The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPL(fl/fl/Nes)) but not postnatal neuronal forebrain-restricted SPL deletion (SPL(fl/fl/CaMK)) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPL(fl/fl/Nes) mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.
Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.
Photolysis of 6-bromo-7-hydroxycoumarinyl-caged ceramide was used to generate ceramide with spatial and temporal control in supported lipid bilayers prepared from mixtures of caged ceramide and phospholipids. The caged ceramide molecules are randomly distributed in fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, and upon photolysis with long wavelength UV light small ordered ceramide domains are formed that phase separate from the bulk fluid membrane. Irradiation of a spatially restricted area leads to the transient formation of ceramide-enriched gel phase domains that equilibrate via lipid diffusion with the surrounding unirradiated membrane. Photorelease of C16-ceramide in supported bilayers prepared from POPC, caged ceramide and the ganglioside GM1 (90:10:1 molar ratio) results in partitioning of a ganglioside-protein complex into the ceramide-enriched domains, modeling some aspects of ceramide’s behavior in cells. The photo-uncaging strategy used here for delivery of ceramide in bilayers provides a novel and useful alternative to the enzymatic generation of ceramide in sphingomyelin-containing membranes. The ability to control membrane phase separation behavior and the clustering of membrane-anchored proteins illustrates the potential of photo-uncaging for studying the compartmentalization of ceramide in cellular membranes.
Background We investigated the hypothesis that postconditioning by FTY720 (FTY) in isolated perfused mouse hearts is independent of the sphingosine 1-phosphate (S1P) pathway. Material and Methods Ex vivo hearts were exposed to postconditioning (POST) by either ischemia or FTY720. Protection against ischemia/reperfusion (IR) injury was measured by recovery of left ventricular developed pressure (LVDP) and infarct size. Results FTY effectively postconditioned (POST) ex vivo hearts against ischemia/reperfusion (IR) injury as measured by recovery of LVDP and a low infarct size. FTY protection, unlike S1P but like sphingosine (Sph), was insensitive to inhibition of S1P G-Protein Coupled Receptors (GPCRs) or inhibition of PI3 kinase. Protection by FTY and Sph was however blocked by inhibitors of PKA and PKG. Thus, FTY follows the same cardioprotective pathway as Sph. This was further supported by studies of FTY POST in knockout (KO) mice lacking the SphK2 form of Sph kinase that is needed for phosphorylation of FTY to an S1P analog. In the absence of SphK2, FTY (and Sph) POST was still cardioprotective. This differed from the effect of SphK2 KO on protection by ischemic POST (IPOST). IPOST was not effective in KO hearts. To see if the GPCR signaling pathway to protection is normal in KO hearts, we looked at POST by GPCR agonists S1P and adenosine. Both provided effective protection even in KO hearts suggesting that the problem with IPOST in KO hearts is a low level of S1P available for release during IPOST. Thus, pharmacologic POST with FTY or Sph, like adenosine and S1P, is unaffected in the KO. Conclusions FTY720 administered in vivo might behave in a dual manner showing both S1P-like effects and sphingosine-like effects. It appears that the latter may have been overlooked and may be the more important in aging hearts.
Sphingomyelins are important phospholipids in plasma membranes of most cells. Because of their dominantly saturated nature, they affect the lateral structure of membranes, and contribute to the regulation of cholesterol distribution within membranes, and in cells. However, the abundance of molecular species present in cells also implies that sphingomyelins have other, more specific functions. Many of these functions are currently unknown, but are under extensive study. Mostly model membrane studies have shown that sphingomyelins (and other sphingolipids), in contrast to glycerophospholipids, have important hydrogen-bonding properties which in several important ways confer specific functional properties to this abundant class of membrane phospholipids. The often very asymmetric nature of sphingomyelins, arising from mismatch in length between the long chain base and N-acyl chains, also impose specific properties (e.g., interdigitation) to sphingomyelins not seen with glycerophospholipids. In this review, the latest sphingomyelin literature will be scrutinized, and an effort will be made to correlate the molecular structure of sphingomyelin with functional properties. In particular, the effects of head group properties, interfacial hydrogen bonding, long chain base hydroxylation, N-acyl chain hydroxylation, and N-acyl chain methyl branching will be discussed.
