nSMase2 Activity Research Articles

Inhibition of neutral sphingomyelinase 2 reduces extracellular vesicle release from neurons, oligodendrocytes, and activated microglial cells following acute brain injury

Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteinsin a growing number of neurological diseases. Given this, there is rising interest in developing inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis. Our group recently discovered phenyl(R)‐(1‐(3‐(3,4‐dimethoxyphenyl)‐2,6‐dimethylimidazo[1,2‐b]pyridazin‐8‐yl)pyrrolidin‐3‐yl)carbamate (PDDC), the first potent, selective, orally-available, and brain-penetrable nSMase2 inhibitor, capable of dose-dependently reducing EVs release in vitro and in vivo. Herein, using multiplexed Surface Plasmon Resonance imaging (SPRi), we evaluated which brain cell-derived EVs were affected by PDDC following acute brain injury. Mice were fed PDDC-containing chow at doses which gave steady PDDC brain exposures exceeding its nSMase2 IC50. Mice were then administered an intra-striatal IL-1β injection and two hours later plasma and brain were collected. IL-1β injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1β-induced injury selectively increased plasma levels of CD171 + and PLP1 + EVs; this EV increase was normalized by PDDC. In contrast, GLAST1 + EVs were unchanged by IL-1β or PDDC. IL-1β injection selectively increased EVs released from activated versus non-activated microglia, indicated by the CD11b+/IB4 + ratio. The increase in EVs from CD11b + microglia was dramatically attenuated with PDDC. Taken together, our data demonstrate that following acute injury, brain nSMase2 activity is elevated. EVs released from neurons, oligodendrocytes, and activated microglial are increased in plasma and inhibition of nSMase2 with PDDC reduced these IL-1β-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury.

Regulatory T cells reduce endothelial neutral sphingomyelinase 2 to prevent T-cell migration into tumors.

Endothelial cells are key regulators of transendothelial migration and their secretion of chemokines and expression of adhesion molecules facilitates lymphocyte entry into tissues. Previously, we demonstrated that Tregs can reduce transendothelial migration of T cells into tumors by decreasing endothelial CXCL10 secretion, but the mechanism by which this occurs is still not known. In this study, we aimed to define how Tregs decrease transendothelial migration into tumors. mRNA sequencing of intestinal tumor endothelial cells from Treg depleted mice identified neutral sphingomyelinase 2 (nSMase2) as a gene downregulated in the presence of Tregs. nSMase2 is expressed in human umbilical vein endothelial cells (HUVECs) and was decreased after coculture with Tregs. Furthermore, blocking of nSMase2 activity in vitro decreased VCAM1, CX3CL1, and CXCL10 expression in HUVECs, mirroring the same decrease found in Treg cocultures. In the APCmin/+ mouse model of intestinal cancer, nSMase2 is lower in tumor endothelial cells than in unaffected small intestine and chronic treatment with a nSMase2 inhibitor suppressed the increased migration that is otherwise seen in the absence of Tregs. We conclude that nSMase2 is an important mediator in endothelial cells supporting transendothelial migration, which may be targeted by Tregs to reduce T-cell migration into tumors.

Neutral sphingomyelinase 2 regulates inflammatory responses in monocytes/macrophages induced by TNF-α

Abstract Obesity is associated with elevated levels of TNF-α and proinflammatory CD11c monocytes/macrophages. TNF-α mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase 2 (nSMase2; product of the sphingomyelin phosphodiesterase 3 gene, SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether the nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-α. In this study, we demonstrate that the disruption of nSMase2 activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF-α mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of IL-1b and MCP-1. However, inhibition aSMase activity did not attenuate CD11c expression and secretion of IL-1b and MCP-1. Phosphorylation of JNK, p38 and NF-κB resulting from TNF-α stimulation was also attenuated by the inhibition of nSMase. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. Our human data show that SMPD3 gene expression was not only found to be elevated in obese individuals but also positively corelate with TNF-α elevated expression. These findings indicate that nSMase acts, in a part, as a master switch in the TNF-α mediated inflammatory responses in monocytes/macrophages.

