Cellular Ceramide Levels Research Articles

Ceramide synthesis regulates biogenesis and packaging of exosomal MALAT1 from adipose derived stem cells, increases dermal fibroblast migration and mitochondrial function

BackgroundThe function of exosomes, small extracellular vesicles (sEV) secreted from human adipose-derived stem cells (ADSC), is becoming increasingly recognized as a means of transferring the regenerative power of stem cells to injured cells in wound healing. Exosomes are rich in ceramides and long noncoding RNA (lncRNA) like metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). We identified putative ceramide responsive cis-elements (CRCE) in MALAT1. We hypothesized that CRCE respond to cellular ceramide levels to regulate sEV MALAT1 packaging. MALAT1 levels by many cells exceed those of protein coding genes and it’s expression is equally high in exosomes. Ceramide also regulates exosome synthesis, however, the contents of exosome cargo via sphingomyelinase and ceramide synthase pathways has not been demonstrated.MethodsADSC were treated with an inhibitor of sphingomyelinase, GW4869, and stimulators of ceramide synthesis, C2- and C6-short chain ceramides, prior to collection of conditioned media (CM). sEV were isolated from CM, and then used to treat human dermal fibroblast (HDF) cultures in cell migration scratch assays, and mitochondrial stress tests to evaluate oxygen consumption rates (OCR).ResultsInhibition of sphingomyelinase by treatment of ADSC with GW4869 lowered levels of MALAT1 in small EVs. Stimulation of ceramide synthesis using C2- and C6- ceramides increased cellular, EVs levels of MALAT1. The functional role of sEV MALAT1 was evaluated in HDF by applying EVs to HDF. Control sEV increased migration of HDF, and significantly increased ATP production, basal and maximal respiration OCR. sEV from GW4869-treated ADSC inhibited cell migration and maximal respiration. However, sEV from C2- and C6-treated cells, respectively, increased both functions but not significantly above control EV except for maximal respiration. sEV were exosomes except when ADSC were treated with GW4869 and C6-ceramide, then they were larger and considered microvesicles.ConclusionsCeramide synthesis regulates MALAT1 EV content. Sphingomyelinase inhibition blocked MALAT1 from being secreted from ADSC EVs. Our report is consistent with those of MALAT1 increasing cell migration and mitochondrial MALAT1 altering maximal respiration in cells. Since MALAT1 is important for exosome function, it stands that increased exosomal MALAT1 should be beneficial for wound healing as shown with these assays.8tyb17USzV7szeprSwPAz5Video

1638-P: Oxidized Lipids Cause Ceramide Accumulation and Insulin Resistance in Skeletal Muscle

Accumulation of muscle lipids promotes insulin resistance and type 2 diabetes (T2D). Ceramides are important muscle lipids that decrease insulin sensitivity and it was previously shown that accumulation of ceramides in the sarcolemma negatively impacts insulin sensitivity in humans. The mechanisms explaining accumulation of lipids in specific compartments in skeletal muscle are unknown, but one possibility is through circulating oxidized lipids. POVPC, a major circulating oxidized phospholipid, was exposed to rat myotubes (1-50 μM) for 4h and the total cellular ceramide levels in myotubes measured by LC-MS/MS. Total ceramide content increased dose dependently at POVPC doses in the physiological range (5 μM, p <0.05). To determine the mechanism of POVPC induced ceramide accumulation, ceramide was measured in myotubes after treatment with POVPC (25 μM) in the presence of inhibitors of neutral and acid sphingomyelinase (GW4869 and ARC39), ceramide synthase (fumonisin B1) and serine palmitoyltransferase (myriocin). The individually administered inhibitors in the presence of POVPC each showed a decrease (41-55%) in ceramide accumulation. When all of inhibitors were administered together along with POVPC, an almost complete inhibition of ceramide accumulation (95%) was observed indicating that the sphingomyelinase, de novo, and salvage pathways of ceramide synthesis are activated by POVPC. The role of oxidized lipids on insulin sensitivity was examined after treatment of rat myotubes with 1-25 μM POVPC. Increasing doses of POVPC at doses as low as 1 μM significantly decreased insulin sensitivity in vitro (p <0.05). These results reveal circulating oxidized lipids promote ceramide accumulation and insulin resistance in muscle cells and may help explain variability in total and localized muscle ceramide levels between individuals. These data also suggest that therapies designed to lower circulating oxidized lipids could be beneficial for the treatment and prevention of T2D. Disclosure K.Zemski berry: None. B.C.Bergman: Research Support; Eli Lilly and Company. Funding National Institutes of Health (P30DK048520); University of Colorado (25N0350)

