Sphingolipid Modulation Research Articles

Hepatic deletion of serine palmitoyl transferase 2 impairs ceramide/sphingomyelin balance, bile acids homeostasis and leads to liver damage in mice

Ceramides (Cer) have been shown as lipotoxic inducers, which disturb numerous cell-signaling pathways, leading to metabolic disorders such as type 2 diabetes. In this study, we aimed to determine the role of de novo hepatic ceramide synthesis in energy and liver homeostasis in mice. We generated mice lacking serine palmitoyltransferase 2 (Sptlc2), the rate limiting enzyme of ceramide de novo synthesis, in liver under albumin promoter. Liver function, glucose homeostasis, bile acid (BA) metabolism and hepatic sphingolipids content were assessed using metabolic tests and LC-MS. Despite lower expression of hepatic Sptlc2, we observed an increased concentration of hepatic Cer, associated with a 10-fold increase in neutral sphingomyelinase 2 (nSMase2) expression, and a decreased sphingomyelin content in the liver. Sptlc2ΔLiv mice were protected against obesity induced by high fat diet and displayed a defect in lipid absorption. In addition, an important increase in tauro-muricholic acid was associated with a downregulation of the nuclear BA receptor FXR target genes. Sptlc2 deficiency also enhanced glucose tolerance and attenuated hepatic glucose production, while the latter effect was dampened in presence of nSMase2 inhibitor. Finally, Sptlc2 disruption promoted apoptosis, inflammation and progressive development of hepatic fibrosis, worsening with age. Our data suggest a compensatory mechanism to regulate hepatic ceramides content from sphingomyelin hydrolysis, with deleterious impact on liver homeostasis. In addition, our results show the involvement of hepatic sphingolipid modulation in BA metabolism and hepatic glucose production in an insulin-independent manner, which highlight the still under-researched role of ceramides in many metabolic functions.

Treatment with the Glycosphingolipid Modulator THI Rescues Myelin Integrity in the Striatum of R6/2 HD Mice

Huntington's disease is one of the most common dominantly inherited neurodegenerative disorders caused by an expansion of a polyglutamine (polyQ) stretch in the N-terminal region of huntingtin (Htt). Among all the molecular mechanisms, affected by the mutation, emerging evidence proposes glycosphingolipid dysfunction as one of the major determinants. High levels of sphingolipids have been found to localize in the myelin sheaths of oligodendrocytes, where they play an important role in myelination stability and functions. In this study, we investigated any potential existing link between sphingolipid modulation and myelin structure by performing both ultrastructural and biochemical analyses. Our findings demonstrated that the treatment with the glycosphingolipid modulator THI preserved myelin thickness and the overall structure and reduced both area and diameter of pathologically giant axons in the striatum of HD mice. These ultrastructural findings were associated with restoration of different myelin marker protein, such as myelin-associated glycoprotein (MAG), myelin basic protein (MBP) and 2', 3' Cyclic Nucleotide 3'-Phosphodiesterase (CNP). Interestingly, the compound modulated the expression of glycosphingolipid biosynthetic enzymes and increased levels of GM1, whose elevation has been extensively reported to be associated with reduced toxicity of mutant Htt in different HD pre-clinical models. Our study further supports the evidence that the metabolism of glycosphingolipids may represent an effective therapeutic target for the disease.

Targeting Sphingolipids for Cancer Therapy.

Sphingolipids are an extensive class of lipids with different functions in the cell, ranging from proliferation to cell death. Sphingolipids are modified in multiple cancers and are responsible for tumor proliferation, progression, and metastasis. Several inhibitors or activators of sphingolipid signaling, such as fenretinide, safingol, ABC294640, ceramide nanoliposomes (CNLs), SKI-II, α-galactosylceramide, fingolimod, and sonepcizumab, have been described. The objective of this review was to analyze the results from preclinical and clinical trials of these drugs for the treatment of cancer. Sphingolipid-targeting drugs have been tested alone or in combination with chemotherapy, exhibiting antitumor activity alone and in synergism with chemotherapy in vitro and in vivo. As a consequence of treatments, the most frequent mechanism of cell death is apoptosis, followed by autophagy. Aslthough all these drugs have produced good results in preclinical studies of multiple cancers, the outcomes of clinical trials have not been similar. The most effective drugs are fenretinide and α-galactosylceramide (α-GalCer). In contrast, minor adverse effects restricted to a few subjects and hepatic toxicity have been observed in clinical trials of ABC294640 and safingol, respectively. In the case of CNLs, SKI-II, fingolimod and sonepcizumab there are some limitations and absence of enough clinical studies to demonstrate a benefit. The effectiveness or lack of a major therapeutic effect of sphingolipid modulation by some drugs as a cancer therapy and other aspects related to their mechanism of action are discussed in this review.

