Acid Sphingomyelinase Controls Early Phases of Skeletal Muscle Regeneration by Shaping the Macrophage Phenotype.

Skeletal muscle regeneration is a complex process involving crosstalk between immune cells and myogenic precursor cells, i.e., satellite cells. In this scenario, macrophage recruitment in damaged muscles is a mandatory step for tissue repair since pro-inflammatory M1 macrophages promote the activation of satellite cells, stimulating their proliferation and then, after switching into anti-inflammatory M2 macrophages, they prompt satellite cells' differentiation into myotubes and resolve inflammation. Here, we show that acid sphingomyelinase (ASMase), a key enzyme in sphingolipid metabolism, is activated after skeletal muscle injury induced in vivo by the injection of cardiotoxin. ASMase ablation shortens the early phases of skeletal muscle regeneration without affecting satellite cell behavior. Of interest, ASMase regulates the balance between M1 and M2 macrophages in the injured muscles so that the absence of the enzyme reduces inflammation. The analysis of macrophage populations indicates that these events depend on the altered polarization of M1 macrophages towards an M2 phenotype. Our results unravel a novel role of ASMase in regulating immune response during muscle regeneration/repair and suggest ASMase as a supplemental therapeutic target in conditions of redundant inflammation that impairs muscle recovery.

PDL1-positive exosomes suppress antitumor immunity by inducing tumor-specific CD8+ T cell exhaustion during metastasis.

Metastasis is the main cause of death in individuals with cancer. Immune checkpoint blockade (ICB) can potentially reverse CD8+ cytotoxic T lymphocytes (CTLs) dysfunction, leading to significant remission in multiple cancers. However, the mechanism underlying the development of CTL exhaustion during metastatic progression remains unclear. Here, we established an experimental pulmonary metastasis model with melanoma cells and discovered a critical role for melanoma-released exosomes in metastasis. Using genetic knockdown of nSMase2 and Rab27a, 2 key enzymes for exosome secretion, we showed that high levels of effector-like tumor-specific CD8+ T cells with transitory exhaustion, instead of terminal exhaustion, were observed in mice without exosomes; these cells showed limited inhibitory receptors and strong proliferation and cytotoxicity. Mechanistically, the immunosuppression of exosomes depends on exogenous PD-L1, which can be largely rescued by pretreatment with antibody blockade. Notably, we also found that exosomal PD-L1 acts as a promising predictive biomarker for ICB therapies during metastasis. Together, our findings suggest that exosomal PD-L1 may be a potential immunotherapy target, suggesting a new curative therapy for tumor metastasis.

Altered Macrophage Function Associated with Crystalline Lung Inflammation in Acid Sphingomyelinase Deficiency.

Deficiency of ASM (acid sphingomyelinase) causes the lysosomal storage Niemann-Pick disease (NPD). Patients with NPD type B may develop progressive interstitial lung disease with frequent respiratory infections. Although several investigations using the ASM-deficient (ASMKO) mouse NPD model revealed inflammation and foamy macrophages, there is little insight into the pathogenesis of NPD-associated lung disease. Using ASMKO mice, we report that ASM deficiency is associated with a complex inflammatory phenotype characterized by marked accumulation of monocyte-derived CD11b+ macrophages and expansion of airspace/alveolar CD11c+ CD11b- macrophages, both with increased size, granularity, and foaminess. Both the alternative and classical pathways were activated, with decreased in situ phagocytosis of opsonized (Fc-coated) targets, preserved clearance of apoptotic cells (efferocytosis), secretion of Th2 cytokines, increased CD11c+/CD11b+ cells, and more than a twofold increase in lung and plasma proinflammatory cytokines. Macrophages, neutrophils, eosinophils, and noninflammatory lung cells of ASMKO lungs also exhibited marked accumulation of chitinase-like protein Ym1/2, which formed large eosinophilic polygonal Charcot-Leyden-like crystals. In addition to providing insight into novel features of lung inflammation that may be associated with NPD, our report provides a novel connection between ASM and the development of crystal-associated lung inflammation with alterations in macrophage biology.

