Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway.

Neurotransmitter release is mediated by ceramide, which is generated by sphingomyelin hydrolysis. In the present study, we examined whether synaptosomal-associated protein 25 (SNAP-25) is involved in ceramide production and exocytosis. Neutral sphingomyelinase 2 (nSMase2) was partially purified from bovine brain and we found that SNAP-25 was enriched in the nSMase2-containing fractions. In rat synaptosomes and PC12 cells, the immunoprecipitation pellet of anti-SNAP-25 antibody showed higher nSMase activity than the immunoprecipitation pellet of anti-nSMase2 antibody. In PC12 cells, SNAP-25 was colocalized with nSMase2. Transfection of SNAP-25 small interfering RNA (siRNA) significantly inhibited nSMase2 translocation to the plasma membrane. A23187-induced ceramide production was concomitantly reduced in SNAP-25 siRNA-transfected PC12 cells compared with that in scrambled siRNA-transfected cells. Moreover, transfection of SNAP-25 siRNA inhibited dopamine release, whereas addition of C6-ceramide to the siRNA-treated cells moderately reversed this inhibition. Additionally, nSMase2 inhibition reduced dopamine release. Collectively, our results indicate that SNAP-25 interacts with nSMase2 during ceramide production, which mediates exocytosis and neurotransmitter release.

Effect of Robinia pseudoacacia Leaf Extract on Interleukin-1ß-mediated Tumor Angiogenesis.

BACKGROUND/AIM: Interleukin (IL)-1ß is a pro-inflammatory cytokine that has recently been established as a stimulator of angiogenesis via regulation of proangiogenic factor expression in the tumor microenvironment. This study aimed to demonstrate the inhibitory effects of Robinia pseudoacacia leaf extract (RP) on IL-1ß-mediated tumor angiogenesis. MATERIALS AND METHODS: Secreted embryonic alkaline phosphatase (SEAP) reporter gene assay, ex vivo and in vitro tube formation assay, western blot, and quantitative PCR were used to analyze the inhibitory effect of RP on IL-1ß-mediated angiogenesis. RESULTS: RP inhibited secretion of SEAP, blocked IL-1ß signaling, and inhibited IL-1ß-mediated angiogenesis in ex vivo and in vitro assays. RP inhibited nuclear translocation of NF-ĸB by suppressing phosphorylation of IL-1ß signaling protein kinases and inhibited mRNA expression of IL-1ß-induced pro-angiogenic factors including VEGFA, FGF2, ICAM1, CXCL8, and IL6. CONCLUSION: RP suppressed IL-1ß-mediated angiogenesis and, thus, could be a promising agent in anticancer therapy.

Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases.

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LCMS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 588.6 → 264.4 for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide.

Autophagy is essential for optimal cell function and survival, and the entire process accompanies membrane dynamics. Ceramides are produced by different enzymes at different cellular membrane sites and mediate differential signaling. However, it remains unclear which ceramide-producing pathways/enzymes participate in autophagy regulation under physiological conditions such as nutrient starvation, and what the underlying mechanisms are. In this study, we demonstrate that among ceramide-producing enzymes, neutral sphingomyelinase 2 (nSMase2) plays a key role in autophagy during nutrient starvation. nSMase2 was rapidly and stably activated upon starvation, and the enzymatic reaction in the Golgi apparatus facilitated autophagy through the activation of p38 MAPK and inhibition of mTOR. Moreover, nSMase2 played a protective role against cellular damage depending on autophagy. These findings suggest that nSMase2 is a novel regulator of autophagy and provide evidence that Golgi-localized ceramides participate in cytoprotective autophagy against starvation.

Dopamine transporter trafficking is regulated by neutral sphingomyelinase 2/ceramide kinase.

Dopamine (DA) reuptake is the primary mechanism to terminate dopaminergic transmission in the synaptic cleft. The dopamine transporter (DAT) has an important role in the regulation of DA reuptake. This study provides anatomical and physiological evidence that DAT recycling is regulated by ceramide kinase via the sphingomyelin pathway. First, the results show that DAT and neutral sphingomyelinase 2 (nSMase2) were successfully co-precipitated from striatal samples and were colocalized in the mouse striatum or PC12 cells. We also identified a protein-protein interaction between nSMase2 and DAT through in situ proximity ligation assay experiments in the mouse striatum. Second, dopamine (DA) stimulated the formation of ceramide and increased nSMase activity in PC12 cells, while treatment with a cell-permeable ceramide-1-phosphate (C1P) increased DA uptake. Third, we used inhibitors and siRNA to inhibit nSMase2 and ceramide kinase and observed the effects on DAT recycling in PC12 cells. Treatment with ceramide kinase inhibitor K1, or nSMase inhibitor GW4869, decreased DA uptake in PC12 cells, although the application of FB1, a ceramide synthase inhibitor, did not affect DA uptake. Transfection of nSMase2 and CERK siRNA decreased DAT surface level in PC12 cells. These results suggested that SM-derived C1P affects cell surface levels of DAT.

P2X1 Receptor-Mediated Ca2+ Influx Triggered by DA-9801 Potentiates Nerve Growth Factor-Induced Neurite Outgrowth.

Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake.

Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH-optimum and Mg(2+)-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.

C(6)-ceramide enhances phagocytic activity of Kupffer cells through the production of endogenous ceramides.

Ceramide has been suggested to be not only a tumor-suppressive lipid but also a regulator of phagocytosis. We examined whether exogenous cell-permeable C(6)-ceramide enhances the phagocytic activity of Kupffer cells (KCs) and affects the level of cellular ceramides. Rat KCs were isolated by collagenase digestion and differential centrifugation, using Percoll system. Phagocytic activity was measured by FACS analysis after incubating KCs with fluorescence-conjugated latex beads, and the level of cellular ceramide was analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). In this study we found that permeable C(6)-ceramide increases the cellular levels of endogenous ceramides via a sphingosine-recycling pathway leading to enhanced phagocytosis by KCs.