Expression Of Acid Sphingomyelinase Research Articles

Acid sphingomyelinase mediates ferroptosis induced by high glucose via autophagic degradation of GPX4 in type 2 diabetic osteoporosis

BackgroundFerroptosis has been implicated in the pathological process of type 2 diabetic osteoporosis (T2DOP), although the specific underlying mechanisms remain largely unknown. This study aimed to clarify the role and possible mechanism of acid sphingomyelinase (ASM)-mediated osteoblast ferroptosis in T2DOP.MethodsWe treated hFob1.19 cells with normal glucose (NG) and different concentrations of high glucose (HG, 26.25 mM, 35 mM, or 43.75 mM) for 48 h. We then measured cell viability and osteogenic function, quantified ferroptosis and autophagy levels, and measured the levels of ASM and ceramide in the cells. To further investigate the specific mechanism, we examined these indicators by knocking down ASM expression, hydroxychloroquine (HCQ) treatment, or N-acetylcysteine (NAC) treatment. Moreover, a T2DOP rat model was induced and microcomputed tomography was used to observe the bone microstructure. We also evaluated the serum levels of iron metabolism-associated factors, ceramide and lipid peroxidation (LPO) and measured the expression of ASM, LC3 and GPX4 in bone tissues.ResultsHG inhibited the viability and osteogenic function of osteoblasts by inducing ferroptosis in a concentration-dependent manner. Furthermore, the expression of ASM and ceramide and autophagy levels were increased by HG treatment, and these factors were required for the HG-induced reactive oxygen species (ROS) generation and LPO. Similarly, inhibiting intracellular ROS also reduced HG-induced ASM activation and autophagy. ASM-mediated activation of autophagy was crucial for HG-induced degradation of GPX4, and inhibiting ASM improved osteogenic function by decreasing HG-induced autophagy, GPX4 degradation, LPO and subsequent ferroptosis. We also found that inhibiting ASM could alleviated ferroptosis and autophagy and improved osteogenic function in a T2DOP rat model.ConclusionASM-mediated autophagy activation induces osteoblast ferroptosis under HG conditions through the degradation of GPX4, providing a novel mechanistic insight into the treatment and prevention of T2DOP.

Sphingolipid Catabolism and Glycerophospholipid Levels Are Altered in Erythrocytes and Plasma from Multiple Sclerosis Patients.

Multiple sclerosis (MS) is an autoimmune, inflammatory, degenerative disease of the central nervous system. Changes in lipid metabolism have been suggested to play important roles in MS pathophysiology and progression. In this work we analyzed the lipid composition and sphingolipid-catabolizing enzymes in erythrocytes and plasma from MS patients and healthy controls. We observed reduction of sphingomyelin (SM) and elevation of its products—ceramide (CER) and shingosine (SPH). These changes were supported by the detected up-regulation of the activity of acid sphingomyelinase (ASM) in MS plasma and alkaline ceramidase (ALCER) in erythrocytes from MS patients. In addition, Western blot analysis showed elevated expression of ASM, but not of ALCER. We also compared the ratios between saturated (SAT), unsaturated (UNSAT) and polyunsaturated fatty acids and suggest, based on the significant differences observed for this ratio, that the UNSAT/SAT values could serve as a marker distinguishing erythrocytes and plasma of MS from controls. In conclusion, the application of lipid analysis in the medical practice would contribute to definition of more precise diagnosis, analysis of disease progression, and evaluation of therapeutic strategies. Based on the molecular changes of blood lipids in neurodegenerative pathologies, including MS, clinical lipidomic analytical approaches could become a promising contemporary tool for personalized medicine.

Inhibitory Effect of Podocyte‐Specific Silencing of Acid Sphingomyelinase Gene on NLRP3 Inflammasome Activation and Glomerular Injury During Hyperhomocysteinemia