Acid Sphingomyelinase Contributes to the Control of Mycobacterial Infection via a Signaling Cascade Leading from Reactive Oxygen Species to Cathepsin D.

Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most severe diseases worldwide. The initial pulmonary localization of the pathogen often develops into systemic infection with high lethality. The present work investigated the role of sphingolipids, specifically the function of acid sphingomyelinase (Asm) and ceramide, in infection of murine macrophages in vitro and mice in vivo with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In vitro, we investigated macrophages from wild-type (wt) and Asm deficient (Asm-/-) mice to define signaling events induced by BCG infection and mediated by Asm. We demonstrate that infection of wt macrophages results in activation of Asm, which increases reactive oxygen species (ROS) via stimulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. ROS promote BCG degradation by cathepsin D. Asm deficiency in macrophages abrogates these effects. In vivo studies reveal that wt mice rapidly control BCG infection, while Asm-/- mice fail to control the infection and kill the bacteria. Transplantation of wt macrophages into Asm-/- mice reversed their susceptibility to BCG, demonstrating the importance of Asm in macrophages for defense against BCG. These findings indicate that Asm is important for the control of BCG infection.

Oral, dental, and craniofacial features in chronic acid sphingomyelinase deficiency.

The aim of this study was to evaluate the oral, dental, and craniofacial features of individuals affected by the chronic forms of acid sphingomyelinase deficiency (ASMD). This study comprised a sample of adult and pediatric patients (n = 8) with chronic ASMD. The individuals underwent oral examinations to evaluate the occurrence of caries, as well as full-mouth periodontal examinations, to assess the occurrence and severity of periodontal diseases. Panoramic and profile radiographs were obtained to analyze dental conditions and craniofacial parameters. Participants also answered questionnaires to identify systemic impairment, parafunctional habits, and bruxism. Dental anomalies of size, shape, and number were found, with agenesis and microdontia being the predominant findings. The average of caries experience was 11.75 (±8.1). Only one patient had periodontal health and all adult individuals had periodontitis at different stages and degrees. Bruxism was found in 87.5% of the sample. The convex profile and maxillary and mandibular retrusion were the most relevant findings in the cephalometric analysis. It is concluded that individuals with chronic ASMD, in addition to several systemic manifestations, present significant modifications in their oral health, from a greater occurrence of dental anomalies, caries, periodontal disease, in addition to skeletal changes.

AISF update on the diagnosis and management of adult-onset lysosomal storage diseases with hepatic involvement.

Lysosomal storage diseases (LSDs) are a heterogeneous group of inherited disorders caused by loss-of-function mutations in genes encoding for lysosomal enzymes/proteins. The consequence is a progressive accumulation of substrates in these intracellular organelles, resulting in cellular and tissue damage. The overall incidence is about 1/8000 live births, but is likely underestimated. LSDs are chronic progressive multi-systemic disorders, generally presenting with visceromegaly, and involvement of the central nervous system, eyes, the skeleton, and the respiratory and cardiovascular systems. The age at onset and phenotypic expression are highly variable, according to the specific enzymatic defect and tissues involved, the residual activity, and the disease-causing genotype. Enzyme-replacement therapies and substrate-reduction therapies have recently become available, leading to the improvement in symptoms, disease progression and quality of life of affected individuals. Liver involvement and hepatosplenomegaly are frequent features of LSDs and a hallmark of adult-onset forms, frequently leading to medical attention. LSDs should therefore be considered in the differential diagnosis of liver disease with organomegaly. The present document will provide a short overview of adult-onset LSDs with hepatic involvement, highlighting the specificities and systemic manifestations of the ones most frequently encountered in clinical practice, which may hint at the correct diagnosis and the appropriate treatment.

Homozygous pArg610del Mutation Unusually Associated With Severe Delay of Growth in 2 Acid Sphingomyelinase Deficiency-affected Sibs.

