Enzymes of sphingolipid metabolism as transducers of metabolic inputs.

Sphingolipids (SLs) constitute a discrete subdomain of metabolism, and they display both structural and signaling functions. Accumulating evidence also points to intimate connections between intermediary metabolism and SL metabolism. Given that many SLs exhibit bioactive properties (i.e. transduce signals), these raise the possibility that an important function of SLs is to relay information on metabolic changes into specific cell responses. This could occur at various levels. Some metabolites are incorporated into SLs, whereas others may initiate regulatory or signaling events that, in turn, modulate SL metabolism. In this review, we elaborate on the former as it represents a poorly appreciated aspect of SL metabolism, and we develop the hypothesis that the SL network is highly sensitive to several specific metabolic changes, focusing on amino acids (serine and alanine), various fatty acids, choline (and ethanolamine), and glucose.

A comprehensive measure of Golgi sphingolipid flux using NBD C6-ceramide: evaluation of sphingolipid inhibitors.

Measurements of sphingolipid metabolism are most accurately performed by LC-MS. However, this technique is expensive, not widely accessible, and without the use of specific probes, it does not provide insight into metabolic flux through the pathway. Employing the fluorescent ceramide analogue NBD-C6-ceramide as a tracer in intact cells, we developed a comprehensive HPLC-based method that simultaneously measures the main nodes of ceramide metabolism in the Golgi. Hence, by quantifying the conversion of NBD-C6-ceramide to NBD-C6-sphingomyelin, NBD-C6-hexosylceramides, and NBD-C6-ceramide-1-phosphate (NBD-C1P), the activities of Golgi resident enzymes sphingomyelin synthase 1, glucosylceramide synthase, and ceramide kinase (CERK) could be measured simultaneously. Importantly, the detection of NBD-C1P allowed us to quantify CERK activity in cells, a usually difficult task. By applying this method, we evaluated the specificity of commonly used sphingolipid inhibitors and discovered that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, which targets glucosylceramide synthase, and fenretinide (4HPR), an inhibitor for dihydroceramide desaturase, also suppress CERK activity. This study demonstrates the benefit of an expanded analysis of ceramide metabolism in the Golgi, and it provides a qualitative and easy-to-implement method.

Drug Targeting of Acyltransferases in the Triacylglyceride and 1-O-AcylCeramide Biosynthetic Pathways.

Acyltransferase enzymes (EC 2.3.) are a large group of enzymes that transfer acyl groups to a variety of substrates. This review focuses on fatty acyltransferases involved in the biosynthetic pathways of glycerolipids and sphingolipids and how these enzymes have been pharmacologically targeted in their biologic context. Glycerolipids and sphingolipids, commonly treated independently in their regulation and biologic functions, are put together to emphasize the parallelism in their metabolism and bioactive roles. Furthermore, a newly considered signaling molecule, 1-O-acylceramide, resulting from the acylation of ceramide by DGAT2 enzyme, is discussed. Finally, the implications of DGAT2 as a putative ceramide acyltransferase (CAT) enzyme, with a putative dual role in TAG and 1-O-acylceramide generation, are explored. SIGNIFICANCE STATEMENT: This manuscript reviews the current status of drug development in lipid acyltransferases. These are current targets in metabolic syndrome and other diseases, including cancer. A novel function for a member in this group of lipids has been recently reported in cancer cells. The responsible enzyme and biological implications of this added member are discussed.

Biological function, topology, and quantification of plasma membrane Ceramide.

Over the past 30 years, a growing body of evidence has revealed the regulatory role of the lipid ceramide in various cellular functions. The structural diversity of ceramide, resulting in numerous species, and its distinct distribution within subcellular compartments may account for its wide range of functions. However, our ability to study the potential role of ceramide in specific subcellular membranes has been limited. Several works have shown mitochondrial, Golgi, and plasma membrane ceramide to mediate signaling pathways independently. These results have started to shift the focus on ceramide signaling research toward specific membrane pools. Nonetheless, the challenge arises from the substantial intracellular ceramide content, hindering efforts to quantify its presence in particular membranes. Recently, we have developed the first method capable of detecting and quantifying ceramide in the plasma membrane, leading to unexpected results such as detecting different pools of ceramide responding to drug concentration or time. This review summarizes the historical context that defined the idea of pools of ceramide, the studies on plasma membrane ceramide as a bioactive entity, and the tools available for its study, especially the new method to detect and, for the first time, quantify plasma membrane ceramide. We believe this method will open new avenues for researching sphingolipid signaling and metabolism.