Elevated Estrogen Induces Senescence in Male Endothelial Cells Via Neutral Sphingomyelinase-Mediated Ceramide Formation

Prevalence of cardiovascular disease is higher among trans-females undergoing estrogen therapy compared to cis-females and males. Our previous work has shown that chronic exposure to elevated estrogen induces microvascular endothelial dysfunction in arterioles collected from healthy biological males, however the mechanism contributing to dysfunction is unknown. Conflicting evidence has shown that estrogen can both promote or prevent cell senescence, a phenotypic change characterized by replicative arrest and endothelial dysfunction. Since estrogen is capable of activating neutral sphingomyelinase (NSmase), an enzyme that increases cellular levels of the pro-senescent sphingolipid ceramide, we hypothesized that elevated estrogen induces endothelial cell senescence in biological males through the activation of NSmase. To study this, primary male human umbilical vein endothelial cells (HUVECs) were treated with increasing doses of 17-beta estradiol (E2; 0.5nM to 100nM) for 24hrs with or without a neutral sphingomyelinase inhibitor (GW4869 5μM). Cells were also exposed to C2-ceramide (5μM) or inactive dihidro-C2-ceramide (5μM) and maintained for 48hrs in 1% serum EBM-2 media prior to beta-galactosidase staining (biomarker of senescence). Treatment groups were imaged using 20x bright-field microscopy. Total number of positive beta-galactosidase-stained cells were counted and normalized to total number of cells per image field (mean%±SEM reported; n=3 patients for all); counters were blinded to treatment group. One-Way ANOVA with alpha<0.05 was used to compare between groups. Male HUVECs exhibited no change in percent positive cells with 0.5nM E2 treatment (25±11.4% vs 21.5±2.4% vehicle control), however had significantly higher percentage of positive cells when exposed to higher doses of E2 (1nM, 45.±11.5%; 50nM, 48.1±6.1%; 100nM, 45.5±2.6%; p<0.05 all). Treatment with ceramide (40.44±7.8% vs 11.2±4.3% control, p<0.05) but not dihydroceramide (14.89±12.1%) increased percentage of positive cells. Inhibition of neutral sphingomyelinase alone did not influence percent positivity (12.4±2.7% vs 11.2±4.3% control), however in comparison to 100nM E2 treatment, those treated with GW4869 and 100nM E2 exhibited a lower percentage of positive cells (45.5±2.6% 100nM E2 vs 19.22±7.0% E2+GW4869, p<0.05). These results suggest that elevated estrogen may cause an increase in endothelial cell senescence among biological males through the upregulation of neutral sphingomyelinase-mediated ceramide formation. Our work adds mechanistic insight by which estrogen may promote endothelial dysfunction among trans-females undergoing estrogen therapy and proposes the inhibition of NSmase-mediated ceramide formation as a novel therapeutic avenue for reducing CVD risk associated with gender transition. This work is supported by National Institutes of Health (NHLBI) K08 HL141562 (JKF) and American Heart Association Predoctoral Fellowship 909315 (GSK). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ormdl3 regulation of specific ceramides is dispensable for mouse β-cell function and glucose homeostasis under obesogenic conditions.

Chronic elevation of sphingolipids contributes to β-cell failure. ORMDL3 has been identified as a key regulator of sphingolipid homeostasis, however, its function in pancreatic β-cell pathophysiology remains unclear. Here, we generated a mouse model lacking Ormdl3 within pancreatic β-cells (Ormdl3 β-/-). We show that loss of β-cell Ormdl3 does not alter glucose tolerance, insulin sensitivity, insulin secretion, islet morphology, or cellular ceramide levels on standard chow diet. When challenged with a high fat diet, while Ormdl3 β-/- mice did not exhibit any alteration in metabolic parameters or islet architecture, lipidomics analysis revealed significantly higher levels of very long chain ceramides in their islets. Taken together, our results reveal that loss of Ormdl3 alone is not sufficient to impinge upon β-cell function or whole-body glucose and insulin homeostasis, however, β-cell-specific loss of Ormdl3 does significantly alter levels of specific sphingolipid species in islets upon high fat feeding.