Acid Sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)‐induced NLRP3 inflammasome activation in podocytes and glomerular sclerosis. The present study was designed to test whether somatic podocyte‐specific silencing of ASM gene (its mouse code is Smpd1) attenuates hHcy‐induced NLRP3 inflammasome activation and associated exosome release in podocytes and thereby suppresses glomerular inflammatory response and injury. We first constructed a floxed Smpd1 shRNA construct (Vigene Bioscience, Cat# IS100001) that has both RFP and GFP as reporter genes, where RFP is expressed if there is no cre excision. If cre excision occurs, GFP is expressed. This floxed Smpd1 shRNA was transfected into podocytes isolated from WT/Podocre mice, which decreased ASM mRNA and protein levels by 90% and 60%, respectively. This suggests efficient gene silencing in podocytes with cre gene. When this floxed Smpd1 shRNA was transfected into mouse kidney by intravenous infusion and ultrasound‐microbubble directed introduction, ASM expression decreased by 65% in WT/Podocre mice and 51% in Smpd1trg/Podocre mice after 8 weeks of shRNA transfection. It was also found that transfection of floxed Smpd1 shRNA substantially attenuated hHcy‐induced increase in NLRP3 inflammasome product, IL‐1β. By nanoparticle tracking analysis, floxed Smpd1 shRNA transfection was shown to significantly block hHcy‐induced elevation of urinary exosome excretion, an indicative of NLRP3 inflammasome product release from the cells. In Smpd1trg/Podocre mice, hHcy was found to induce exaggerated NLRP3 inflammasome activation, exosome excretion and glomerular inflammatory response such as immune cell infiltration compared to WT/WT mice. This exaggerated glomerular pathological response to hHcy was also significantly suppressed by podocyte‐specific Smpd1 gene silencing. Besides, podocyte‐specific Smpd1 gene deletion substantially attenuated proteinuria and albuminuria. Taken together, our results suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and associated exosome release and that podocyte‐specific silencing of ASM gene may be an important therapeutic strategy for treatment of glomerular injury and ultimate sclerosis during hHcy.

Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes.

Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood–retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1β, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 μM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

Skin of atopic dermatitis patients shows disturbed β-glucocerebrosidase and acid sphingomyelinase activity that relates to changes in stratum corneum lipid composition

Patients with Atopic Dermatitis (AD) suffer from inflamed skin and skin barrier defects. Proper formation of the outermost part of the skin, the stratum corneum (SC), is crucial for the skin barrier function. In this study we analyzed the localization and activity of lipid enzymes β-glucocerebrosidase (GBA) and acid sphingomyelinase (ASM) in the skin of AD patients and controls. Localization of both the expression and activity of GBA and ASM in the epidermis of AD patients was altered, particularly at lesional skin sites. These changes aligned with the altered SC lipid composition. More specifically, abnormal localization of GBA and ASM related to an increase in specific ceramide subclasses [AS] and [NS]. Moreover we related the localization of the enzymes to the amounts of SC ceramide subclasses and free fatty acids (FFAs).We report a correlation between altered localization of active GBA and ASM and a disturbed SC lipid composition. Localization of antimicrobial peptide beta-defensin-3 (HBD-3) and AD biomarker Thymus and Activation Regulated Chemokine (TARC) also appeared to be diverging in AD skin compared to control. This research highlights the relation between correct localization of expressed and active lipid enzymes and a normal SC lipid composition for a proper skin barrier.

Pharmacological Inhibition of Acid Sphingomyelinase Ameliorates Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is one of the most common autoimmune disorders of the central nervous system (CNS) and the leading cause of neurological disability among young adults in the Western world. We have previously shown that the acid sphingomyelinase plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. We induced adoptively transferred EAE in wildtype and acid sphingomyelinase-deficient mice. In addition, we immunized mice with MOGaa35-55 to induce active EAE and treated the mice with amitriptyline, a functional inhibitor of the acid sphingomyelinase. We investigated symptoms of EAE, blood-brain barrier integrity and neuroinflammation. In the model of adoptively transferred EAE we demonstrate that expression of acid sphingomyelinase in the recipients rather than on transferred encephalitogenic T cells contributes to the clinical development of EAE symptoms. To test if pharmacological targeting of acid sphingomyelinase can be explored for the development of novel therapies for MS, we inhibited acid sphingomyelinase with amitriptyline in mice in which EAE was induced by active immunization. We demonstrate that pharmacological inhibition of acid sphingomyelinase using amitriptyline protects against the development of EAE and markedly attenuates the characteristic detrimental neuroinflammatory response. The studies identify the acid sphingomyelinase as a novel therapeutic target for treating MS patients.

The Transfer of Sphingomyelinase Contributes to Drug Resistance in Multiple Myeloma.

