Recycling of Sphingosine Is Regulated by the Concerted Actions of Sphingosine-1-phosphate Phosphohydrolase 1 and Sphingosine Kinase 2

Abstract

In yeast, the long-chain sphingoid base phosphate phosphohydrolase Lcb3p is required for efficient ceramide synthesis from exogenous sphingoid bases. Similarly, in this study, we found that incorporation of exogenous sphingosine into ceramide in mammalian cells was regulated by the homologue of Lcb3p, sphingosine-1-phosphate phosphohydrolase 1 (SPP-1), an endoplasmic reticulum resident protein. Sphingosine incorporation into endogenous long-chain ceramides was increased by SPP-1 overexpression, whereas recycling of C 6 -ceramide into long-chain ceramides was not altered. The increase in ceramide was inhibited by fumonisin B 1 , an inhibitor of ceramide synthase, but not by ISP-1, an inhibitor of serine palmitoyltransferase, the rate-limiting step in the de novo biosynthesis of ceramide. Mass spectrometry analysis revealed that SPP-1 expression increased the incorporation of sphingosine into all ceramide acyl chain species, particularly enhancing C16:0, C18:0, and C20:0 long-chain ceramides. The increased recycling of sphingosine into ceramide was accompanied by increased hexosylceramides and, to a lesser extent, sphingomyelins. Sphingosine kinase 2, but not sphingosine kinase 1, acted in concert with SPP-1 to regulate recycling of sphingosine into ceramide. Collectively, our results suggest that an evolutionarily conserved cycle of phosphorylation-dephosphorylation regulates recycling and salvage of sphingosine to ceramide and more complex sphingolipids.Sphingolipids are a structurally diverse family of membrane lipids. Several lines of evidence have implicated metabolites of sphingolipids such as ceramide, sphingosine, and sphingosine 1-phosphate (S1P) 5 in diverse cellular processes (1, 2). Ceramide and sphingosine have been implicated in pathways involving stress responses, cell differentiation, apoptosis, and cell cycle arrest (1). Unlike ceramide and sphingosine, S1P promotes cell growth and survival and inhibits apoptosis (2, 3). Because of their inter-convertibility and opposing effects, the dynamic balance between S1P and ceramide/sphingosine has been proposed to be an important factor that determines cell fate (2). Accumulating evidence suggests that the S1P/ceramide balance is ultimately regulated by the relative activities of enzymes controlling the turnover of these sphingolipid metabolites. However, the molecular mechanisms involved in the regulation of intracellular levels of these sphingolipids are not yet fully understood. Cellular levels of S1P are kept low by tight spatio-temporal regulation of its synthesis and degradation. Sphingosine kinases (SphKs) catalyze the synthesis of S1P by phosphorylation of sphingosine. Two distinct SphK isoforms, SphK1 and SphK2, have been cloned and characterized in mammals (2). Diverse external stimuli, particularly growth and survival factors, stimulate SphK1, generating S1P that has been implicated in their mitogenic and anti-apoptotic effects (4, 5). In contrast to SphK1, rather than promoting growth and survival, overexpression of SphK2 suppressed growth and...

Ceramide starves cells to death by downregulating nutrient transporter proteins

“…Because of their relatively high solubility in aqueous media, short-chain ceramides have been used extensively to study the effects of ceramide on cells. Exogenous, short-chain ceramides are converted into long-chain ceramides within the cell (21,22). We performed HPLC ESI-MS/MS to evaluate whether the levels and types of long-chain ceramide produced in C2-ceramide-treated cells were similar to those produced when ceramide was generated endogenously (Fig.…”

Sphingosine 1-Phosphate Lyase Deficiency Disrupts Lipid Homeostasis in Liver

“…Further, the lack of histologic and morphologic evidence of lysosomal storage was suggestive that lysosomal degradation of sphingolipids was not impaired and, thus, not responsible for the elevated lipids in the lyase-deficient mice. Instead, the increased levels of sphingolipids is more compatible with their generation by the sphingolipid recycling or salvage pathway that has been shown to utilize S1P as a starting point (5,15). In the recycling pathway, S1P is dephosphorylated by S1P phosphatases, which reside in the endoplasmic reticulum, to produce sphingosine.…”

“…It is only through the S1P lyase reaction that sphingolipid substrates are able to irreversibly exit the metabolic pathway. S1P, which is synthesized by sphingosine kinase, if not degraded by the S1P lyase, may be dephosphorylated by S1P phosphatase to generate sphingosine that is reutilized in sphingolipid metabolism through a recycling or salvage pathway (5). Alternatively, S1P can be exported out of the cell, where it is then available for interactions with cell surface receptors (6).…”

Sphingosine-1-phosphate Phosphatase 2 Regulates Pancreatic Islet β-Cell Endoplasmic Reticulum Stress and Proliferation

“…Disruption of ceramide synthesis, by depletion of ceramide synthase 2, also leads to activation of the UPR (32). SPPase activity is required for efficient recycling of sphingoid bases into the ceramide synthesis pathway (7). In some mammalian cells, recycling can account for more than half of complex sphingolipid synthesis (33).…”

“…In an alternative catabolic route catalyzed by S1P phosphatases, the resulting sphingosine product can be derivatized with a fatty acid by ceramide synthase to produce ceramide by the sphingolipid salvage or recycling pathway (7). The metabolism of S1P constitutes a key point in the sphingolipid pathway in which substrate may either leave or be retained within the pathway, thus regulating the homeostatic flux of three bioactive sphingolipid metabolites: S1P, sphingosine, and ceramide.…”