BACKGROUND: Typically, patients with Acid Sphingomyelinase Deficiency (ASMD) because of p.Arg610del mutation, have mild phenotype with normal linear growth. OBSERVATION: We reported the case of 2 Tunisian brothers who have been referred for splenomegaly, polyadenopathies, pubertal, and growth delay. Molecular testing of SMPD1 gene revealed the presence of a homozygous p.Arg610del mutation. Lysosphingomyelin and its isoform-509 were both increased confirming ASMD for both cases. Growth hormone deficiency was highly suspected but growth hormone response after stimulating tests was acceptable for both patients. CONCLUSIONS: There is no correlation between phenotype-genotype in case of p.Arg610del mutation that could be associated to a severe delay of growth.

Small-hairpin RNA and pharmacological targeting of neutral sphingomyelinase prevent diaphragm weakness in rats with heart failure and reduced ejection fraction.

Heart failure with reduced ejection fraction (HFREF) increases neutral sphingomyelinase (NSMase) activity and mitochondrial reactive oxygen species (ROS) emission and causes diaphragm weakness. We tested whether a systemic pharmacological NSMase inhibitor or short-hairpin RNA (shRNA) targeting NSMase isoform 3 (NSMase3) would prevent diaphragm abnormalities induced by HFREF caused by myocardial infarction. In the pharmacological intervention, we used intraperitoneal injection of GW4869 or vehicle. In the genetic intervention, we injected adeno-associated virus serotype 9 (AAV9) containing shRNA targeting NSMase3 or a scrambled sequence directly into the diaphragm. We also studied acid sphingomyelinase-knockout mice. GW4869 prevented the increase in diaphragm ceramide content, weakness, and tachypnea caused by HFREF. For example, maximal specific forces (in N/cm2) were vehicle [sham 31 ± 2 and HFREF 26 ± 2 ( P < 0.05)] and GW4869 (sham 31 ± 2 and HFREF 31 ± 1). Respiratory rates were (in breaths/min) vehicle [sham 61 ± 3 and HFREF 84 ± 11 ( P < 0.05)] and GW4869 (sham 66 ± 2 and HFREF 72 ± 2). AAV9-NSMase3 shRNA prevented heightening of diaphragm mitochondrial ROS and weakness [in N/cm2, AAV9-scrambled shRNA: sham 31 ± 2 and HFREF 27 ± 2 ( P < 0.05); AAV9-NSMase3 shRNA: sham 30 ± 1 and HFREF 30 ± 1] but displayed tachypnea. Both wild-type and ASMase-knockout mice with HFREF displayed diaphragm weakness. Our study suggests that activation of NSMase3 causes diaphragm weakness in HFREF, presumably through accumulation of ceramide and elevation in mitochondrial ROS. Our data also reveal a novel inhibitory effect of GW4869 on tachypnea in HFREF likely mediated by changes in neural control of breathing.

Deficiency of alkaline SMase enhances dextran sulfate sodium-induced colitis in mice with upregulation of autotaxin.

Intestinal alkaline SMase (Alk-SMase) cleaves phosphocholine from SM, platelet-activating factor (PAF), and lysophosphatidylcholine. We recently found that colitis-associated colon cancer was 4- to 5-fold enhanced in Alk-SMase KO mice. Here, we further studied the pathogenesis of colitis induced by dextran sulfate sodium (DSS) in WT and KO mice. Compared with WT mice, KO mice demonstrated greater body weight loss, more severe bloody diarrhea, broader inflammatory cell infiltration, and more serious epithelial injury. Higher levels of PAF and lower levels of interleukin (IL)10 were identified in KO mice 2 days after DSS treatment. A greater and progressive increase of lysophosphatidic acid (LPA) was identified. The change was associated with increased autotaxin expression in both small intestine and colon, which was identified by immunohistochemistry study, Western blot, and sandwich ELISA. The upregulation of autotaxin coincided with an early increase of PAF. IL6 and TNFα were increased in both WT and KO mice. At the later stage (day 8), significant decreases in IL6, IL10, and PAF were identified, and the decreases were greater in KO mice. In conclusion, deficiency of Alk-SMase enhances DSS-induced colitis by mechanisms related to increased autotaxin expression and LPA formation. The early increase of PAF might be a trigger for such reactions.