Ceramide as an endothelial cell surface receptor and a lung-specific lipid vascular target for circulating ligands.

The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.

A simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane.

The role of ceramide in biological functions is typically based on the elevation of cellular ceramide, measured by LC-MS in the total cell lysate. However, it has become increasingly appreciated that ceramide in different subcellular organelles regulates specific functions. In the plasma membrane, changes in ceramide levels might represent a small percentage of the total cellular ceramide, evading MS detection but playing a critical role in cell signaling. Importantly, there are currently no efficient techniques to quantify ceramide in the plasma membrane. Here, we developed a method to measure the mass of ceramide in the plasma membrane using a short protocol that is based on the hydrolysis of plasma membrane ceramide into sphingosine by the action of exogenously applied bacterial recombinant neutral ceramidase. Plasma membrane ceramide content can then be determined by measuring the newly generated sphingosine at a stoichiometry of 1:1. A key step of this protocol is the chemical fixation of cells to block cellular sphingolipid metabolism, especially of sphingosine to sphingosine 1-phosphate. We confirmed that chemical fixation does not disrupt the lipid composition at the plasma membrane, which remains intact during the time of the assay. We illustrate the power of the approach by applying this protocol to interrogate the effects of the chemotherapeutic compound doxorubicin. Here we distinguished two pools of ceramide, depending on the doxorubicin concentration, consolidating different reports. In summary, we have developed the first approach to quantify ceramide in the plasma membrane, allowing the study of new avenues in sphingolipid compartmentalization and function.

Compartmentalization of Sphingolipid metabolism: Implications for signaling and therapy.

Sphingolipids (SLs) are a family of bioactive lipids implicated in a variety of cellular processes, and whose levels are controlled by an interlinked network of enzymes. While the spatial distribution of SL metabolism throughout the cell has been understood for some time, the implications of this for SL signaling and biological outcomes have only recently begun to be fully explored. In this review, we outline the compartmentalization of SL metabolism and describe advances in tools for investigating and probing compartment-specific SL functions. We also briefly discuss the implications of SL compartmentalization for cell signaling and therapeutic approaches to targeting the SL network.

Sublethal doxorubicin promotes migration and invasion of breast cancer cells: role of Src Family non-receptor tyrosine kinases.

BACKGROUND: Doxorubicin (Dox) is a widely used chemotherapy, but its effectiveness is limited by dose-dependent side effects. Although lower Dox doses reduce this risk, studies have reported higher recurrence of local disease with no improvement in survival rate in patients receiving low doses of Dox. To effectively mitigate this, a better understanding of the adverse effects of suboptimal Dox doses is needed. METHODS: Effects of sublethal dose of Dox on phenotypic changes were assessed with light and confocal microscopy. Migratory and invasive behavior were assessed by wound healing and transwell migration assays. MTT and LDH release assays were used to analyze cell growth and cytotoxicity. Flow cytometry was employed to detect cell surface markers of cancer stem cell population. Expression and activity of matrix metalloproteinases were probed with qRT-PCR and zymogen assay. To identify pathways affected by sublethal dose of Dox, exploratory RNAseq was performed and results were verified by qRT-PCR in multiple cell lines (MCF7, ZR75-1 and U-2OS). Regulation of Src Family kinases (SFK) by key players in DNA damage response was assessed by siRNA knockdown along with western blot and qRT-PCR. Dasatinib and siRNA for Fyn and Yes was employed to inhibit SFKs and verify their role in increased migration and invasion in MCF7 cells treated with sublethal doses of Dox. RESULTS: The results show that sublethal Dox treatment leads to increased migration and invasion in otherwise non-invasive MCF7 breast cancer cells. Mechanistically, these effects were independent of the epithelial mesenchymal transition, were not due to increased cancer stem cell population, and were not observed with other chemotherapies. Instead, sublethal Dox induces expression of multiple SFK-including Fyn, Yes, and Src-partly in a p53 and ATR-dependent manner. These effects were validated in multiple cell lines. Functionally, inhibiting SFKs with Dasatinib and specific downregulation of Fyn suppressed Dox-induced migration and invasion of MCF7 cells. CONCLUSIONS: Overall, this study demonstrates that sublethal doses of Dox activate a pro-invasive, pro-migration program in cancer cells. Furthermore, by identifying SFKs as key mediators of these effects, our results define a potential therapeutic strategy to mitigate local invasion through co-treatment with Dasatinib.