ORMDL3 and Asthma: Linking Sphingolipid Regulation to Altered T Cell Function.

Orosomucoid like 3 (ORMDL3) encodes an ER-resident transmembrane protein that regulates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme for sphingolipid biosynthesis in cells. A decade ago, several genome wide association studies revealed single nucleotide polymorphisms associated with increased ORMDL3 protein expression and susceptibility to allergic asthma. Since that time, numerous studies have investigated how altered ORMDL3 expression might predispose to asthma and other autoimmune/inflammatory diseases. In this brief review, we focus on growing evidence suggesting that heightened ORMDL3 expression specifically in CD4+ T lymphocytes, the central orchestrators of adaptive immunity, constitutes a major underlying mechanism of asthma pathogenesis by skewing their differentiation and function. Furthermore, we explore how sphingolipid modulation in T cells might be responsible for these effects, and how further studies may interrogate this intriguing hypothesis.

At last - linking ORMDL3 polymorphisms, decreased sphingolipid synthesis, and asthma susceptibility.

Asthma is a common chronic respiratory disease that has a heritable component. Polymorphisms in the endoplasmic reticular protein orosomucoid-like protein 3 (ORMDL3), which regulates sphingolipid homeostasis, have been strongly linked with childhood-onset asthma. Despite extensive investigation, a link between ORMDL3 asthma-risk genotypes and altered sphingolipid synthesis has been lacking. In this issue of the JCI, Ono et al. establish a clear association between nonallergic childhood asthma, lower whole-blood sphingolipids, and asthma-risk 17q21 genotypes. These results demonstrate that genetic variants in ORMDL3 may confer a risk of developing childhood asthma through dysregulation of sphingolipid synthesis. As such, modulation of sphingolipids may represent a promising avenue of therapeutic development for childhood asthma.

Evaluation of Xie etal.: Sphingolipid Modulation Activates Proteostasis Programs to Govern Human Hematopoietic Stem Cell Self-Renewal.

This article shows an example of the peer review process for "Sphingolipid Modulation Activates Proteostasis Programs to Govern Human Hematopoietic Stem Cell Self-Renewal" (Xie etal., 2019).

Vitamin E δ-tocotrienol inhibits TNF-α-stimulated NF-κB activation by up-regulation of anti-inflammatory A20 via modulation of sphingolipid including elevation of intracellular dihydroceramides

Nuclear factor-κB (NF-κB) regulates inflammation and cell survival, and is considered a potential target for anti-inflammatory and anti-cancer therapy. δ-Tocotrienol (δTE), a vitamin E form, has been shown to inhibit NF-κB, but the mechanism underlying this action is not clear. In the present study, we show that δTE inhibited TNF-α-induced activation of NF-κB and LPS-stimulated IL-6 in a dose- and time-dependent manner in Raw 264.7 macrophages. δTE potently inhibited TNF-α-induced phosphorylation of transforming growth factor β-activated kinase 1 (TAK1), an upstream kinase essential for the activation of NF-κB. Interestingly, δTE significantly increased the expression of A20 and to a less extent, cylindromatosis (CYLD), both of which are inhibitors of NF-κB. The importance of induction of A20 in δTE's anti-NF-κB effect is validated in A20 knockout cells where δTE's inhibition of NF-κB was largely diminished. In pursuit of the cause for A20 induction, we found that δTE treatment caused rapid and persistent elevation of dihydroceramides, while decreased ceramides initially but increased ceramides during prolonged treatment. These changes of sphingolipids were accompanied by increased cellular stress markers. Importantly, δTE's induction of A20 and inhibition of NF-κB activation were partially counteracted by myriocin, a potent inhibitor of de novo synthesis of sphingolipids, indicating a critical role of sphingolipid modulation in δTE-mediated effects. Since dihydroceramide has been shown to induce A20 and inhibit NF-κB in RAW cells, we conclude that that δTE inhibits NF-κB activation by enhancing its negative regulator A20 as a result of modulating sphingolipids especially elevation of dihydroceramides.