Ceramide Metabolism in Cardiovascular Disease: A Network With High Therapeutic Potential.

Growing evidence suggests that ceramides play an important role in the development of atherosclerotic and valvular heart disease. Ceramides are biologically active sphingolipids that are produced by a complex network of enzymes. Lowering cellular and tissue levels of ceramide by inhibiting the ceramide-producing enzymes counteracts atherosclerotic and valvular heart disease development in animal models. In vascular tissues, ceramides are produced in response to hyperglycemia and TNF (tumor necrosis factor)-α signaling and are involved in NO-signaling and inflammation. In humans, elevated blood ceramide levels are associated with cardiovascular events. Furthermore, important cardiovascular risk factors, such as obesity and diabetes, have been linked to ceramide accumulation. This review summarizes the basic mechanisms of how ceramides drive cardiovascular disease locally and links these findings to the intriguing results of human studies on ceramides as biomarkers for cardiovascular events. Moreover, we discuss the current state of interventions to therapeutically influence vascular ceramide metabolism, both locally and systemically.

A novel C-terminal DxRSDxE motif in ceramide synthases involved in dimer formation

Ceramide is a lipid moiety synthesized via the enzymatic activity of ceramide synthases (CerSs), six of which have been identified in mammalian cells, and each of which uses a unique subset of acyl-CoAs for ceramide synthesis. The CerSs are part of a larger gene family, the Tram-Lag-CLN8 domain family. Here, we identify a unique, C-terminal motif, the DxRSDxE motif, which is only found in CerSs and not in other Tram-Lag-CLN8 family members. Deletion of this motif in either CerS2 or in CerS6 did not affect the ability of either enzyme to generate ceramide using both an in vitro assay and metabolic labeling, but deletion of this motif did affect the activity of CerS2 when coexpressed with CerS6. Surprisingly, transfection of cells with either CerS2 or CerS6 lacking the motif did not result in changes in cellular ceramide levels. We found that CerS2 and CerS6 interact with each other, as shown by immunoprecipitation, but deletion of the DxRSDxE motif impeded this interaction. Moreover, proteomics analysis of cells transfected with CerS6Δ338–344 indicated that deletion of the C-terminal motif impacted cellular protein expression, and in particular, the levels of ORMDL1, a negative regulator of sphingolipid synthesis. We suggest that this novel C-terminal motif regulates CerS dimer formation and thereby impacts ceramide synthesis.

N-Palmitoyl Serinol Stimulates Ceramide Production through a CB1-Dependent Mechanism in In Vitro Model of Skin Inflammation.

Ceramides, a class of sphingolipids containing a backbone of sphingoid base, are the most important and effective structural component for the formation of the epidermal permeability barrier. While ceramides comprise approximately 50% of the epidermal lipid content by mass, the content is substantially decreased in certain inflammatory skin diseases, such as atopic dermatitis (AD), causing improper barrier function. It is widely accepted that the endocannabinoid system (ECS) can modulate a number of biological responses in the central nerve system, prior studies revealed that activation of endocannabinoid receptor CB1, a key component of ECS, triggers the generation of ceramides that mediate neuronal cell fate. However, as the impact of ECS on the production of epidermal ceramide has not been studied, we here investigated whether the ECS stimulates the generation of epidermal ceramides in an IL-4-treated in vitro model of skin inflammation using N-palmitoyl serinol (PS), an analog of the endocannabinoid N-palmitoyl ethanolamine. Accordingly, an IL-4-mediated decrease in cellular ceramide levels was significantly stimulated in human epidermal keratinocytes (KC) following PS treatment through both de novo ceramide synthesis- and sphingomyelin hydrolysis-pathways. Importantly, PS selectively increases ceramides with long-chain fatty acids (FAs) (C22–C24), which mainly account for the formation of the epidermal barrier, through activation of ceramide synthase (CerS) 2 and Cer3 in IL-4-mediated inflamed KC. Furthermore, blockade of cannabinoid receptor CB1 activation by AM-251 failed to stimulate the production of total ceramide as well as long-chain ceramides in response to PS. These studies demonstrate that an analog of endocannabinoid, PS, stimulates the generation of specific ceramide species as well as the total amount of ceramides via the endocannabinoid receptor CB1-dependent mechanism, thereby resulting in the enhancement of epidermal permeability barrier function.