The doxorubicin-induced cell motility network is under the control of the ceramide-activated protein phosphatase 1 alpha.

We have recently reported that a specific pool of ceramide, located in the plasma membrane, mediated the effects of sublethal doses of the chemotherapeutic compound doxorubicin on enhancing cancer cell migration. We identified neutral sphingomyelinase 2 (nSMase2) as the enzyme responsible to generate this bioactive pool of ceramide. In this work, we explored the role of members of the protein phosphatases 1 family (PP1), and we identified protein phosphatase 1 alpha isoform (PP1 alpha) as the specific PP1 isoform to mediate this phenotype. Using a bioinformatics approach, we build a functional interaction network based on phosphoproteomics data on plasma membrane ceramide. This led to the identification of several ceramide-PP1 alpha downstream substrates. Studies on phospho mutants of ezrin (T567) and Scrib (S1378/S1508) demonstrated that their dephosphorylation is sufficient to enhance cell migration. In summary, we identified a mechanism where reduced doses of doxorubicin result in the dysregulation of cytoskeletal proteins and enhanced cell migration. This mechanism could explain the reported effects of doxorubicin worsening cancer metastasis in animal models.

Ceramide synthase 2-C24:1 -ceramide axis limits the metastatic potential of ovarian cancer cells.

Regulation of sphingolipid metabolism plays a role in cellular homeostasis, and dysregulation of these pathways is involved in cancer progression. Previously, our reports identified ceramide as an anti-metastatic lipid. In the present study, we investigated the biochemical alterations in ceramide-centered metabolism of sphingolipids that were associated with metastatic potential. We established metastasis-prone sublines of SKOV3 ovarian cancer cells using an in vivo selection method. These cells showed decreases in ceramide levels and ceramide synthase (CerS) 2 expression. Moreover, CerS2 downregulation in ovarian cancer cells promoted metastasis in vivo and potentiated cell motility and invasiveness. Moreover, CerS2 knock-in suppressed the formation of lamellipodia required for cell motility in this cell line. In order to define specific roles of ceramide species in cell motility controlled by CerS2, the effect of exogenous long- and very long-chain ceramide species on the formation of lamellipodia was evaluated. Treatment with distinct ceramides increased cellular ceramides and had inhibitory effects on the formation of lamellipodia. Interestingly, blocking the recycling pathway of ceramides by a CerS inhibitor was ineffective in the suppression of exogenous C24:1 -ceramide for the formation of lamellipodia. These results suggested that C24:1 -ceramide, a CerS2 metabolite, predominantly suppresses the formation of lamellipodia without the requirement for deacylation/reacylation. Moreover, knockdown of neutral ceramidase suppressed the formation of lamellipodia concomitant with upregulation of C24:1 -ceramide. Collectively, the CerS2-C24:1 -ceramide axis, which may be countered by neutral ceramidase, is suggested to limit cell motility and metastatic potential. These findings may provide insights that lead to further development of ceramide-based therapy and biomarkers for metastatic ovarian cancer.

Targeting acid ceramidase inhibits YAP/TAZ signaling to reduce fibrosis in mice.

Hepatic stellate cells (HSCs) drive hepatic fibrosis. Therapies that inactivate HSCs have clinical potential as antifibrotic agents. We previously identified acid ceramidase (aCDase) as an antifibrotic target. We showed that tricyclic antidepressants (TCAs) reduce hepatic fibrosis by inhibiting aCDase and increasing the bioactive sphingolipid ceramide. We now demonstrate that targeting aCDase inhibits YAP/TAZ activity by potentiating its phosphorylation-mediated proteasomal degradation via the ubiquitin ligase adaptor protein ß-TrCP. In mouse models of fibrosis, pharmacologic inhibition of aCDase or genetic knockout of aCDase in HSCs reduces fibrosis, stromal stiffness, and YAP/TAZ activity. In patients with advanced fibrosis, aCDase expression in HSCs is increased. Consistently, a signature of the genes most down-regulated by ceramide identifies patients with advanced fibrosis who could benefit from aCDase targeting. The findings implicate ceramide as a critical regulator of YAP/TAZ signaling and HSC activation and highlight aCDase as a therapeutic target for the treatment of fibrosis.

Ceramide launches an acute anti-adhesion pro-migration cell signaling program in response to chemotherapy.