Increased liver tumor formation in neutral sphingomyelinase-2-deficient mice.

Sphingolipids are key signaling lipids in cancer. Genome-wide studies have identified neutral SMase-2 (nSMase2), an enzyme generating ceramide from SM, as a potential repressor for hepatocellular carcinoma. However, little is known about the sphingolipids regulated by nSMase2 and their roles in liver tumor development. We discovered growth of spontaneous liver tumors in 27.3% (9 of 33) of aged male nSMase2-deficient (fro/fro) mice. Lipidomics analysis showed a marked increase of SM in the tumor. Unexpectedly, tumor tissues presented with more than a 7-fold increase of C16-ceramide, concurrent with upregulation of ceramide synthase 5. The fro/fro liver tumor, but not adjacent tissue, exhibited substantial accumulation of lipid droplets, suggesting that nSMase2 deficiency is associated with tumor growth and increased neutral lipid generation in the tumor. Tumor tissue expressed significantly increased levels of CD133 and EpCAM mRNA, two markers of liver cancer stem-like cells (CSCs) and higher levels of phosphorylated signal transducer and activator of transcription 3, an essential regulator of stemness. CD133(+) cells showed strong labeling for SM and ceramide. In conclusion, these results suggest that SMase-2 deficiency plays a role in the survival or proliferation of CSCs, leading to spontaneous tumors, which is associated with tumor-specific effects on lipid homeostasis.

Staphylococcus aureus α-Toxin Induces Inflammatory Cytokines via Lysosomal Acid Sphingomyelinase and Ceramides.

BACKGROUND/AIMS: Staphylococcus aureus (S. aureus) infections are a major clinical problem and range from mild skin and soft-tissue infections to severe and even lethal infections such as pneumonia, endocarditis, sepsis, osteomyelitis, and toxic shock syndrome. Toxins that are released from S. aureus mediate many of these effects. Here, we aimed to identify molecular mechanisms how α-toxin, a major S. aureus toxin, induces inflammation. METHODS: Macrophages were isolated from the bone marrow of wildtype and acid sphingomyelinase-deficient mice, stimulated with S. aureus α-toxin and activation of the acid sphingomyelinase was quantified. The subcellular formation of ceramides was determined by confocal microscopy. Release of cathepsins from lysosomes, activation of inflammasome proteins and formation of Interleukin-1ß (IL-1ß) and Tumor Necrosis Factor-α (TNF-α) were analyzed by western blotting, confocal microscopy and ELISA. RESULTS: We demonstrate that S. aureus α-toxin activates the acid sphingomyelinase in ex vivo macrophages and triggers a release of ceramides. Ceramides induced by S. aureus α-toxin localize to lysosomes and mediate a release of cathepsin B and D from lysosomes into the cytoplasm. Cytosolic cathepsin B forms a complex with Nlrc4. Treatment of macrophages with α-toxin induces the formation of IL-1ß and TNF-α. These events are reduced or abrogated, respectively, in cells lacking the acid sphingomyelinase and upon treatment of macrophages with amitriptyline, a functional inhibitor of acid sphingomyelinase. Pharmacological inhibition of cathepsin B prevented activation of the inflammasome measured as release of IL-1ß, while the formation of TNF-α was independent of cathepsin B. CONCLUSION: We demonstrate a novel mechanism how bacterial toxins activate the inflammasome and mediate the formation and release of cytokines: S. aureus α-toxin triggers an activation of the acid sphingomyelinase and a release of ceramides resulting in the release of lysosomal cathepsin B and formation of pro-inflammatory cytokines.

Limited benefits of presymptomatic cord blood transplantation in neurovisceral acid sphingomyelinase deficiency (ASMD) intermediate type.