Novel Human Neutral Sphingomyelinase 2 Inhibitors as Potential Therapeutics for Alzheimer's Disease.

Neutral sphingomyelinase 2 (nSMase2) catalyzes the cleavage of sphingomyelin to phosphorylcholine and ceramide, an essential step in the formation and release of exosomes from cells that is critical for intracellular communication. Chronic increase of brain nSMase2 activity and related exosome release have been implicated in various pathological processes, including the progression of Alzheimer's disease (AD), making nSMase2 a viable therapeutic target. Recently, we identified phenyl (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate 1 (PDDC), the first nSMase2 inhibitor that possesses both favorable pharmacodynamics and pharmacokinetic (PK) parameters, including substantial oral bioavailability, brain penetration, and significant inhibition of exosome release from the brain in vivo. Herein we demonstrate the efficacy of 1 (PDDC) in a mouse model of AD and detail extensive structure-activity relationship (SAR) studies with 70 analogues, unveiling several that exert similar or higher activity against nSMase2 with favorable pharmacokinetic properties.

The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles.

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA-profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2), and LC3-dependent recruitment of Factor-associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.

Lipid Metabolism in Late-Onset Alzheimer's Disease Differs from Patients Presenting with Other Dementia Phenotypes.

Abnormal cerebrospinal fluid (CSF) levels of β-amyloid peptides (Aβ42) and Tau and cognitive decline are typical characteristics of Alzheimer’s disease (AD). Since dysregulation in lipid metabolism accompanies abnormal amyloid formation, we quantified glycerophospholipids (GP) and sphingolipids (SP) in CSF fractions from participants with late-onset AD (LOAD, n = 29) or with Other Dementia (OD, n = 10) to determine if alterations in lipid metabolism account for pathological differences. Aβ42 and total Tau levels were determined using a sandwich ELISA. Liposomal-based fluorescent assays were used to measure phospholipase A2 (PLA2) and acid or neutral sphingomyelinase (aSMase, nSMase) activities. Supernatant fluid (SF) and nanoparticle (NP) lipids were quantified using LC-MS/MS. Although CSF Aβ42 and Tau levels are similar, phosphatidylserine (PS) in SF and ceramide (CM) levels in NP are significantly higher in OD compared with LOAD. The aSMase but not the nSMase activity is higher in OD. PLA2 activity in CSF from OD subjects positively correlates with several GP classes in SF and NP fractions but not in LOAD fractions. Our data indicate differences in CSF lipid metabolism between dementia variants. Higher levels of inflammatory and apoptotic lipids may induce faster neuronal death, resulting in the earlier cognitive decline in patients with OD phenotypes.

Suppression of tau propagation using an inhibitor that targets the DK-switch of nSMase2

Targeting of molecular pathways involved in the cell-to-cell propagation of pathological tau species is a novel approach for development of disease-modifying therapies that could block tau pathology and attenuate cognitive decline in patients with Alzheimer's disease and other tauopathies. We discovered cambinol through a screening effort and show that it is an inhibitor of cell-to-cell tau propagation. Our in vitro data demonstrate that cambinol inhibits neutral sphingomyelinase 2 (nSMase2) enzyme activity in dose response fashion, and suppresses extracellular vesicle (EV) production while reducing tau seed propagation. Our in vivo testing with cambinol shows that it can reduce the nSMase2 activity in the brain after oral administration. Our molecular docking and simulation analysis reveals that cambinol can target the DK-switch in the nSMase2 active site.