Ceramide contributes to pathogenesis and may be targeted for therapy in VCP inclusion body myopathy.

Knock-in homozygote VCPR155H/R155H mutant mice are a lethal model of valosin-containing protein (VCP)-associated inclusion body myopathy associated with Paget disease of bone, frontotemporal dementia and amyotrophic lateral sclerosis. Ceramide (d18:1/16:0) levels are elevated in skeletal muscle of the mutant mice, compared to wild-type controls. Moreover, exposure to a lipid-enriched diet reverses lethality, improves myopathy and normalizes ceramide levels in these mutant mice, suggesting that dysfunctions in lipid-derived signaling are critical to disease pathogenesis. Here, we investigated the potential role of ceramide in VCP disease using pharmacological agents that manipulate the ceramide levels in myoblast cultures from VCP mutant mice and VCP patients. Myoblasts from wild-type, VCPR155H/+ and VCPR155H/R155H mice, as well as patient-induced pluripotent stem cells (iPSCs), were treated with an inhibitor of ceramide degradation to increase ceramide via acid ceramidase (ARN082) for proof of principle. Three chemically distinct inhibitors of ceramide biosynthesis via serine palmitoyl-CoA transferase (L-cycloserine, myriocin or ARN14494) were used as a therapeutic strategy to reduce ceramide in myoblasts. Acid ceramidase inhibitor, ARN082, elevated cellular ceramide levels and concomitantly enhanced pathology. Conversely, inhibitors of ceramide biosynthesis L-cycloserine, myriocin and ARN14494 reduced ceramide production. The results point to ceramide-mediated signaling as a key contributor to pathogenesis in VCP disease and suggest that manipulating this pathway by blocking ceramide biosynthesis might exert beneficial effects in patients with this condition. The ceramide pathway appears to be critical in VCP pathogenesis, and small-molecule inhibitors of ceramide biosynthesis might provide therapeutic benefits in VCP and related neurodegenerative diseases.

Sphingolipids in Non-Alcoholic Fatty Liver Disease and Hepatocellular Carcinoma: Ceramide Turnover.

Non-alcoholic fatty liver disease (NAFLD) has emerged as one of the main causes of chronic liver disease worldwide. NAFLD comprises a group of conditions characterized by the accumulation of hepatic lipids that can eventually lead to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), the fifth most common cancer type with a poor survival rate. In this context, several works have pointed out perturbations in lipid metabolism and, particularly, changes in bioactive sphingolipids, as a hallmark of NAFLD and derived HCC. In the present work, we have reviewed existing literature about sphingolipids and the development of NAFLD and NAFLD-derived HCC. During metabolic syndrome, considered a risk factor for steatosis development, an increase in ceramide and sphigosine-1-phosphate (S1P) have been reported. Likewise, other reports have highlighted that increased sphingomyelin and ceramide content is observed during steatosis and NASH. Ceramide also plays a role in liver fibrosis and cirrhosis, acting synergistically with S1P. Finally, during HCC, metabolic fluxes are redirected to reduce cellular ceramide levels whilst increasing S1P to support tumor growth.

Fludarabine-resistance associates with ceramide metabolism and leukemia stem cell development in chronic lymphocytic leukemia.

Fludarabine (flu) -containing regimens such as flu, cyclophosphamide and rituximab have been established as one of the standard first line therapy in medically-fit chronic lymphocytic leukemia (CLL) patients. Therefore, flu-refractory (primary flu-insensitivity or flu-caused relapse) remains a major problem causing treatment failure for CLL patients. We isolated the peripheral blood mononuclear cells (PBMCs) from CLL patients and treated with flu to find flu-refractory cases, and established flu-resistant clonal cells to study molecular mechanism of flu-resistance. By comparing parental MEC-2 cells, a human CLL cell line, we found that flu-resistant clonal cells were significantly increased lethal dose 50 of flu concentration, and up-regulated expression of P-glycoprotein, a drug-resistant marker, glucosylceramide synthase (GCS), an enzyme that can convert ceramide to glucosylceramide, and CD34, a leukemia stem cell marker. Overexpression of GCS leads to promptly elimination of cellular ceramide levels and accumulation of glucosylceramide, which reduces apoptosis and promotes survival and proliferation of flu-resistant clonal cells. Furthermore, we demonstrated that the accumulation of glucosylceramide can be blocked by PDMP to restore flu-sensitivity in flu-resistant clonal cells. We also found that elevating glucosylceramide levels in flu-resistant clonal cells was associated with up-regulation of GCS and CD34 expression. Importantly, overexpression of GCS or CD34 was also determined in flu-refractory PBMCs. Our results show that flu-resistance is associated with the alteration of ceramide metabolism and the development of leukemia stem cell-like cells. The flu-resistance can be reversed by GCS inhibition as a novel strategy for overcoming drug resistance.