P075 Inflammation-induced cancer vs. cancer-induced inflammation is dependent on S1P lyase

The dichotomy of inflammation-induced cancer vs. cancer-induced inflammation is still enigmatic. Since sphingolipids are involved in both tumour cell survival and immune cell differentiation, migration and cytokine pattern we hypothesised their essential role in both mechanism of cancer development. We employed inducible S1P lyase (SGPL1) knockdown bone marrow-chimeric mice in a colitis associated colon cancer model leading to compartment-specific accumulation of sphingolipid levels. We found that, independent of the compartment of SGPL1 deficiency, haematopoietic or extra-haematopoietic, DSS/AOM-treated mice demonstrated tumours with s1pr1, stat3, and IL-6 elevation. However, compartment-specific actions of the SGPL1-S1P axis contributed both morphologically and genetically to a very distinct development of inflammation and carcinogenesis. Haematopoietic SGPL1 knockdown forced severe inflammatory colitis with delayed carcinogenesis characterised by pdcd4-down-regulation. In contrast, extra-haematopoietic SGPL1 knockdown triggered a much more rapid occurrence of epithelial-driven sphingosine kinase 1-positive tumours, exhibiting kras, egfr and IL23-p19 signatures and a weak but distinct Th2 driven immune microenvironment. Moreover, we found that SGPL1 derived from haematopoietic cells is sufficient for S1P-driven lymphocyte sequestration. In this study we demonstrate that compartment-specific sphingolipid modulation clarifies the dichotomy of inflammation-induced cancer vs. cancer-induced inflammation.

Resveratrol and pterostilbene inhibit TNF‐α stimulated activation of NF‐kB and its upstream TAK1 in macrophages via modulation of sphingolipids

Nuclear factor‐kB (NF‐kB) plays a key role in inflammatory responses and cancer development. Inhibition of NF‐kB has been shown to attenuate inflammation and cancer progression. Resveratrol (Res), a polyphenol rich in grapes and red wine, has been reported to inhibit NF‐kB signaling in macrophages. However, the underlying mechanism is not completely understood. Pterostilbene (Pte), a natural dimethylated analog of Res found in blueberries, is believed to have higher bioavailability than Res. While there are few studies on the effect of Pte on NF‐kB signaling, here we compared the effect of Res and Pte on modulating NF‐kB signaling and investigated potential underlying mechanisms in Raw 264.7 macrophages. Both Res and Pte inhibited TNF‐α induced phosphorylation of IkBα in a time‐ and dose‐ dependent manner. Res showed stronger inhibitory effect than Pte in this activity. Consistently, Res was stronger than Pte in blocking TNF‐α induced degradation of IkBα and inhibiting p65 phosphorylation in the nucleus. Furthermore, we found that both Res and Pte potently inhibited TNF‐α‐induced phosphorylation of TAK1, a key upstream signaling for NF‐kB activation. In search for the underlying mechanisms, we observed that Res treatment induced intracellular elevation of ceramides, sphingosine and dihydrosphingosine. Intriguingly, co‐treatment of C17 sphingosine and C8 ceramide attenuated TNF‐α‐induced phosphorylation of IkBα, mimicking the effect of Res. Our study demonstrates that Res inhibits NF‐kB and its upstream signaling TAK1 possibly via modulation of sphingolipid metabolism. Studies are undertaken to further investigate modulation effect of Pte on sphingolipid metabolism.

Vitamin E metabolite 13′-carboxychromanols inhibit pro-inflammatory enzymes, induce apoptosis and autophagy in human cancer cells by modulating sphingolipids and suppress colon tumor development in mice

Vitamin E forms are substantially metabolized to various carboxychromanols including 13′-carboxychromanols (13′-COOHs) that are found at high levels in feces. However, there is limited knowledge about functions of these metabolites. Here we studied δT-13′-COOH and δTE-13′-COOH, which are metabolites of δ-tocopherol and δ-tocotrienol, respectively. δTE-13′-COOH is also a natural constituent of a traditional medicine Garcinia Kola. Both 13′-COOHs are much stronger than tocopherols in inhibition of pro-inflammatory and cancer promoting cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), and in induction of apoptosis and autophagy in colon cancer cells. The anticancer effects by 13′-COOHs appeared to be partially independent of inhibition of COX-2/5-LOX. Using liquid chromatography tandem mass spectrometry, we found that 13′-COOHs increased intracellular dihydrosphingosine and dihydroceramides after short-time incubation in HCT-116 cells, and enhanced ceramides while decreased sphingomyelins during prolonged treatment. Modulation of sphingolipids by 13′-COOHs was observed prior to or coinciding with biochemical manifestation of cell death. Pharmaceutically blocking the increase of these sphingolipids partially counteracted 13′-COOH-induced cell death. Further, 13′-COOH inhibited dihydroceramide desaturase without affecting the protein expression. In agreement with these mechanistic findings, δTE-13′-COOH significantly suppressed the growth and multiplicity of colon tumor in mice. Our study demonstrates that 13′-COOHs have anti-inflammatory and anticancer activities, may contribute to in vivo anticancer effect of vitamin E forms and are promising novel cancer prevention agents.

Vitamin E γ-Tocotrienol Inhibits Cytokine-Stimulated NF-κB Activation by Induction of Anti-Inflammatory A20 via Stress Adaptive Response Due to Modulation of Sphingolipids.

NF-κB plays a central role in pathogenesis of inflammation and cancer. Many phytochemicals, including γ-tocotrienol (γTE), a natural form of vitamin E, have been shown to inhibit NF-κB activation, but the underlying mechanism has not been identified. In this study, we show that γTE inhibited cytokine-triggered activation of NF-κB and its upstream regulator TGF-β-activated kinase-1 in murine RAW 264.7 macrophages and primary bone marrow-derived macrophages. In these cells, γTE induced upregulation of A20, an inhibitor of NF-κB. Knockout of A20 partially diminished γTE's anti-NF-κB effect, but γTE increased another NF-κB inhibitor, Cezanne, in A20(-/-) cells. In search of the reason for A20 upregulation, we found that γTE treatment increased phosphorylation of translation initiation factor 2, IκBα, and JNK, indicating induction of endoplasmic reticulum stress. Liquid chromatography-tandem mass spectrometry analyses revealed that γTE modulated sphingolipids, including enhancement of intracellular dihydroceramides, sphingoid bases in de novo synthesis of the sphingolipid pathway. Chemical inhibition of de novo sphingolipid synthesis partially reversed γTE's induction of A20 and the anti-NF-κB effect. The importance of dihydroceramide increase is further supported by the observation that C8-dihydroceramide mimicked γTE in upregulating A20, enhancing endoplasmic reticulum stress, and attenuating TNF-triggered NF-κB activation. Our study identifies a novel anti-NF-κB mechanism where A20 is induced by stress-induced adaptive response as a result of modulation of sphingolipids, and it demonstrates an immunomodulatory role of dihydrocermides.

Gamma‐tocotrienol inhibits cytokine‐stimulated NF‐kB activation by inducing A20 and modulating sphingolipids

Nuclear factor‐kB (NF‐kB) plays a central role in inflammation‐associated diseases and cancer. Gamma‐tocotrienol (γTE), a natural form of vitamin E, is known to inhibit NF‐kB activation, but the underlying mechanism has not been identified. Here we show that γTE inhibited cytokine‐triggered activation of NF‐kB and its upstream regulator transforming growth factor β‐activated kinase‐1 in macrophages. gTE induced up‐regulation of A20, an inhibitor of NF‐kB. Knockout of A20 slightly diminished gTE's anti‐NF‐kB effect but gTE increased other NF‐kB inhibitors like Cezanne and phosphatse DUSP14 in A20‐/‐ cells. In search of the reason for A20 upregulation, we found that gTE increased phosphorylation of translation initiation factor 2 (eIF2a), IkBa and JNK, indicating induction of ER stress. Meanwhile, gTE led to accumulation of dihydroceramides, sphingoid bases in de novo synthesis of sphingolipid pathway. Chemical inhibition of de novo sphingolipid synthesis reversed gTE's anti‐NF‐kB effect. The importance of sphingolipid modulation is further supported by the observation that C8‐dihydroceramide mimicked gTE in up‐regulating A20, enhancing ER stress and attenuating TNF‐triggered NF‐kB activation. Our study identifies a novel anti‐NF‐kB mechanism via induction of A20 and modulation of sphingolipids, and demonstrates an immune‐modulatory role of dihydrocermide.

Sphingolipid Modulation: A Strategy for Cancer Therapy

Sphingolipids are membrane lipids that play important roles in the regulation of cell functions and homeostasis. Alterations in their metabolism have been associated with several pathologies. For this reason, therapeutic strategies based on the design of small molecules to restore sphingolipid levels to their physiological condition have rapidly emerged. In addition, some of these new chemical entities, even if they fail to succeed along the pipeline, can become valuable pharmacological tools for the study of sphingolipid function. Implications of altered sphingolipid metabolism in cancer progression have allowed the identification of new targets for the development of potential anticancer agents. Based on these premises, this review is focused on the most recent achievements in the field, with special attention to the development of small molecules, mainly enzyme inhibitors, able to disrupt some of the key sphingolipid metabolic pathways implicated in cancer progression. On the other hand, metabolic dysregulation can also be modulated by the use of sphingolipid analogs, which can alter the sphingolipid balance driving cells to death or survival and thus becoming useful candidates for subsequent drug development.

Dihydroceramide accumulation and reactive oxygen species are distinct and nonessential events in 4-HPR-mediated leukemia cell death

4-(Hydroxyphenyl)retinamide (4-HPR) is a synthetic retinoid with a strong apoptotic effect towards different cancer cell lines in vitro, and it is currently tested in clinical trials. Increases of reactive oxygen species (ROS) and modulation of endogenous sphingolipid levels are well-described events observed upon 4-HPR treatment, but there is still a lack of understanding of their relationship and their contribution to cell death. LC-MS analysis of sphingolipids revealed that in human leukemia CCRF-CEM and Jurkat cells, 4-HPR induced dihydroceramide but not ceramide accumulation even at sublethal concentrations. Myriocin prevented the 4-HPR-induced dihydroceramide accumulation, but it did not prevent the loss of viability and increase of intracellular ROS production. On the other hand, ascorbic acid, Trolox, and vitamin E reversed 4-HPR effects on cell death but not dihydroceramide accumulation. NDGA, described as a lipoxygenase inhibitor, exerted a significantly higher antioxidant activity than vitamin E and abrogated 4-HPR-mediated ROS. It did not however rescue cellular viability. Taken together, this study demonstrates that early changes observed upon 4-HPR treatment, i.e., sphingolipid modulation and ROS production, are mechanistically independent events. Furthermore, the results indicate that 4-HPR-driven cell death may occur even in the absence of dihydroceramide or ROS accumulation. These observations should be taken into account for an improved design of drug combinations.

Abstract 2342: Sphingosine Kinase & Sphingosine 1-phosphate Modulation On Microglia And Brain Derived Endothelial Cells

Sphingolipid metabolites, have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes. In particular, sphingosine-1-phosphate (S1P) the product of sphingosine phosphorylation, through transactivation of five cognate G protein-coupled receptors namely endothelial cell differentiation gene receptors (EDGs/S1P1-5), promotes proliferation, differentiation, enhances cell survival and inhibits apoptosis in different cellular systems. Modulation of sphingolipids and their receptors has been shown in some stroke models to be neuroprotective and anti-inflammatory. We undertook this study to elucidate the role of S1P receptor subtypes and sphinosine kinase (SPHK) in models of brain derived endothelial cells and microglia. We studied mono and co-cultures where endotoxin (LPS) activated microglia stimulate endothelial cell death. After documenting SPHK1/ 2 expression in microglia by immunofluorescence, we found that an agonist of SPHK ((2-(p-Hydrozyanilino)-4(p-chlorophenyl) tiazole, SKI) preserved microvascular endothelial cells, and markedly prevented microglia-induced endothelial cell injury. Furthermore, SKI inhibited iNOS and NO generation in microglia by both endotoxin as well as a second TLR4 agonist (Kdo2-Lipid (ADi[3-deoxy-D-manno-octulosonyl]-lipid A) (*P<0.01). S1P (1) agonists SEW-1 and FTY720 and endogenous ligand S1P had no significant effect in on microglia-mediated cell injury or NO induction by LPS. However, preincubation with the S1P (3) antagonist ( CAY10444 ) dose dependently prevented NO accumulation, while JTE-013 (S1P2 antagonist) & VPC23019 (S1P1 antagonist) were effective in preventing NO release. Our findings indicate that sphingosine phosphorylation promotes immune responses in microglia and its blockade, especially SPHK1 and to lesser extent S1P (3) attenuated these responses. This inhibition also led to decreased cell death due to immune cell activation. Our data suggests that targeting aspects of the sphingolipid pathway may be a new strategy to inhibit microglia activation and preserving endothelial cell integrity in relevant cerebrovascular pathologies such as stroke, trauma, or sepsis

Pharmacological Modulation of Sphingolipids and Role in Disease and Cancer Cell Biology

Sphingolipids comprise a family of bioactive lipids that exert antagonizing roles in diverse cellular functions such as cell proliferation, growth arrest or apoptosis. Synthesized in the ER/Golgi, sphingolipids are subsequently distributed to different compartments, most predominantly in the plasma membrane, where they integrate signaling platforms. In addition to its precursor role in the synthesis of complex glycosphingolipids, ceramide has been identified as a cell death effector and its generation increases in response to apoptotic stimuli including stress, radiation, chemotherapy, and death ligands. In contrast, sphingosine-1-phosphate (S1P) has been mainly characterized as an antiapoptotic sphingolipid mediating cell proliferation and survival. Thus, the relative balance between ceramide and SIP has important implications in disease pathogenesis, and therefore the pharmacological modulation of enzymes involved in regulation of the ceramide to SIP ratio could constitute a novel therapeutic approach for the treatment of human diseases and cancer.

The role of cholesterol and sphingolipids in chemokine receptor function and HIV-1 envelope glycoprotein-mediated fusion

BackgroundHIV-1 entry into cells is a multifaceted process involving target cell CD4 and the chemokine receptors, CXCR4 or CCR5. The lipid composition of the host cell plays a significant role in the HIV fusion process as it orchestrates the appropriate disposition of CD4 and co-receptors required for HIV-1 envelope glycoprotein (Env)-mediated fusion. The cell membrane is primarily composed of sphingolipids and cholesterol. The effects of lipid modulation on CD4 disposition in the membrane and their role in HIV-1 entry have extensively been studied. To focus on the role of lipid composition on chemokine receptor function, we have by-passed the CD4 requirement for HIV-1 Env-mediated fusion by using a CD4-independent strain of HIV-1 Env.ResultsCell fusion mediated by a CD4-independent strain of HIV-1 Env was monitored by observing dye transfer between Env-expressing cells and NIH3T3 cells bearing CXCR4 or CCR5 in the presence or absence of CD4. Chemokine receptor signaling was assessed by monitoring changes in intracellular [Ca2+] mobilization induced by CCR5 or CXCR4 ligand. To modulate target membrane cholesterol or sphingolipids we used Methyl-β-cyclodextrin (MβCD) or 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), respectively. Treatment of the target cells with these agents did not change the levels of CD4 or CXCR4, but reduced levels of CCR5 on the cell surface. Chemokine receptor signalling was inhibited by cholesterol removal but not by treatment with PPMP. HIV-1 Env mediated fusion was inhibited by >50% by cholesterol removal. Overall, PPMP treatment appeared to slow down the rates of CD4-independent HIV-1 Env-mediated Fusion. However, in the case of CXCR4-dependent fusion, the differences between untreated and PPMP-treated cells did not appear to be significant.ConclusionAlthough modulation of cholesterol and sphingolipids has similar effects on CD4 -dependent HIV-1 Env-mediated fusion, sphingolipid modulation had little effect on CD4-independent HIV-1 Env-mediated fusion. Chemokine receptor function remained intact following treatment of cells with PPMP. Therefore such treatment may be considered a more suitable agent to inhibit CD4 dependent HIV-1 infection.