Multiple myeloma (MM) is well-known for the development of drug resistance, leading to relapse. Therefore, finding novel treatment strategies remains necessary. By performing a lipidomics assay on MM patient plasma, we aimed to identify new targets. We observed a dysregulation in the sphingolipid metabolism, with the upregulation of several ceramides and downregulation of sphingomyelin. This imbalance suggests an increase in sphingomyelinase, the enzyme responsible for hydrolyzing sphingomyelin into ceramide. We confirmed the upregulation of acid sphingomyelinase (ASM) in primary MM cells. Furthermore, we observed an increase in ASM expression in MM cell lines treated with melphalan or bortezomib, as well as in their exosomes. Exosomes high in ASM content were able to transfer the drug-resistant phenotype to chemosensitive cells, hereby suggesting a tumor-protective role for ASM. Finally, inhibition of ASM by amitriptyline improved drug sensitivity in MM cell lines and primary MM cells. In summary, this study is the first to analyze differences in plasma lipid composition of MM patients and match the observed differences to an upregulation of ASM. Moreover, we demonstrate that amitriptyline is able to inhibit ASM and increase sensitivity to anti-myeloma drugs. This study, therefore, provides a rational to include ASM-targeting-drugs in combination strategies in myeloma patients.

T Cell-Specific Overexpression of Acid Sphingomyelinase Results in Elevated T Cell Activation and Reduced Parasitemia During Plasmodium yoelii Infection.

The enzyme acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and is thereby involved in several cellular processes such as differentiation, proliferation, and apoptosis in different cell types. However, the function of ASM in T cells is still not well characterized. Here, we used T cell-specific ASM overexpressing mice (t-ASM/CD4cre) to clarify the impact of cell-intrinsic ASM activity on T cell function in vitro and in vivo. We showed that t-ASM/CD4cre mice exhibit decreased frequencies of Foxp3+ T regulatory cells (Tregs) within the spleen. Enforced T cell-specific ASM expression resulted in less efficient induction of Tregs and promoted differentiation of CD4+CD25− naïve T cells into IFN-γ producing Th1 cells in vitro. Further analysis revealed that ASM-overexpressing T cells from t-ASM/CD4cre mice show elevated T cell receptor (TCR) signaling activity accompanied with increased proliferation upon stimulation in vitro. Plasmodium yoelii infection of t-ASM/CD4cre mice resulted in enhanced T cell activation and was associated with reduced parasitemia in comparison to infected control mice. Hence, our results provide evidence that ASM activity modulates T cell function in vitro and in vivo.

Levels of Acid Sphingomyelinase (ASM) in Caenorhabditis elegans in Microgravity

Abstract Both Amyotrophic Lateral Sclerosis (ALS) patients and astronauts in spaceflight suffer from muscle atrophy. Previous research suggests that the enzyme acid sphingomyelinase (ASM) may be involved in the pathogenesis of ALS, but it is not known if ASM influences muscle atrophy in microgravity. In this study, Caenorhabditis elegans (C. elegans) were exposed to microgravity conditions on the International Space Station (ISS) within the confines of a Fluid Mixing Enclosure (FME). Return of the FME yielded 72,050 live nematodes, the first demonstration of C. elegans survival of space travel in an FME. After the nematodes returned to Earth, in much larger numbers than seen in previous FME experiments, the size and ASM expression levels in experimental worms were compared to control Earth-bound worms. C. elegans that returned from the ISS were larger in both length and cross-sectional area than the control worms, and they exhibited decreased expression of ASM-1 and ASM-2 proteins. Further research must be conducted to elucidate the role of ASM in muscle atrophy, as there were many limitations to this study. Understanding the role of ASM in muscle atrophy may lead to the discovery of novel targets for treatment of both ALS and muscle atrophy in microgravity. This study was a student led initiative and undertaken as a project within the Student Spaceflight Experiments Program (SSEP), under the auspices of the National Center for Earth and Space Science Education and the Arthur C. Clarke Institute for Space Education.