Anti-GM1 ganglioside antibodies modulate membrane-associated sphingomyelin metabolism by altering neutral sphingomyelinase activity

Previous studies have shown that patients with Guillain-Barré syndrome express autoantibodies against ganglioside GM1 (GM1), although its pathogenic significance for the development of the disease remains to be elucidated.nSMase2 is the best characterized neutral sphingomyelinase (nSMase) found in neuronal cells. Activation of this enzyme leads to ceramide production, which is a known second messenger of the cell-death program in neuronal cells.We have explored the effects of anti-GM1 antibodies on sphingomyelin metabolism of PC12 cells stably transfected with human trk cDNA (PCtrk cells) by determining their effects on nSMase2 activity. The data we present here strongly suggest that anti-GM1 caused a significant change in sphingomyelin content of the membrane fraction in PCtrk cells. Both nSMase2 activity and the level of nSMase2 protein were significantly decreased by anti-GM1 treatment of PCtrk cells, while acidic SMase activities remained unchanged.Our results indicate, for the first time, that anti-GM1 may produce profound impacts on lipid metabolism in neuronal cell membranes.

Myocardial reparative functions of exosomes from mesenchymal stem cells are enhanced by hypoxia treatment of the cells via transferring microRNA-210 in an nSMase2-dependent way

Hypoxia treatment enhances paracrine effect of mesenchymal stem cells (MSCs). The aim of this study was to investigate whether exosomes from hypoxia-treated MSCs (ExoH) are superior to those from normoxia-treated MSCs (ExoN) for myocardial repair. Mouse bone marrow-derived MSCs were cultured under hypoxia or normoxia for 24 h, and exosomes from conditioned media were intramyocardially injected into infarcted heart of C57BL/6 mouse. ExoH resulted in significantly higher survival, smaller scar size and better cardiac functions recovery. ExoH conferred increased vascular density, lower cardiomyocytes (CMs) apoptosis, reduced fibrosis and increased recruitment of cardiac progenitor cells in the infarcted heart relative to ExoN. MicroRNA analysis revealed significantly higher levels of microRNA-210 (miR-210) in ExoH compared with ExoN. Transfection of a miR-210 mimic into endothelial cells (ECs) and CMs conferred similar biological effects as ExoH. Hypoxia treatment of MSCs increased the expression of neutral sphingomyelinase 2 (nSMase2) which is crucial for exosome secretion. Blocking the activity of nSMase2 resulted in reduced miR-210 secretion and abrogated the beneficial effects of ExoH. In conclusion, hypoxic culture augments miR-210 and nSMase2 activities in MSCs and their secreted exosomes, and this is responsible at least in part for the enhanced cardioprotective actions of exosomes derived from hypoxia-treated cells.

Enriched Expression of Neutral Sphingomyelinase 2 in the Striatum is Essential for Regulation of Lipid Raft Content and Motor Coordination

Sphingomyelinases are a family of enzymes that hydrolyze sphingomyelin to generate phosphocholine and ceramide. The brain distribution and function of neutral sphingomyelinase 2 (nSMase2) were elucidated in this study. nSMase2 mRNA expression was greatest in the striatum, followed by the prefrontal cortex, hippocampus, cerebellum, thalamus, brainstem, and olfactory bulb. The striatum had the highest level of nSMase2 protein expression, followed by the prefrontal cortex, thalamus, hippocampus, brainstem, and cerebellum. Dense immunolabeling was observed in the striatum, including the caudate-putamen, while moderately dense staining was found in the olfactory bulb and cerebral neocortex. Electron microscopy of the caudate-putamen showed nSMase2 immunoreaction product was present in small diameter dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled axon terminals containing small round vesicles; and characteristics of glutamatergic axons. Lipidomic analysis of the striatum showed increase in long chain sphingomyelins, SM36:1 and SM38:1 after inhibition of nSMase activity. Quantitative proteomic analysis of striatal lipid raft fraction showed many proteins were downregulated by more than 2-fold after inhibition or antisense knockdown of nSMase; consistent with the notion that nSMase2 activity is important for aggregation or clustering of proteins in lipid rafts. Inhibition or antisense knockdown of nSMase2 in the caudate-putamen resulted in motor deficits in the rotarod and narrow beam tests; as well as decreased acoustic startle and improved prepulse inhibition of the startle reflex. Together, results indicate an important function of nSMase2 in the striatum.