Acid sphingomyelinase (ASM) is a lipid hydrolase that cleaves the sphingolipid, sphingomyelin, into ceramide. Mutations in the ASM gene (SMPD1) result in the rare lysosomal storage disorder, Niemann-Pick disease (NPD). In addition to its role in NPD, over the past two decades, the importance of sphingolipids, and ASM in particular, in normal physiology and the pathophysiology of numerous common diseases also has become known. For example, altered sphingolipid metabolism occurs in many cancers, generally reducing the levels of the pro-apoptotic lipid, ceramide, and/or elevating the levels of the proliferative lipid, sphingosine-1-phosphate (S1P). These changes likely contribute to the tumorigenicity and/or metastatic capacity of the cancer. In addition, many cancer therapies induce ceramide-mediated death, and cancer cells have evolved novel mechanisms to overcome this effect. In the present review, we discuss sphingolipid metabolism in cancer, and specifically the potential for pharmacological modulation using ASM. Of note, recombinant human ASM (rhASM) has been produced for human use and is being evaluated as a treatment for NPD. Thus, its use for cancer therapy could be rapidly evaluated in the clinic after appropriate animal model studies have been completed. As this enzyme was initially studied in the context of NPD, we start with a brief overview of the history of ASM and NPD, followed by a discussion of the role of ASM in cancer biology, and then summarize emerging preclinical efficacy studies using rhASM as an adjunct in the treatment of solid tumors.
Renal fibrosis is defined as the excessive deposition and modification of extracellular matrix (ECM) in the renal parenchyma in response to injury and inflammation, resulting in renal function loss. This condition is common to many chronic kidney diseases (CKD) that occur under diverse pathological conditions, such as diabetic and hypertensive nephropathy. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in regulation of cardiovascular functions and pathogenesis of various cardiovascular diseases. S1P has also been seen as an important regulator of fibrotic diseases, playing significant roles in the differentiation of fibroblasts to myofibroblasts and in the induction of inflammatory responses in early stages of fibrotic diseases. This mini review summarizes recent research findings on the importance of the sphingosine kinase-1 (SphK1)/S1P/S1P receptor (S1PR) axis in interaction with other classic fibrotic signaling pathways and the immune inflammatory response to reveal novel therapeutic targets for treatment of renal fibrosis.
Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine-1-phosphate (S1P). In the present study, we evaluated the role of AC-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared to other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human subjects with Stage II melanoma. Immunofluorescence studies revealed that the subcellular localization of AC differs between melanocytes (where it is found in both cytosol and nucleus) and melanoma cells (where it is primarily localized to cytosol). In addition to having high AC levels, melanoma cells generate lower amounts of ceramides than normal melanocytes do. This down-regulation in ceramide production appears to result from suppression of de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC50 =12 nM) and stable inhibitor. AC inhibition increased cellular ceramide levels, decreased S1P levels, and acted synergistically with several, albeit not all, antitumoral agents. The results suggest that AC-controlled sphingolipid metabolism may play an important role in the control of melanoma proliferation.
Activation of the lysosomal ceramide-producing enzyme acid sphingomyelinase (ASM) by various stresses is centrally involved in cell death and has been implicated in autophagy. We set out to investigate the role of the baseline ASM activity in maintaining physiological functions of lysosomes, focusing on lysosomal nutrient-sensing complex (LYNUS), a lysosomal membrane-anchored multiprotein complex that includes the mammalian target of rapamycin (mTOR) and the transcription factor EB (TFEB). ASM inhibition with imipramine or SMPD1 siRNA in human lung cells, or by transgenic Smpd1+/- haploinsufficiency of mouse lungs, markedly reduced mTOR- and P70-S6 kinase Thr 389- phosphorylation and modified TFEB in a pattern consistent with its activation. Inhibition of baseline ASM activity significantly increased autophagy with preserved degradative potential. Pulse labeling of sphingolipid metabolites revealed that ASM inhibition markedly decreased sphingosine and sphingosine-1 phosphate (S1P) levels at the level of ceramide hydrolysis. These findings suggest that ASM functions to maintain physiological mTOR signaling and inhibit autophagy and implicate sphingosine and/or S1P in the control of lysosomal function.
The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery to pathological EMT and may yield new biomarkers and therapeutic targets.