Chemotherapy has been reported to upregulate sphingomylinases and increase cellular ceramide, often linked to the induction to cell death. In this work, we show that sublethal doses of doxorubicin and vorinostat still increased cellular ceramide, which was located predominantly at the plasma membrane. To interrogate possible functions of this specific pool of ceramide, we used recombinant enzymes to mimic physiological levels of ceramide at the plasma membrane upon chemotherapy treatment. Using mass spectrometry and network analysis, followed by experimental confirmation, the results revealed that this pool of ceramide acutely regulates cell adhesion and cell migration pathways with weak connections to commonly established ceramide functions (eg, cell death). Neutral sphingomyelinase 2 (nSMase2) was identified as responsible for the generation of plasma membrane ceramide upon chemotherapy treatment, and both ceramide at the plasma membrane and nSMase2 were necessary and sufficient to mediate these "side" effects of chemotherapy on cell adhesion and migration. This is the first time a specific pool of ceramide is interrogated for acute signaling functions, and the results define plasma membrane ceramide as an acute signaling effector necessary and sufficient for regulation of cell adhesion and cell migration under chemotherapeutical stress.

Replacing plasma membrane outer leaflet lipids with exogenous lipid without damaging membrane integrity.

We recently introduced a MαCD-based method to efficiently replace virtually the entire population of plasma membrane outer leaflet phospholipids and sphingolipids of cultured mammalian cells with exogenous lipids (Li et al, (2016) Proc. Natl. Acad. Sci USA 113:14025-14030). Here, we show if the lipid-to- MαCD ratio is too high or low, cells can round up and develop membrane leakiness. We found that this cell damage can be reversed/prevented if cells are allowed to recover from the exchange step by incubation in complete growth medium. After exchange and transfer to complete growth medium cell growth was similar to that of untreated cells. In some cases, cell damage was also prevented by carrying out exchange at close to room temperature (rather than at 37°C). Exchange with lipids that do (sphingomyelin) or do not (unsaturated phosphatidylcholine) support a high level of membrane order in lipid vesicles had the analogous effect on plasma membrane order, confirming exogenous lipid localization in the plasma membrane. Importantly, changes in lipid composition and plasma membrane properties after exchange and recovery persisted for several hours. Thus, it should be possible to use lipid exchange to investigate the effect of plasma membrane lipid composition upon several aspects of membrane structure and function.

Probing compartment-specific sphingolipids with targeted bacterial sphingomyelinases and ceramidases.

Sphingolipids contribute to the regulation of cell and tissue homeostasis, and disorders of sphingolipid metabolism lead to diseases such as inflammation, stroke, diabetes, and cancer. Sphingolipid metabolic pathways involve an array of enzymes that reside in specific subcellular organelles, resulting in the formation of many diverse sphingolipids with distinct molecular species based on the diversity of the ceramide (Cer) structure. In order to probe compartment-specific metabolism of sphingolipids in this study, we analyzed the Cer and SM species preferentially produced in the inner plasma membrane (PM), Golgi apparatus, ER, mitochondria, nucleus, and cytoplasm by using compartmentally targeted bacterial SMases and ceramidases. The results showed that the length of the acyl chain of Cer becomes longer according to the progress of Cer from synthesis in the ER to the Golgi apparatus, then to the PM. These findings suggest that each organelle shows different properties of SM-derived Cers consistent with its emerging distinct functions in vitro and in vivo.

Visualizing bioactive ceramides.

In the last 30 years, ceramides have been found to mediate a myriad of biological processes. Ceramides have been recognized as bioactive molecules and their metabolizing enzymes are attractive targets in cancer therapy and other diseases. The molecular mechanism of action of cellular ceramides are still not fully established, with insights into roles through modification of lipid rafts, creation of ceramide platforms, ceramide channels, or through regulation of direct protein effectors such as protein phosphatases and kinases. Recently, the 'Many Ceramides' hypothesis focuses on distinct pools of subcellular ceramides and ceramide species as potential defined bioactive entities. Traditional methods that measure changes in ceramide levels in the whole cell, such as mass spectrometry, fluorescent ceramide analogues, and ceramide antibodies, fail to differentiate specific bioactive species at the subcellular level. However, a few ceramide binding proteins have been reported, and a smaller subgroup within these, have been shown to translocate to ceramide-enriched membranes, revealing these localized pools of bioactive ceramides. In this review we want to discuss and consolidate these works and explore the possibility of defining these binding proteins as new tools are emerging to visualize bioactive ceramides in cells. Our goal is to encourage the scientific community to explore these ceramide partners, to improve techniques to refine the list of these binding partners, making possible the identification of specific domains that recognize and bind ceramides to be used to visualize the 'Many Ceramides' in the cell.

Functions of neutral ceramidase in the Golgi apparatus.

Ceramidases hydrolyze ceramides into sphingosine and fatty acids, with sphingosine being further metabolized into sphingosine-1-phosphate (S1P); thus, ceramidases control the levels of these bioactive sphingolipids in cells and tissues. Neutral ceramidase (nCDase) is highly expressed in colorectal tissues, and a recent report showed that nCDase activity is involved in Wnt/ß-catenin signaling. In addition, the inhibition of nCDase decreases the development and progression of colorectal tumor growth. Here, to determine the action of nCDase in colorectal cancer cells, we focused on the subcellular localization and metabolic functions of this enzyme in HCT116 cells. nCDase was found to be located in both the plasma membrane and in the Golgi apparatus, but it had minimal effects on basal levels of ceramide, sphingosine, or S1P. Cells overexpressing nCDase were protected from the cell death and Golgi fragmentation induced by C6-ceramide, and they showed reduced levels of C6-ceramide and higher levels of S1P and sphingosine. Furthermore, compartment-specific metabolic functions of the enzyme were probed using C6-ceramide and Golgi-targeted bacterial SMase (bSMase) and bacterial ceramidase (bCDase). The results showed that Golgi-specific bCDase also demonstrated resistance against the cell death stimulated by C6-ceramide, and it catalyzed the metabolism of ceramides and produced sphingosine in the Golgi. Targeting bSMase to the Golgi resulted in increased levels of ceramide that were attenuated by the expression of nCDase, also supporting its ability to metabolize Golgi-generated ceramide. These results are critical in understanding the functions of nCDase actions in colorectal cancer cells as well as the compartmentalized pathways of sphingolipid metabolism.

Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism.

The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (-50 ± 3%) and sphingosine 1-phosphate (S1P, -40 ± 4%), which ended up to reduction in cell motility (-46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility.

Sphingolipids in mitochondria.

Sphingolipids are bioactive lipids found in cell membranes that exert a critical role in signal transduction. In recent years, it has become apparent that sphingolipids participate in growth, senescence, differentiation and apoptosis. The anabolism and catabolism of sphingolipids occur in discrete subcellular locations and consist of a strictly regulated and interconnected network, with ceramide as the central hub. Altered sphingolipid metabolism is linked to several human diseases. Hence, an advanced knowledge of how and where sphingolipids are metabolized is of paramount importance in order to understand the role of sphingolipids in cellular functions. In this review, we provide an overview of sphingolipid metabolism. We focus on the distinct pathways of ceramide synthesis, highlighting the mitochondrial ceramide generation, transport of ceramide to mitochondria and its role in the regulation of mitochondrial-mediated apoptosis, mitophagy and implications to disease. We will discuss unanswered questions and exciting future directions. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

Inhibitors of the sphingomyelin cycle: Sphingomyelin synthases and sphingomyelinases.

Sphingolipids are a class of bioactive lipids, which are key modulators of an increasing number of physiologic and pathophysiologic processes that include cell cycle, apoptosis, angiogenesis, stress and inflammatory responses. Sphingomyelin is an important structural component of biological membranes, and one of the end-points in the synthesis of sphingolipids. Mainly synthetized in the Golgi apparatus, sphingomyelin is transported to all other biological membranes. Upon stimulation, sphingomyelin can be hydrolyzed to ceramide by 5 different sphingomyelinases. The diversity and cellular topology of ceramide allow it to exert multiple biologies. Furthermore, ceramide can be metabolized to many other bioactive sphingolipids. Ceramide, coming from sphingomyelin or other complex sphingolipids, can be hydrolyzed to sphingosine, which can easily change cellular localization. In turn, sphingosine can be recycled to ceramide and to sphingomyelin in the endoplasmic reticulum, completing the sphingomyelin cycle. Our understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from studying the enzymes that regulate these sphingolipids, and their manipulation. The use of pharmacologic inhibitors has been critical for their study, as well as being promising bullets for disease treatment. Some of these diseases involving the sphingomyelin cycle include cancer, inflammation, atherosclerosis, diabetes and some rare diseases such as Niemann-Pick disease. This review will focus on the enzymes involved in the sphingomyelin cycle, their history, and their involvement in pathophysiological processes. Finally, it will describe in details all the small molecules that are being used to inhibit these enzymes and their use in therapeutics.