The hyaluronic acid inhibitor 4-methylumbelliferone is an NSMase2 activator—role of Ceramide in MU anti-tumor activity

Increased synthesis of hyaluronic acid (HA) is often associated with increased metastatic potential and invasivity of tumor cells. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis, and has been studied as a potential anti-tumor drug to inhibit the growth of primary tumors and distant metastasis of tumor cells. Although several studies reported that the anticancer effects of MU are mediated by inhibition of HA signaling, the mechanism still needs to be clarified. In a previous study we demonstrated the regulation of HA synthesis by ceramide, and now show how MU activated neutral sphingomyelinase2 (NSMase2) generates ceramides and mediates MU induced inhibition of HA synthesis, cell migration and invasion, and apoptosis of tumor cells. Using a HA enriched mouse oligodendroglioma cell line G26-24 we found that MU elevated the activity of NSMase2 and increased ceramide levels, which in turn increased phosphatase PP2A activity. Further, the activated PP2A reduced phosphorylation of Akt, decreased activities of HA synthase2 (HAS2) and calpains, and inhibited both the synthesis of HA, and the migration and invasion of G26-24 tumor cells. In addition, MU mediated ceramide stimulated activation of p53 and caspase-3, reduced SIRT1 expression and decreased G26-24 viability. The mechanism of the MU anticancer therefore initially involves NSMase2/ceramide/PP2A/AKT/HAS2/caspase-3/p53/SIRT1 and the calpain signaling pathway, suggesting that ceramides play a key role in the ability of a tumor to become aggressively metastatic and grow.

Neutral sphingomyelinase 2 is required for cytokine-induced skeletal muscle calpain activation.

Calpain contributes to infection-induced diaphragm dysfunction but the upstream mechanism(s) responsible for calpain activation are poorly understood. It is known, however, that cytokines activate neutral sphingomyelinase (nSMase) and nSMase has downstream effects with the potential to increase calpain activity. We tested the hypothesis that infection-induced skeletal muscle calpain activation is a consequence of nSMase activation. We administered cytomix (20 ng/ml TNF-α, 50 U/ml IL-1β, 100 U/ml IFN-γ, 10 μg/ml LPS) to C2C12 muscle cells to simulate the effects of infection in vitro and studied mice undergoing cecal ligation puncture (CLP) as an in vivo model of infection. In cell studies, we assessed sphingomyelinase activity, subcellular calcium levels, and calpain activity and determined the effects of inhibiting sphingomyelinase using chemical (GW4869) and genetic (siRNA to nSMase2 and nSMase3) techniques. We assessed diaphragm force and calpain activity and utilized GW4869 to inhibit sphingomyelinase in mice. Cytomix increased cytosolic and mitochondrial calcium levels in C2C12 cells (P < 0.001); addition of GW4869 blocked these increases (P < 0.001). Cytomix also activated calpain, increasing calpain activity (P < 0.02), and the calpain-mediated cleavage of procaspase 12 (P < 0.001). Procaspase 12 cleavage was attenuated by either GW4869 (P < 0.001), BAPTA-AM (P < 0.001), or siRNA to nSMase2 (P < 0.001) but was unaffected by siRNA to nSMase3. GW4869 prevented CLP-induced diaphragm calpain activation and diaphragm weakness in mice. These data suggest that nSMase2 activation is required for the development of infection-induced diaphragm calpain activation and muscle weakness. As a consequence, therapies that inhibit nSMase2 in patients may prevent infection-induced skeletal muscle dysfunction.

Src regulates cigarette smoke-induced ceramide generation via neutral sphingomyelinase 2 in the airway epithelium.

