FTY720 Inhibits Ceramide Synthases and Up-regulates Dihydrosphingosine 1-Phosphate Formation in Human Lung Endothelial Cells

Abstract

Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent sphingosine 1-phosphate (S1P)-like action through S1P 1,3-5 receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with L-[U-13 C,15 N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, sphingosine, and S1P but increased levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent K i of 2.15 M. FTY720-induced up-regulation of DHS1P level was mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells. FTY7202 is a synthetic analog of sphingosine and is currently being studied as a potent immunosuppressive and immunomodulatory agent (1-3). FTY720-induced immunosuppression is ascribed, in part, to its protective effect on endothelial cell barrier function that results in inhibition of lymphocyte egress from lymph nodes and down-regulation of innate and adaptive immune responses (4). As endothelial cells predominantly express the sphingosine 1-phosphate 1 (S1P 1 ) receptor and its activation initiates signaling that results in the assembly of VEcadherin-based adherens junctions (5), it is thought that the phosphorylation of FTY720 and the binding of FTY720-P to the S1P 1 receptor determine its effect on vasculature (1). Recently it became evident that the action of FTY720 is more complex as several other direct protein targets were identified. Thus, FTY720 was found to bind to and inhibit the cannabinoid CB1 receptor (6), to inhibit cytosolic phospholipase A 2 (cPLA 2 ), and to counteract ceramide 1-phosphate-induced cPLA 2 activation (7). Additionally FTY720 but not FTY720-P was shown to inhibit S1P lyase (8), which degrades ...

Stimulation of Sphingosine 1-Phosphate Signaling as an Alveolar Cell Survival Strategy in Emphysema

“…Ceramide, dihydroceramide, and S1P and dihydro-S1P were measured as described (24)(25)(26) and detailed in the online supplement.…”

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: A protective role of autophagy

“…It is notable that in most instances phosphorylation of FTY720 was not required for its cytotoxic effect, indicating the involvement of S1P receptor-independent mechanisms which are starkly different from the immunosuppresive property of FTY720. 16 To date, a number of molecular targets have been suggested for the unphosphorylated form of FTY720, including cytosolic phospholipase A2, 17 protein phosphatase 2a (PP2a), 18 protein kinase Cδ, 14 ceramide synthase 19 and sphingosine kinase 1 (SphK1). 20 These downstream targets appear to be associated with the ability of FTY720 to suppress cell growth and/or induce cell death.…”

Down-regulation of S1P1 Receptor Surface Expression by Protein Kinase C Inhibition

“…But S1P-lyase inhibition was not evident in the in vivo setting (23), and possible contributions of PLA2, SK1/2, and ceramide synthase for the in vivo efficacy of FTY720 were not even tested (21,22,24). This study clearly identifies FTY720 as a pan-PKC inhibitor not only in vitro but also in vivo at physiologically relevant concentrations (Figs.…”

“…FTY720 was shown to directly affect several different cellular enzymes including PLA2 (22), S1P-lyase (23), SK1/2 (21), and ceramide synthases (24). But S1P-lyase inhibition was not evident in the in vivo setting (23), and possible contributions of PLA2, SK1/2, and ceramide synthase for the in vivo efficacy of FTY720 were not even tested (21,22,24).…”

“…FTY720 phosphate (FTY-P) not only activates S1P 1 but also three of the four remaining S1P receptors except S1P 2 (6,17). Furthermore FTY720 is cell-permeable and targets many other intracellular enzymes related to sphingolipid metabolism like sphingosine kinases 1 and 2 (SK1/2) (21), phospholipase A2 (PLA 2 ) (22), S1P-lyase (23), and ceramide synthases (24). Because we originally observed a different specificity for FTY720-induced inhibition of S1P receptors compared with FTY-P induced stimulation (7), we aimed to study potential cellular targets of FTY720 involved in S1P 1 receptor surface expression that are modulated independently from its phosphorylation to FTY-P.…”