Acid sphingomyelinase promotes mitochondrial dysfunction due to glutamate-induced regulated necrosis

Inhibiting the glutamate/cystine antiporter system xc-, a key antioxidant defense machinery in the CNS, could trigger a novel form of regulated necrotic cell death, ferroptosis. The underlying mechanisms of system xc--dependent cell demise were elucidated using primary oligodendrocytes (OLs) treated with glutamate to block system xc- function. Pharmacological analysis revealed ferroptosis as a major contributing factor to glutamate-initiated OL death. A sphingolipid profile showed elevations of ceramide species and sphingosine that were preventable by inhibiting of an acid sphingomyelinase (ASM) activity. OL survival was enhanced by both downregulating ASM expression and blocking ASM activity. Glutamate-induced ASM activation seems to involve posttranscriptional mechanisms and was associated with a decreased GSH level. Further investigation of the mechanisms of OL response to glutamate revealed enhanced reactive oxygen species production, augmented lipid peroxidation, and opening of the mitochondrial permeability transition pore that were attenuated by hindering ASM. Of note, knocking down sirtuin 3, a deacetylase governing the mitochondrial antioxidant system, reduced OL survival. The data highlight the importance of the mitochondrial compartment in regulated necrotic cell death and accentuate the novel role of ASM in disturbing mitochondrial functions during OL response to glutamate toxicity, which is essential for pathobiology in stroke and traumatic brain injury.

Increase in acid sphingomyelinase level in human retinal endothelial cells and CD34+ circulating angiogenic cells isolated from diabetic individuals is associated with dysfunctional retinal vasculature and vascular repair process in diabetes

Diabetic retinopathy is a microvascular disease that results from retinal vascular degeneration and defective repair due to diabetes-induced endothelial progenitor dysfunction. Understanding key molecular factors involved in vascular degeneration and repair is paramount for developing effective diabetic retinopathy treatment strategies. We propose that diabetes-induced activation of acid sphingomyelinase (ASM) plays essential role in retinal endothelial and CD34+ circulating angiogenic cell (CAC) dysfunction in diabetes. Human retinal endothelial cells (HRECs) isolated from control and diabetic donor tissue and human CD34+ CACs from control and diabetic patients were used in this study. ASM messenger RNA and protein expression were assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. To evaluate the effect of diabetes-induced ASM on HRECs and CD34+ CACs function, tube formation, CAC incorporation into endothelial tubes, and diurnal release of CD34+ CACs in diabetic individuals were determined. ASM expression level was significantly increased in HRECs isolated from diabetic compared with control donor tissue, as well as CD34+ CACs and plasma of diabetic patients. A significant decrease in tube area was observed in HRECs from diabetic donors compared with control HRECs. The tube formation deficiency was associated with increased expression of ASM in diabetic HRECs. Moreover, diabetic CD34+ CACs with high ASM showed defective incorporation into endothelial tubes. Diurnal release of CD34+ CACs was disrupted with the rhythmicity lost in diabetic patients. Collectively, these findings support that diabetes-induced ASM upregulation has a marked detrimental effect on both retinal endothelial cells and CACs.

Altered Sphingolipid Balance in Capillary Cerebral Amyloid Angiopathy

The majority of patients with Alzheimer's disease (AD) exhibit amyloid-β (Aβ) deposits at the brain vasculature, a process referred to as cerebral amyloid angiopathy (CAA). In over 51% of AD cases, Aβ also accumulates in cortical capillaries, which is termed capillary CAA (capCAA). It has been postulated that the presence of capCAA in AD is a specific subtype of AD, although underlying mechanisms are not yet fully understood. Sphingolipids (SLs) are implicated in neurodegenerative disorders, including AD. However, to date it remains unknown whether alterations in the SL pathway are involved in capCAA pathogenesis and if these differ from AD. To determine whether AD cases with capCAA have an altered SL profile compared to AD cases without capCAA. Immunohistochemistry was performed to assess the expression and localization of ceramide, acid sphingomyelinase (ASM), and sphingosine-1-phosphate receptors (S1P1, S1P3). In addition, we determined the concentrations of S1P as well as different chain-lengths of ceramides using HPLC-MS/MS. Immunohistochemical analysis revealed an altered expression of ceramide, ASM, and S1P receptors by reactive astrocytes and microglial cells specifically associated with capCAA. Moreover, a shift in the balance of ceramides with different chain-lengths and S1P content is observed in capCAA. Here we provide evidence of a deregulated SL balance in capCAA. The increased levels of ASM and ceramide in activated glia cells suggest that the SL pathway is involved in the neuroinflammatory response in capCAA pathogenesis. Future research is needed to elucidate the role of S1P in capCAA.

Structural and functional analysis of the ASM p.Ala359Asp mutant that causes acid sphingomyelinase deficiency

Niemann-Pick disease (NPD) type A and B are recessive hereditary disorders caused by deficiency in acid sphingomyelinase (ASM). The p.Ala359Asp mutation has been described in several patients but its functional and structural effects in the protein are unknown. In order to characterize this mutation, we modeled the three-dimensional ASM structure using the recent available crystal of the mammalian ASM as a template. We found that the p.Ala359Asp mutation is localized in the hydrophobic core and far from the sphingomyelin binding site. However, energy function calculations using statistical potentials indicate that the mutation causes a decrease in ASM stability. Therefore, we investigated the functional effect of the p.Ala359Asp mutation in ASM expression, secretion, localization and activity in human fibroblasts. We found a 3.8% residual ASM activity compared to the wild-type enzyme, without changes in the other parameters evaluated. These results support the hypothesis that the p.Ala359Asp mutation causes structural alterations in the hydrophobic environment where ASM is located, decreasing its enzymatic activity. A similar effect was observed in other previously described NPDB mutations located outside the active site of the enzyme. This work shows the first full size ASM mutant model describe at date, providing a complete analysis of the structural and functional effects of the p.Ala359Asp mutation over the stability and activity of the enzyme.

Abstract P259: Endothelial Acid Sphingomyelinase Gene Determines Inflammasome Activation and Atherosclerotic Lesions in Carotid Arteries During Hypercholesterolemia

Nucleotide oligomerization domain (NOD)-like receptor protein with pyrin domain containing 3 (Nlrp3) inflammasome has been reported to be activated by atherogenic factors, thereby triggering endothelial injury and consequent atherosclerotic lesions in the arterial wall. However, the mechanism activating and regulating Nlrp3 inflammasomes remains poorly understood. The present study tested whether membrane raft (MR) signaling platforms associated with acid sphingomyelinase (ASM) and its product ceramide (Ce) importantly contribute to the activation of Nlrp3 inflammasomes and atherosclerotic lesions during hypercholesterolemia (HC). By confocal microscopy and biochemical analyses, we demonstrated the formation and activation of Nlrp3 inflammasomes in the intima of the carotid arteries of Asm +/+ mice with HC (as shown by a 2-fold increase in caspase-1 activity and a 6-fold enhancement of IL-1β positive stain areas), but not in Asm -/- mice. In endothelium-specific ASM transgenic mice (EC-Asm trg ), this inflammasome formation and activation were enhanced. Correspondingly, HC-induced increases in IL-1β production, ASM expression, Ce level and MR-gp91 phox clustering in the carotid intima were abolished in Asm -/- mice, but enhanced in EC-Asm trg mice. Functionally, endothelium-dependent vasodilation (EDVD) in carotid arteries in vivo (by ultrasound flowmetry) and in vitro (in perfused artery) was impaired by HC in Asm +/+ mice by 33% and 54%, respectively. This endothelial dysfunction was not observed in Asm -/- mice. The endothelial tight junction protein, ZO-1 was reduced by HC in both Asm +/+ and EC-Asm trg mice, but not in Asm -/- mice. It was also found that HC-increased neointimal formation, T-cell infiltration, and fibrosis in 2-week partially ligated carotid arteries (PLCA) occurred in Asm +/+ mice, but not in Asm -/- mice with HC. EC-Asm trg mice even exhibited more severe inflammatory and atherosclerotic lesions. All these results suggest that Asm gene and related MR clustering are essential to endothelial inflammasome activation and dysfunction in carotid arteries, ultimately determining the extent of atherosclerotic lesions.

Control of autophagy maturation by acid sphingomyelinase in mouse coronary arterial smooth muscle cells: protective role in atherosclerosis.

Recent studies have indicated a protective role of autophagy in regulating vascular smooth muscle cells homeostasis in atherogenesis, but the mechanisms controlling autophagy, particularly autophagy maturation, are poorly understood. Here, we investigated whether acid sphingomyelinase (ASM)-regulated lysosome function is involved in autophagy maturation in coronary arterial smooth muscle cells (CASMCs) in the pathogenesis of atherosclerosis. In coronary arterial wall of ASM-deficient (Smpd1⁻/⁻) mice on Western diet, there were high expression levels of both LC3B, a robust marker of autophagosomes (APs), and p62, a selective autophagy substrate, compared with those in wild-type (Smpd1⁺/⁺) mice. By Western blotting and flow cytometry, atherogenic stimulation of Smpd1⁺/⁺ CASMCs with 7-ketocholesterol was found to significantly enhance LC3B expression and increase the content of both APs and autophagolysosomes (APLs). In Smpd1⁻/⁻ CASMCs, such 7-ketocholesterol-induced increases in LC3B and p62 expression and APs were further augmented, but APLs formation was abolished. Analysis of fluorescence resonance energy transfer between fluorescence-labeled LC3B and Lamp1 (lysosome marker) showed that 7-ketocholesterol markedly induced fusion of APs with lysosomes in Smpd1⁺/⁺ CASMCs, which was abolished in Smpd1⁻/⁻ CASMCs. Moreover, 7-ketocholesterol-induced expression of cell dedifferentiation marker vimentin and proliferation was enhanced in Smpd1⁻/⁻ CASMCs compared with those in Smpd1⁺/⁺ CASMCs. Lastly, overexpression of ASM further increased APLs formation in Smpd1⁺/⁺ CASMCs and restored APLs formation in Smpd1⁻/⁻ CASMCs indicating that increased ASM expression is highly correlated with enhanced APLs formation. Taken together, our data suggest that the control of lysosome trafficking and fusion by ASM is essential to a normal autophagic flux in CASMCs, which implicates that the deficiency of ASM-mediated regulation of autophagy maturation may result in imbalance of arterial smooth muscle cell homeostasis and thus serve as an important atherogenic mechanism in coronary arteries. Acid sphingomyelinase (ASM) controls autophagy maturation in smooth muscle cells. ASM maintains smooth muscle cell homeostasis and its contractile phenotype. ASM plays a protective role in smooth muscle dysfunction and atherosclerosis.

Transformation-Associated Changes in Sphingolipid Metabolism Sensitize Cells to Lysosomal Cell Death Induced by Inhibitors of Acid Sphingomyelinase

Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome-destabilizing experimental anticancer agent siramesine inhibits ASM by interfering with the binding of ASM to its essential lysosomal cofactor, bis(monoacylglycero)phosphate. Like siramesine, several clinically relevant ASM inhibitors trigger cancer-specific lysosomal cell death, reduce tumor growth in vivo, and revert multidrug resistance. Their cancer selectivity is associated with transformation-associated reduction in ASM expression and subsequent failure to maintain sphingomyelin hydrolysis during drug exposure. Taken together, these data identify ASM as an attractive target for cancer therapy.

Glioma Cell Death Induced by Irradiation or Alkylating Agent Chemotherapy Is Independent of the Intrinsic Ceramide Pathway

Background/AimsResistance to genotoxic therapy is a characteristic feature of glioma cells. Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and glucosylceramide synthase (GCS) catalyzes ceramide metabolism. Increased ceramide levels have been suggested to enhance chemotherapy-induced death of cancer cells.MethodsMicroarray and clinical data for ASM and GCS in astrocytomas WHO grade II–IV were acquired from the Rembrandt database. Moreover, the glioblastoma database of the Cancer Genome Atlas network (TCGA) was used for survival data of glioblastoma patients. For in vitro studies, increases in ceramide levels were achieved either by ASM overexpression or by the GCS inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) in human glioma cell lines. Combinations of alkylating chemotherapy or irradiation and ASM overexpression, PPMP or exogenous ceramide were applied in parental cells. The anti-glioma effects were investigated by assessing proliferation, metabolic activity, viability and clonogenicity. Finally, viability and clonogenicity were assessed in temozolomide (TMZ)-resistant cells upon treatment with PPMP, exogenous ceramide, alkylating chemotherapy, irradiation or their combinations.ResultsInterrogations from the Rembrandt and TCGA database showed a better survival of glioblastoma patients with low expression of ASM or GCS. ASM overexpression or PPMP treatment alone led to ceramide accumulation but did not enhance the anti-glioma activity of alkylating chemotherapy or irradiation. PPMP or exogenous ceramide induced acute cytotoxicity in glioblastoma cells. Combined treatments with chemotherapy or irradiation led to additive, but not synergistic effects. Finally, no synergy was found when TMZ-resistant cells were treated with exogenous ceramide or PPMP alone or in combination with TMZ or irradiation.ConclusionModulation of intrinsic glioma cell ceramide levels by ASM overexpression or GCS inhibition does not enhance the anti-glioma activity of alkylating chemotherapy or irradiation.