We previously demonstrated that the neutral sphingomyelinase (nSMase) 2 is the sole sphingomyelinase activated during cigarette smoke (CS)-induced oxidative stress of human airway epithelial cells, leading to ceramide generation and subsequent apoptosis of affected cells. Since then, we reported that nSMase2 is a phosphoprotein, the degree of enzymatic activity and stability of which are dictated by its degree of phosphorylation. Simultaneously, the non-receptor tyrosine kinase and proto-oncogene Src has increasingly become a target of interest in both smoking-related lung injury, such as chronic obstructive pulmonary disease, and lung cancer. Within this context, we tested and now present Src as a regulator of ceramide generation via modulation of nSMase2 phosphorylation and activity during CS-induced oxidative stress. Specifically, we provide evidence that Src activity is necessary for both CS-induced ceramide accumulation in vivo (129/Sv mice) and in vitro (human airway epithelial cells) and for nSMase2 activity during CS-induced oxidative stress. Moreover, because nSMase2 is exclusively phosphorylated on serines, we show that this occurs through Src-dependent activation of the serine/threonine kinase p38 mitogen-activated protein kinase during oxidative stress. Finally, we provide evidence that Src and p38 mitogen-activated protein kinase activities are critical for regulating nSMase2 phosphorylation. This study provides insights into a molecular target involved in smoking-related lung injury, represented here as nSMase2, and its modulation by the oncogene Src.

Cambinol, a novel inhibitor of neutral sphingomyelinase 2 shows neuroprotective properties.

Ceramide is a bioactive lipid that plays an important role in stress responses leading to apoptosis, cell growth arrest and differentiation. Ceramide production is due in part to sphingomyelin hydrolysis by sphingomyelinases. In brain, neutral sphingomyelinase 2 (nSMase2) is expressed in neurons and increases in its activity and expression have been associated with pro-inflammatory conditions observed in Alzheimer’s disease, multiple sclerosis and human immunodeficiency virus (HIV-1) patients. Increased nSMase2 activity translates into higher ceramide levels and neuronal cell death, which can be prevented by chemical or genetic inhibition of nSMase2 activity or expression. However, to date, there are no soluble, specific and potent small molecule inhibitor tool compounds for in vivo studies or as a starting point for medicinal chemistry optimization. Moreover, the majority of the known inhibitors were identified using bacterial, bovine or rat nSMase2. In an attempt to identify new inhibitor scaffolds, two activity assays were optimized as screening platform using the recombinant human enzyme. First, active hits were identified using a fluorescence-based high throughput compatible assay. Then, hits were confirmed using a 14C sphingomyelin-based direct activity assay. Pharmacologically active compounds and approved drugs were screened using this strategy which led to the identification of cambinol as a novel uncompetitive nSMase2 inhibitor (K i = 7 μM). The inhibitory activity of cambinol for nSMase2 was approximately 10-fold more potent than for its previously known target, silence information regulator 1 and 2 (SIRT1/2). Cambinol decreased tumor necrosis factor-α or interleukin-1 β-induced increases of ceramide and cell death in primary neurons. A preliminary study of cambinol structure and activity allowed the identification of the main structural features required for nSMase2 inhibition. Cambinol and its analogs may be useful as nSMase2 inhibitor tool compounds to prevent ceramide-dependent neurodegeneration.

Changes in the metabolism of sphingolipids after subarachnoid hemorrhage.

We previously described how ceramide (Cer), a mediator of cell death, increases in the cerebrospinal fluid (CSF) of subarachnoid hemorrhage (SAH) patients. This study investigates the alterations of biochemical pathways involved in Cer homeostasis in SAH. Cer, dihydroceramide (DHC), sphingosine-1-phosphate (S1P), and the activities of acid sphingomyelinase (ASMase), neutral sphingomyelinase (NSMase), sphingomyelinase synthase (SMS), S1P-lyase, and glucosylceramide synthase (GCS) were determined in the CSF of SAH subjects and in brain homogenate of SAH rats. Compared with controls (n = 8), SAH patients (n = 26) had higher ASMase activity (10.0 ± 3.5 IF/µl· min vs. 15.0 ± 4.6 IF/µl • min; P = 0.009) and elevated levels of Cer (11.4 ± 8.8 pmol/ml vs. 33.3 ± 48.3 pmol/ml; P = 0.001) and DHC (1.3 ± 1.1 pmol/ml vs. 3.8 ± 3.4 pmol/ml; P = 0.001) in the CSF. The activities of GCS, NSMase, and SMS in the CSF were undetectable. Brain homogenates from SAH animals had increased ASMase activity (control: 9.7 ± 1.2 IF/µg • min; SAH: 16.8 ± 1.6 IF/µg • min; P < 0.05) and Cer levels (control: 3,422 ± 26 fmol/nmol of total lipid P; SAH: 7,073 ± 2,467 fmol/nmol of total lipid P; P < 0.05) compared with controls. In addition, SAH was associated with a reduction of 60% in S1P levels, a 40% increase in S1P-lyase activity, and a twofold increase in the activity of GCS. In comparison, NSMase and SMS activities were similar to controls and SMS activities similar to controls. In conclusion, our results show an activation of ASMase, S1P-lyase, and GCS resulting in a shift in the production of protective (S1P) in favor of deleterious (Cer) sphingolipids after SAH. Additional studies are needed to determine the effect of modulators of the pathways described here in SAH.