An oxanthroquinone derivative that disrupts RAS plasma membrane localization inhibits cancer cell growth

Oncogenic RAS proteins are commonly expressed in human cancer. To be functional, RAS proteins must undergo post-translational modification and localize to the plasma membrane (PM). Therefore, compounds that prevent RAS PM targeting have potential as putative RAS inhibitors. Here we examine the mechanism of action of oxanthroquinone G01 (G01), a recently described inhibitor of KRAS PM localization. We show that G01 mislocalizes HRAS and KRAS from the PM with similar potency and disrupts the spatial organization of RAS proteins remaining on the PM. G01 also inhibited recycling of epidermal growth factor receptor and transferrin receptor, but did not impair internalization of cholera toxin, indicating suppression of recycling endosome function. In searching for the mechanism of impaired endosomal recycling we observed that G01 also enhanced cellular sphingomyelin (SM) and ceramide levels and disrupted the localization of several lipid and cholesterol reporters, suggesting that the G01 molecular target may involve SM metabolism. Indeed, G01 exhibited potent synergy with other compounds that target SM metabolism in KRAS localization assays. Furthermore, G01 significantly abrogated RAS-RAF-MAPK signaling in Madin-Darby canine kidney (MDCK) cells expressing constitutively activated, oncogenic mutant RASG12V. G01 also inhibited the proliferation of RAS-less mouse embryo fibroblasts expressing oncogenic mutant KRASG12V or KRASG12D but not RAS-less mouse embryo fibroblasts expressing oncogenic mutant BRAFV600E. Consistent with these effects, G01 selectively inhibited the proliferation of KRAS-transformed pancreatic, colon, and endometrial cancer cells. Taken together, these results suggest that G01 should undergo further evaluation as a potential anti-RAS therapeutic.

Adiponectin receptor agonist AdipoRon decreased ceramide, and lipotoxicity, and ameliorated diabetic nephropathy

BackgroundAdiponectin is known to take part in the regulation of energy metabolism. AdipoRon, an orally-active synthetic adiponectin agonist, binds to both adiponectin receptors (AdipoR)1/R2 and ameliorates diabetic complications. Among the lipid metabolites, the ceramide subspecies of sphingolipids have been linked to features of lipotoxicity, including inflammation, cell death, and insulin resistance. We investigated the role of AdipoRon in the prevention and development of type 2 diabetic nephropathy. MethodsAdipoRon (30 mg/kg) was mixed into the standard chow diet and provided to db/db mice (db + AdipoRon, n = 8) and age-matched male db/m mice (dm + AdipoRon, n = 8) from 17 weeks of age for 4 weeks. Control db/db (db cont, n = 8) and db/m mice (dm cont, n = 8) were fed a normal diet of mouse chow. ResultsAdipoRon-fed db/db mice showed a decreased amount of albuminuria and lipid accumulation in the kidney with no significant changes in serum adiponectin, glucose, and body weight. Restoring expression of adiponectin receptor-1 and -2 in the renal cortex was observed in db/db mice with AdipoRon administration. Consistent up-regulation of phospho-Thr172 AMP-dependent kinase (AMPK), peroxisome proliferative-activated receptor α (PPARα), phospho-Thr473 Akt, phospho-Ser79Acetyl-CoA carboxylase (ACC), and phospho-Ser1177 endothelial NO synthase (eNOS), and down-regulation of protein phosphatase 2A (PP2A), sterol regulatory element-binding protein-1c (SREBP-1c), and inducible nitric oxide synthase (iNOS) were associated within the same group. AdipoRon lowered cellular ceramide levels by activation of acid ceramidase, which normalized ceramide to sphingosine-1 phosphate (S1P) ratio. In glomerular endothelial cells (GECs) and podocytes, AdipoRon treatment markedly decreased palmitate-induced lipotoxicity, which ultimately ameliorated oxidative stress and apoptosis. ConclusionsAdipoRon may prevent lipotoxicity in the kidney particularly in both GECs and podocytes through an improvement in lipid metabolism, as shown by the ratio of ceramide to sphingosines, and further contribute to prevent deterioration of renal function, independent of the systemic effects of adiponectin. The reduction in oxidative stress and apoptosis by AdipoRon provides protection against renal damage, thereby ameliorating endothelial dysfunction in type 2 diabetic nephropathy.