Filaggrin-dependent secretion of sphingomyelinase protects against staphylococcal α-toxin–induced keratinocyte death

The skin of patients with atopic dermatitis (AD) has defects in keratinocyte differentiation, particularly in expression of the epidermal barrier protein filaggrin. AD skin lesions are often exacerbated by Staphylococcus aureus-mediated secretion of the virulence factor α-toxin. It is unknown whether lack of keratinocyte differentiation predisposes to enhanced lethality from staphylococcal toxins. We investigated whether keratinocyte differentiation and filaggrin expression protect against cell death induced by staphylococcal α-toxin. Filaggrin-deficient primary keratinocytes were generated through small interfering RNA gene knockdown. RNA expression was determined by using real-time PCR. Cell death was determined by using the lactate dehydrogenase assay. Keratinocyte cell survival in filaggrin-deficient (ft/ft) mouse skin biopsies was determined based on Keratin 5 staining. α-Toxin heptamer formation and acid sphingomyelinase expression were determined by means of immunoblotting. We found that filaggrin expression, occurring as the result of keratinocyte differentiation, significantly inhibits staphylococcal α-toxin-mediated pathogenicity. Furthermore, filaggrin plays a crucial role in protecting cells by mediating the secretion of sphingomyelinase, an enzyme that reduces the number of α-toxin binding sites on the keratinocyte surface. Finally, we determined that sphingomyelinase enzymatic activity directly prevents α-toxin binding and protects keratinocytes against α-toxin-induced cytotoxicity. The current study introduces the novel concept that S aureus α-toxin preferentially targets and destroys filaggrin-deficient keratinocytes. It also provides a mechanism to explain the increased propensity for S aureus-mediated exacerbation of AD skin disease.

Contribution of acid sphingomyelinase in the periaqueductal gray region to morphine-induced analgesia in mice

Opioids are the most widely used drugs for long-term pain management, but their use is limited by the development of antinociceptive tolerance. The present study investigated the role of ceramide production through acid sphingomyelinase (ASM) activation in the periaqueductal gray region, a brain region implicated in opioid analgesia and tolerance. Morphine treatment was found, using immunohistochemistry, to increase ASM expression and intracellular ceramide in the periaqueductal gray 30 min after an acute injection (10 mg/kg). The effects of acute morphine treatment on ASM expression and ceramide generation in the periaqueductal gray region were completely blocked by pretreatment with naloxone and by silencing the ASM gene by plasmid-mediated transfection of ASM shRNA. In chronic morphine pellet-implanted mice, ASM expression and ceramide generation in the periaqueductal gray region were also significantly increased. Functionally, selective silencing of the ASM gene by local ASM shRNA transfection reduced the analgesic response to acute morphine, but the data on the effect of ASM shRNA on the development of antinociceptive tolerance were inconclusive. These data provide evidence that ASM activation and ceramide generation in the periaqueductal gray region play a major role in the antinociceptive mechanism of morphine.

C-Abl Is an Upstream Regulator of Acid Sphingomyelinase in Apoptosis Induced by Inhibition of Integrins αvβ3 and αvβ5

Inhibition of integrins αvβ3/αvβ5 by the cyclic function-blocking peptide, RGDfV (Arg-Gly-Asp-Phe-Val) can induce apoptosis in both normal cells and tumor cells. We show that RGDfV induced apoptosis in ECV-304 carcinoma cells, increased activity and mRNA expression of acid sphingomyelinase (ASM), and increased ceramides C16, C18∶0, C24∶0 and C24∶1 while decreasing the corresponding sphingomyelins. siRNA to ASM decreased RGDfV-induced apoptosis as measured by TUNEL, PARP cleavage, mitochondrial depolarization, and caspase-3 and caspase-8 activities, as well as by annexinV in a 3D collagen model. These findings indicate a causal role for ASM in RGDfV-induced apoptosis in ECV-304. We have shown that c-Abl, a non-receptor tyrosine kinase, also mediates RGDfV-induced apoptosis. However, c-Abl, has not been previously linked to ASM in any system. Here we show that STI-571 (imatinib, inhibitor of c-Abl) inhibited RGDfV-induced ASM activity. Furthermore, STI-571 and c-Abl-siRNA both inhibited RGDfV-induced increase in ASM mRNA, but ASM-siRNA did not affect c-Abl phosphorylation or expression, supporting that c-Abl regulates the RGDfV-induced increase in ASM expression. These studies implicate ASM as a mediator of apoptosis induced by inhibition of integrins αvβ3/αvβ5, and for the first time place c-Abl as an upstream regulator of ASM expression and activity.