Annexin II-dependent actin remodelling evoked by hydrogen peroxide requires the metalloproteinase/sphingolipid pathway

Actin remodeling is a dynamic process associated with cell shape modification occurring during cell cycle and proliferation. Oxidative stress plays a role in actin reorganization via various systems including p38MAPK. Beside, the mitogenic response evoked by hydrogen peroxide (H2O2) in fibroblasts and smooth muscle cells (SMC) involves the metalloproteinase (MMPs)/sphingomyelinase 2 (nSMase2) signaling pathway. The aim of this work was to investigate whether this system plays a role in actin remodeling induced by H2O2.Low H2O2 dose (5µM) rapidly triggered a signaling cascade leading to nSMase2 activation, src and annexin 2 (AnxA2) phosphorylation, and actin remodeling, in fibroblasts and SMC. These events were blocked by pharmacological inhibitors of MMPs (Ro28-2653) and p38MAPK (SB203580), and were lacking in MMP2−/− and in nSMase2-mutant (fro) fibroblasts. Likewise, H2O2 was unable to induce actin remodeling in fro and MMP2−/− fibroblasts or in cells pretreated with p38MAPK, or MMP inhibitors. Finally we show that nSMase2 activation by H2O2, depends on MMP2 and p38MAPK, and is required for the src-dependent phosphorylation of AnxA2, and actin remodeling.Taken together, these findings indicate for the first time that AnxA2 phosphorylation and actin remodeling evoked by oxidative stress depend on the sphingolipid pathway, via MMP2 and p38MAPK.

Hyaluronan synthase-2 upregulation protects smpd3-deficient fibroblasts against cell death induced by nutrient deprivation, but not against apoptosis evoked by oxidized LDL

The neutral type 2 sphingomyelinase (nSMase2) hydrolyzes sphingomyelin and generates ceramide, a major bioactive sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (oxidized LDL, hydrogen peroxide and nutrient starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient starvation (fetal calf serum/glucose/pyruvate-free DMEM), whereas wt fibroblasts die after 48h incubation in this medium. In contrast, oxidized LDL and hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor GW4869 were more resistant to starvation-induced apoptosis.The resistance of fro/fro cells to starvation-induced apoptosis is associated with an increased expression of hyaluronan synthase 2 (HAS2) mRNAs and protein, which is inhibited by ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against starvation-induced apoptosis.The protective mechanism of HAS2 involves an increased expression of the heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient starvation that could contribute to the delayed apoptosis of hypertrophic chondrocytes in the growth plate, and emphasize the antiapoptotic properties of HAS2.

Enhanced diacylglycerol production by phospholipase D activation is responsible for abnormal increase in concanavalin A cap formation in polymorphonuclear leukocytes from Chediak-Higashi syndrome (beige) mice.

We previously reported that enhanced ceramide production induces calpain-mediated proteolysis of protein kinase C (PKC) in leukocytes from Chediak-Higashi syndrome (CHS). In the present study, we demonstrated that phospholipase D (PLD) inhibitors ameliorated abnormal increases in concanavalin A (Con A) cap formation in polymorphonuclear leukocytes (PMNs) from beige mouse, an animal model of CHS. PLD activity in PMNs from beige mice enhanced at 30 to 60s after Con A stimulation. In Con A-stimulated beige PMNs, both neutral sphingomyelinase (N-SMase) and acidic sphingomyelinase (A-SMase) activities enhanced, and ceramide levels are also increased. We found that ceramide levels were reversed by the treatment of beige PMNs with propranolol which inhibits phosphatidic acid phosphohydrolase. In addition, we showed that diacylgycerol (DAG) analogs enhance both N-SMase and A-SMase activities in PMNs from normal mice. We subsequently examined the association of CHS1 with PLD, and showed that expression of a truncated mutant of CHS1 in 293T cells induced abnormally rapid activation of PLD after phorbol ester stimulation. Moreover, we showed that specific inhibitors of 14-3-3 proteins, which interact with CHS1/LYST and bind PKC, did not affect abnormal increases in Con A cap formation in beige PMNs. These results suggest that the enhanced DAG production via the PLD pathway is associated with abnormal increases in Con A cap formation in beige PMNs, and that CHS1 may be involved in the regulation of PLD activity.

Advance in research on regulatory mechanism and functions of neutral sphingomyelinse 2

Neutral sphingomyelinase 2 (nSMase2), which located mainly on the plasma membrane, hydrolyzes sphingomyelin into ceramide and plays an important role in the physiological and pathological regulation of cell apoptosis, cell growth arrest, and inflammation. nSMase2 is also involved in the development of Alzheimer's disease and the bone growth.Under neutral pH and the presence of Ca(2+), Mg(2+), and Mn(+), the activity of nSMase2 is induced by oxidative stress through phosphorylation. Furthermore, the induced interaction of anionic phospholipids and the signaling molecules like receptor for activated C-kinase 1/embryonic ectodermal development with nSMase2 are also crucial mechanisms of protein activation. In the review, recent research advances in the structure and function of nSMase2 and its underlying mechanisms are summarized.

Early activation of nSMase2/ceramide pathway in astrocytes is involved in ischemia-associated neuronal damage via inflammation in rat hippocampi

BackgroundCeramide accumulation is considered a contributing factor to neuronal dysfunction and damage. However, the underlying mechanisms that occur following ischemic insult are still unclear.MethodsIn the present study, we established cerebral ischemia models using four-vessel occlusion and oxygen-glucose deprivation methods. The hippocampus neural cells were subjected to immunohistochemistry and immunofluorescence staining for ceramide and neutral sphingomyelinase 2 (nSMase2) levels; immunoprecipitation and immunoblot analysis for nSMase2, receptor for activated C kinase 1 (RACK1), embryonic ectoderm development (EED), p38 mitogen-activated protein kinase (p38MAPK) and phosphorylated p38MAPK expression; SMase assay for nSMase and acid sphingomyelinase (aSMase) activity; real-time reverse transcription polymerase chain reaction for cytokine expression; and Nissl, microtubule-associated protein 2 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining.ResultsWe found considerable production of ceramide in astrocytes, but not in neurons, during early cerebral ischemia. This was accompanied by the induction of nSMase (but not aSMase) activity in the rat hippocampi. The inhibition of nSMase2 activity effectively reduced ceramide accumulation in astrocytes and alleviated neuronal damage to some extent. Meanwhile, the expression levels of proinflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6, were found to be upregulated, which may have played an import role in neuronal damage mediated by the nSMase2/ceramide pathway. Although enhanced binding of nSMase2 with RACK1 and EED were also observed after cerebral ischemia, nSMase2 activity was not blocked by the TNF-α receptor inhibitor through RACK1/EED signaling. p38MAPK, but not protein kinase Cζ or protein phosphatase 2B, was able to induce nSMase2 activation after ischemia. p38MAPK can be induced by A2B adenosine receptor (A2BAR) activity.ConclusionsThese results indicate that the inhibition of ceramide production in astrocytes by targeting A2BAR/p38MAPK/nSMase2 signaling may represent a viable approach for attenuating inflammatory responses and neuronal damage after cerebral ischemia.