3,4,5 ).…”