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated fatty acids, which might play a critical role in regulating their immune functions. Numerous studies have shown that various fatty acids, saturated or unsaturated, may possess different impacts on cell growth and function. However, the approaches used for fatty acid preparation vary, which may lead to non-physiological results. Serum albumin, a natural carrier for fatty acids in mammalian peripheral blood, is recommended for forming a conjugate complex with the sodium salt of fatty acids to study fatty acid function in mammalian cells, thus minimizing the toxicity of fatty acid soap. Thus, a simple, relatively quick heating and sonicating method is developed and presented here for BSA-fatty acid conjugate formation. We describe a protocol using saturated fatty acids, especially stearic acids to induce severe cell death in mouse bone-marrow derived macrophages. We further demonstrate that saturated fatty acid-induced cell death is positively associated with accumulated cellular ceramide levels. This method can be extended for studies of the impact of fatty acid on other mammalian cells.

Trivalent chromium induces autophagy by activating sphingomyelin phosphodiesterase 2 and increasing cellular ceramide levels in renal HK2 cells.

In this study, we examined the role of autophagy in the initiation of lipid increases in renal epithelial HK2 cells. We found that trivalent chromium [Cr(III)] induced autophagy by activating sphingomyelin phosphodiesterase 2 (SMPD2). SMPD2 increases levels of ceramide and other lipids. Confocal immunofluorescence microscopy showed that signals of ceramide overlapped with LC3, suggesting that ceramide might play an important role in the formation of autophagosome. In conclusion, our data indicate that Cr(III) induces autophagy via structural aberration of organelle membrane, in particular by the increase of lipid compositions in addition to autophagy-associated proteins.

Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo

Glucosylceramide synthase (GCS) is a rate-limiting enzyme catalyzing ceramide glycosylation, thereby regulating cellular ceramide levels and the synthesis of glycosphingolipids (GSLs) in cellular membranes. Alterations of GCS not only affect membrane integrity, but also closely correlate with stem cell pluripotency, cancer drug resistance, GSL storage disorders and other diseases. Enzyme activities measured conventionally with currently available ex-vivo methods do not enable reliable assessment of the roles played by GCS in vivo. We report herein a substrate-incorporation method enabling rapid and efficient assessment of GCS in-vivo activity. Upon nanoparticle-based delivery, fluorescent NBD C6-ceramide was efficiently converted to NBD C6-glucosylceramide in live cells or in mouse tissues, whereupon an HPLC assay enabled detection and quantification of NBD C6-glucosylceramide in the low-femtomolar range. The enzyme kinetics of GCS in live cells and mouse liver were well-described by the Michaelis-Menten model. GCS activities were significantly higher in drug-resistant cancer cells and in tumors overexpressing GCS, but reduced after silencing GCS expression or inhibiting this enzyme. Our studies indicate that this rapid and efficient method provides a valuable means for accurately assessing the roles played by GCS in normal vs. pathological states, including ones involving cancer drug resistance.