Acid sphingomyelinase (ASM) deficient Niemann-Pick disease is a lysosomal storage disorder resulting from mutations in the SMPD1 gene. The clinical spectrum distinguishes a severe infantile neurological form (type A), a non-neurological visceral form (type B) and a rare intermediate neurovisceral form. We report the first case of presymptomatic cord blood transplantation in a child with the intermediate type of ASM deficiency due to a homozygous Tyr369Cys mutation, whose affected elder brother had developed neurodevelopmental delay from 19 months of age, and had died from severe visceral complications at the age of 3. In the transplanted propositus, neurological deterioration became evident by 4 years of age; the child was alive at age 8, although severely disabled. Whereas the transplant prevented visceral progression and early death, it could only delay neurocognitive deterioration.

Acid sphingomyelinase deficiency in Western diet-fed mice protects against adipocyte hypertrophy and diet-induced liver steatosis.

OBJECTIVE: Alterations in sphingolipid and ceramide metabolism have been associated with various diseases, including nonalcoholic fatty liver disease (NAFLD). Acid sphingomyelinase (ASM) converts the membrane lipid sphingomyelin to ceramide, thereby affecting membrane composition and domain formation. We investigated the ways in which the Asm knockout (Smpd1-/-) genotype affects diet-induced NAFLD. METHODS: Smpd1-/- mice and wild type controls were fed either a standard or Western diet (WD) for 6 weeks. Liver and adipose tissue morphology and mRNA expression were assessed. Quantitative proteome analysis of liver tissue was performed. Expression of selected genes was quantified in adipose and liver tissue of obese NAFLD patients. RESULTS: Although Smpd1-/- mice exhibited basal steatosis with normal chow, no aggravation of NAFLD-type injury was observed with a Western diet. This protective effect was associated with the absence of adipocyte hypertrophy and the increased expression of genes associated with brown adipocyte differentiation. In white adipose tissue from obese patients with NAFLD, no expression of these genes was detectable. To further elucidate which pathways in liver tissue may be affected by Smpd1-/-, we performed an unbiased proteome analysis. Protein expression in WD-fed Smpd1-/- mice indicated a reduction in Rictor (mTORC2) activity; this reduction was confirmed by diminished Akt phosphorylation and altered mRNA expression of Rictor target genes. CONCLUSION: These findings indicate that the protective effect of Asm deficiency on diet-induced steatosis is conferred by alterations in adipocyte morphology and lipid metabolism and by reductions in Rictor activation.

Acid sphingomyelinase mediates murine acute lung injury following transfusion of aged platelets.

Pulmonary complications from stored blood products are the leading cause of mortality related to transfusion. Transfusion-related acute lung injury is mediated by antibodies or bioactive mediators, yet underlying mechanisms are incompletely understood. Sphingolipids such as ceramide regulate lung injury, and their composition changes as a function of time in stored blood. Here, we tested the hypothesis that aged platelets may induce lung injury via a sphingolipid-mediated mechanism. To assess this hypothesis, a two-hit mouse model was devised. Recipient mice were treated with 2 mg/kg intraperitoneal lipopolysaccharide (priming) 2 h before transfusion of 10 ml/kg stored (1-5 days) platelets treated with or without addition of acid sphingomyelinase inhibitor ARC39 or platelets from acid sphingomyelinase-deficient mice, which both reduce ceramide formation. Transfused mice were examined for signs of pulmonary neutrophil accumulation, endothelial barrier dysfunction, and histological evidence of lung injury. Sphingolipid profiles in stored platelets were analyzed by mass spectrophotometry. Transfusion of aged platelets into primed mice induced characteristic features of lung injury, which increased in severity as a function of storage time. Ceramide accumulated in platelets during storage, but this was attenuated by ARC39 or in acid sphingomyelinase-deficient platelets. Compared with wild-type platelets, transfusion of ARC39-treated or acid sphingomyelinase-deficient aged platelets alleviated lung injury. Aged platelets elicit lung injury in primed recipient mice, which can be alleviated by pharmacological inhibition or genetic deletion of acid sphingomyelinase. Interventions targeting sphingolipid formation represent a promising strategy to increase the safety and longevity of stored blood products.

Acid Sphingomyelinase Inhibition in Stored Erythrocytes Reduces Transfusion-Associated Lung Inflammation.

OBJECTIVE: We aimed to identify the role of the enzyme acid sphingomyelinase in the aging of stored units of packed red blood cells (pRBCs) and subsequent lung inflammation after transfusion. SUMMARY BACKGROUND DATA: Large volume pRBC transfusions are associated with multiple adverse clinical sequelae, including lung inflammation. Microparticles are formed in stored pRBCs over time and have been shown to contribute to lung inflammation after transfusion. METHODS: Human and murine pRBCs were stored with or without amitriptyline, a functional inhibitor of acid sphingomyelinase, or obtained from acid sphingomyelinase-deficient mice, and lung inflammation was studied in mice receiving transfusions of pRBCs and microparticles isolated from these units. RESULTS: Acid sphingomyelinase activity in pRBCs was associated with the formation of ceramide and the release of microparticles. Treatment of pRBCs with amitriptyline inhibited acid sphingomyelinase activity, ceramide accumulation, and microparticle production during pRBC storage. Transfusion of aged pRBCs or microparticles isolated from aged blood into mice caused lung inflammation. This was attenuated after transfusion of pRBCs treated with amitriptyline or from acid sphingomyelinase-deficient mice. CONCLUSIONS: Acid sphingomyelinase inhibition in stored pRBCs offers a novel mechanism for improving the quality of stored blood.

Acid Sphingomyelinase (ASM) is a Negative Regulator of Regulatory T Cell (Treg) Development.

BACKGROUND/AIMS: Regulatory T cell (Treg) is required for the maintenance of tolerance to various tissue antigens and to protect the host from autoimmune disorders. However, Treg may, indirectly, support cancer progression and bacterial infections. Therefore, a balance of Treg function is pivotal for adequate immune responses. Acid sphingomyelinase (ASM) is a rate limiting enzyme involved in the production of ceramide by breaking down sphingomyelin. Previous studies in T-cells have suggested that ASM is involved in CD28 signalling, T lymphocyte granule secretion, degranulation, and vesicle shedding similar to the formation of phosphatidylserine-exposing microparticles from glial cells. However, whether ASM affects the development of Treg has not yet been described. METHODS: Splenocytes, isolated Naive T lymphocytes and cultured T cells were characterized for various immune T cell markers by flow cytometery. Cell proliferation was measured by Carboxyfluorescein succinimidyl ester (CFSE) dye, cell cycle analysis by Propidium Iodide (PI), mRNA transcripts by q-RT PCR and protein expression by Western Blotting respectively. RESULTS: ASM deficient mice have higher number of Treg compared with littermate control mice. In vitro induction of ASM deficient T cells in the presence of TGF-ß and IL-2 lead to a significantly higher number of Foxp3+ induced Treg (iTreg) compared with control T-cells. Further, ASM deficient iTreg has less AKT (serine 473) phosphorylation and Rictor levels compared with control iTreg. Ceramide C6 led to significant reduction of iTreg in both ASM deficient and WT mice. The reduction in iTreg leads to induction of IL-1ß, IL-6 and IL-17 but not IFN-γ mRNA levels. CONCLUSION: ASM is a negative regulator of natural and iTreg.

Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure.

Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes.

Role of Acid Sphingomyelinase-Induced Signaling in Melanoma Cells for Hematogenous Tumor Metastasis.

BACKGROUND: Hematogenous metastasis of malignant tumor cells is a multistep process that requires release of tumor cells from the local tumor mass, interaction of the tumor cells with platelets in the blood, and adhesion of either the activated tumor cells or the complexes of platelets and tumor cells to the endothelial cells of the target organ. We have previously shown that the interaction of melanoma cells with platelets results in the release of acid sphingomyelinase (Asm) from activated platelets. Secreted platelet-derived Asm acts on malignant tumor cells to cluster and activate integrins; such clustering and activation are necessary for tumor cell adhesion to endothelial cells and for metastasis. METHODS: We examined the response of tumor cells to treatment with extracellular sphingomyelinase or co-incubation with wild-type and Asm-deficient platelets. We determined the phosphorylation and activation of several intracellular signaling molecules, in particular p38 kinase (p38K), phospholipase Cx03B3; (PLCx03B3;), ezrin, and extracellular signal-regulated kinases. RESULTS: Incubation of B16F10 melanoma cells with Asm activates p38 MAP kinase (p38K), phospholipase Cx03B3; (PLCx03B3;), ezrin, and extracellular signal-regulated kinases. Co-incubation of B16F10 melanoma cells with wild-type or Asm-deficient platelets showed that the phosphorylation/activation of p38K is dependent on Asm. Pharmacological blockade of p38K prevents activation of ß1 integrin and adhesion in vitro. Most importantly, inhibition of p38K activity in B16F10 melanoma cells prevents tumor cell adhesion and metastasis to the lung in vivo, a finding indicating the importance of p38K for metastasis. CONCLUSIONS: Asm, secreted from activated platelets after tumor cell-platelet contact, induces p38K phosphorylation in tumor cells. This in turn stimulates ß1 integrin activation that is necessary for adhesion and subsequent metastasis of tumor cells. Thus, inhibition of p38K might be a novel target to prevent tumor metastasis.

Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy.

The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Here, we investigated the impact of genetic and drug-induced lysosomal cholesterol (LC) accumulation in APAP hepatotoxicity. Acid sphingomyelinase (ASMase)(-/-) mice exhibit LC accumulation and higher mortality after APAP overdose compared to ASMase(+/+) littermates. ASMase(-/-) hepatocytes display lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes, which decreased mitochondrial quality control. LC accumulation in ASMase(+/+) hepatocytes caused by U18666A reproduces the susceptibility of ASMase(-/-) hepatocytes to APAP and the impairment in the formation of mitochondria-containing autolysosomes. LC extraction by 25-hydroxycholesterol increased APAP-mediated mitophagy and protected ASMase(-/-) mice and hepatocytes against APAP hepatotoxicity, effects that were reversed by chloroquine to disrupt autophagy. The regulation of LC by U18666A or 25-hydroxycholesterol did not affect total cellular sphingomyelin content or its lysosomal distribution. Of relevance, amitriptyline-induced ASMase inhibition in human hepatocytes caused LC accumulation, impaired mitophagy and increased susceptibility to APAP. Similar results were observed upon glucocerebrosidase inhibition by conduritol ß-epoxide, a cellular model of Gaucher disease. These findings indicate that LC accumulation determines susceptibility to APAP hepatotoxicity by modulating mitophagy, and imply that genetic or drug-mediated ASMase disruption sensitizes to APAP-induced liver injury.

Regulation of ceramide generation during macrophage apoptosis by ASMase and de novo synthesis.

The survival of macrophages depends on the presence of specific cytokines that activate survival signaling events, as well as suppressing formation of apoptosis-inducing pathways. We have previously shown that macrophages deprived of macrophage colony stimulating factor (M-CSF) produce ceramide that contributes to apoptosis of these cells, a pathway that is suppressed by exposure to oxidized LDL. In this study we have examined macrophages derived from mice lacking acid sphingomyelinase (ASMase) to ask whether these events are altered due to the impaired ability of these cells to break down sphingomyelin and produce ceramide. We found that these cells do survive better than cells from wild type mice, but they still undergo cell death and some ceramide is formed. We show that the ceramide is being produced by a de novo synthetic pathway. Therefore, ceramide production in M-CSF-deprived macrophages arises from a combination of ASMase activity and de novo synthesis.