Increased ceramide production sensitizes breast cancer cell response to chemotherapy

Advanced breast cancer remains clinically challenging due to its resistance to chemotherapy. To understand the underlying mechanisms of resistance and identify drugable target, the involvement of ceramide metabolism is investigated. Ceramide levels in breast cancer tissues derived from 30 patients with stage IV breast cancer before and after chemotherapy were analyzed using liquid chromatography mass spectrometry. mRNA and protein levels of ceramide enzymes were examined using western blot and QRT-PCR. The effects of ceramide analog were investigated using cellular assays and xenograft tumor model. The results demonstrated that pro-apoptotic ceramide was significantly lower in all patients after chemotherapy, suggesting that downregulation of ceramide is a common feature of breast cancer patients in response to chemotherapy. Molecular characteristics analysis of ceramide indicated C16:0 as the predominant sphingolipid regulated by chemotherapy in breast cancer patients. Mechanistically, ceramide levels were suppressed by chemotherapy via increasing mRNA and protein levels of UDP-glucose ceramide glucosyltransferase (UGCG). Importantly, inhibition of UGCG using siRNA or upregulation of cellular ceramide levels using C2 ceramide alone inhibited proliferation and induced apoptosis of breast cancer cells, and enhanced the inhibitory effects of chemotherapeutic drugs in vitro and in vivo. This study clearly demonstrated that the decreased ceramide production via up-regulating UGCG was involved in the resistance of breast cancer cells to chemotherapy. Stimulating ceramide or decreasing UGCG can potentially be useful for breast cancer treatment.

Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes.

The molecular mechanisms responsible for the development of proteinuria and glomerulosclerosis in radiation nephropathy remain largely unknown. Podocytes are increasingly recognized as key players in the pathogenesis of proteinuria in primary and secondary glomerular disorders. The lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) is a key determinant of podocyte injury and a known off target of the anti-CD20 antibody rituximab (RTX). The current study investigates the role of sphingolipids in radiation-induced podocytopathy. After a single dose of radiation (8 Gy), several ceramide species were significantly elevated. In particular, C16:00, C24:00, and C24:1 ceramides were the most abundant ceramide species detected. These changes were paralleled by a time-dependent drop in SMPDL3b protein, sphingosine, and sphingosine-1-phosphate levels. Interestingly, SMPDL3b-overexpressing podocytes had higher basal levels of sphingosine-1-phosphate and maintained basal ceramide levels after irradiation. Morphologically, irradiated podocytes demonstrated loss of filopodia and remodeling of cortical actin. Furthermore, the actin binding protein ezrin relocated from the plasma membrane to the cytosol as early as 2 h after radiation. In contrast, SMPDL3b overexpressing podocytes were protected from radiation-induced cytoskeletal remodeling. Treatment with RTX before radiation exposure partially protected podocytes from SMPDL3b loss, cytoskeletal remodeling, and caspase 3 cleavage. Our results demonstrate that radiation injury induces early cytoskeletal remodeling, down-regulation of SMPDL3b, and elevation of cellular ceramide levels. Overexpression of SMPDL3b and pretreatment with RTX confer a radioprotective effect in cultured podocytes. These findings indicate a potential role for SMPDL3b and RTX in radiation-induced podocytopathy.-Ahmad, A., Mitrofanova, A., Bielawski, J., Yang, Y., Marples, B., Fornoni, A., Zeidan, Y. H. Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes.

An oncogenic role for sphingosine kinase 2.

While both human sphingosine kinases (SK1 and SK2) catalyze the generation of the pleiotropic signaling lipid sphingosine 1-phosphate, these enzymes appear to be functionally distinct. SK1 has well described roles in promoting cell survival, proliferation and neoplastic transformation. The roles of SK2, and its contribution to cancer, however, are much less clear. Some studies have suggested an anti-proliferative/pro-apoptotic function for SK2, while others indicate it has a pro-survival role and its inhibition can have anti-cancer effects. Our analysis of gene expression data revealed that SK2 is upregulated in many human cancers, but only to a small extent (up to 2.5-fold over normal tissue). Based on these findings, we examined the effect of different levels of cellular SK2 and showed that high-level overexpression reduced cell proliferation and survival, and increased cellular ceramide levels. In contrast, however, low-level SK2 overexpression promoted cell survival and proliferation, and induced neoplastic transformation in vivo. These findings coincided with decreased nuclear localization and increased plasma membrane localization of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis, supporting the use of SK2-specific inhibitors as anti-cancer agents.

Acid Ceramidase in Melanoma: EXPRESSION, LOCALIZATION, AND EFFECTS OF PHARMACOLOGICAL INHIBITION

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. 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 with 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 the 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 sphingosine 1-phosphate 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.

Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane.

K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization.