Purification and Characterization of Mitochondrial Mg2+-Independent Sphingomyelinase from Rat Brain.

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg2+-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg2+, Mn2+, N2+, Cu2+, Zn2+, Fe2+, and Fe3+ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg2+-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

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.

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.

(S)-tetrahydroisoquinoline alkaloid inhibits LPS-induced arachidonic acid release through downregulation of cPLA2 expression.

Sepsis, a systemic inflammatory response syndrome, remains a potentially lethal condition. (S)-1-α-Naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD712) is noted as a drug candidate for sepsis. Many studies have demonstrated its significant anti-inflammatory effects. Here we first examined whether CKD712 inhibits lipopolysaccharide (LPS)-induced arachidonic acid (AA) release in the RAW 264.7 mouse monocyte cell line, and subsequently, its inhibitory mechanisms. CKD712 reversed LPS-associated morphological changes in the RAW 264.7 cells, and inhibited LPS-induced release of AA in a concentrationdependent manner. The inhibition was apparently due to the diminished expression of a cytosolic form of phospholipase A2 (cPLA2) by CKD712, resulting from reduced NF-κB activation. Furthermore, CKD712 inhibited the activation of ERK1/2 and SAP/JNK, but not of p38 MAPK. CKD712 had no effect on the activity or phosphorylation of cPLA2 and on calcium influx. Our results collectively suggest that CKD712 inhibits LPS-induced AA release through the inhibition of a MAPKs/NF-κB pathway leading to reduced cPLA2 expression in RAW 264.7 cells.

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.

Effect of rice cell-derived human granulocyte-macrophage colony-stimulating factor on 5-fluorouracil-induced mucositis in hamsters.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important regulator of the maturation and function of cells in the granulocyte and macrophage lineages, and also plays a significant role in wound healing. In a previous study, we expressed human GM-CSF in rice cells (rice cell-derived human GM-CSF; rhGM-CSF). The purpose of the present study was to evaluate its effect on wound healing in oral mucositis. Oral mucositis was induced in Syrian hamster cheek pouches by 5-fluorouracil treatment and mechanical scratching. Ulcerated areas were treated from days 3 to 14 with an application of 200 µL saline, or of the same volume of a solution containing 0.04, 0.2, or 1 µg/mL rhGM-CSF. Treatment of hamsters with rhGM-CSF reduced the ulcerated areas of the oral mucosa, compared with the control. Early in the healing process, the mucositis tissue layer of the rhGM-CSF-treated group showed significantly decreased myeloperoxidase activity and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells. Treatment with rhGM-CSF also affected expression of inflammatory cytokines in the ulcerative mucosal tissue. These results demonstrate the efficacy of plant-produced rhGM-CSF in wound healing and have significant implications for the development of rhGM-CSF as a therapeutic agent for ulcerative oral mucositis.

Purification and characterization of a cytosolic Ca(2+)-independent phospholipase A(2) from bovine brain.

The Ca(2+)-independent phospholipase A(2) (iPLA(2)) subfamily of enzymes is associated with arachidonic acid (AA) release and the subsequent increase in fatty acid turnover. This phenomenon occurs not only during apoptosis but also during inflammation and lymphocyte proliferation. In this study, we purified and characterized a novel type of iPLA(2) from bovine brain. iPLA(2) was purified 4,174-fold from the bovine brain by a sequential process involving DEAE-cellulose anion exchange, phenyl-5PW hydrophobic interaction, heparin-Sepharose affinity, Sephacryl S-300 gel filtration, Mono S cation exchange, Mono Q anion exchange, and Superose 12 gel filtration. A single peak of iPLA(2) activity was eluted at an apparent molecular mass of 155 kDa during the final Superose 12 gel-filtration step. The purified enzyme had an isoelectric point of 5.3 on two-dimensional gel electrophoresis (2-DE) and was inhibited by arachidonyl trifluoromethyl ketone (AACOCF(3)), Triton X-100, iron, and Ca(2+). However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA(2), and adenosine triphosphate (ATP). The spot with the iPLA(2) activity did not match with any known protein sequence, as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Altogether, these data suggest that the purified enzyme is a novel form of cytosolic iPLA(2).

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.

Identification of a 42-kDa Group IV cPLA2-activating protein, cPLAPγ, as a GTP-binding protein in the bovine brain.

Brain tissue contains multiple forms of Phospholipase A(2) (PLA(2)) whose activities are involved in intracellular and intercellular signalling related to normal functions such as long-term potentiation, neurotransmitter release, cell growth and differentiation. Among them, we focused on regulatory mechanism of cPLA(2)α (Group IVA cytosolic PLA(2)) in brain tissue. In the present study, we report the identification of a cPLA(2)-activating protein (cPLAP) in the bovine brain. cPLAP activity appeared as two major peaks with molecular masses of 200 and 42 kDa in a Superose 12 gel filtration FPLC column. The 42-kDa form of cPLAP, designated cPLAPγ, was further purified using a Mono S FPLC column to near homogeneity and characterized to as a GTP-binding protein (G protein). Metabolic labelling and immunoprecipitation studies revealed that cPLAPγ associates with cPLA(2) in vitro and co-immunoprecipitates with [(35)S]-cPLA(2). Notably, cPLAPγ rendered cPLA(2) fully activated at submicromolar concentrations of Ca(2+). These results suggest that cPLAPγ may act as a G protein, activating cPLA(2)α prior to reaching full intracellular Ca(2+) concentrations.

Identification and evaluation of neutral sphingomyelinase 2 inhibitors.

Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to generate ceramide, an important molecule involved in the regulation of various cellular responses. In this study, we partially purified the neutral sphingomyelinase2 (nSMase2) and identified the inhibitors, D-lyxophytosphingosine and D-arabino-phytosphingosine, which have an inhibitory effect on nSMase2 in a concentration-dependent manner. A Dixon plot of each phytosphingosines revealed their probable inhibitory pattern, i.e., apparent competitive inhibition. These compounds did not inhibit the Mg(2+)-independent neutral SMase activity, although the known nSMase2 inhibitor, GW4869, showed inhibitory effects on Mg(2+)-independent neutral SMase activity. Further, the two phytosphingosines specifically inhibited the ceramide generation regulated by nSMase2.

Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2.

Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C(6)-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A(2) (PLA(2)) inhibitors. Moreover, cytosolic PLA(2) GIVA (cPLA(2) GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells. Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A(2) GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA(2) are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA(2).

Neutral sphingomyelinase 2 induces dopamine uptake through regulation of intracellular calcium.

Ceramide serves as a second messenger produced from sphingomyelin by the activation of sphingomyelinase (SMase). Here, we suggest that neutral SMase 2 (nSMase2) may regulate dopamine (DA) uptake. nSMase2 siRNA-transfected PC12 cells showed lower levels of nSMase activity and ceramide than scramble siRNA-transfected and control cells. Interestingly, transfection of nSMase2 siRNA or pretreatment with the nSMase2-specific inhibitor GW4869 resulted in decreased DA uptake. Reciprocally, exposure of PC12 cells to cell-permeable C(6)-ceramide induced a concentration-dependent increase in DA uptake. Removal of extracellular calcium by EGTA increased DA uptake in scramble-transfected and control cells, but not in nSMase2 siRNA-transfected or GW4869-pretreated cells. Moreover, siRNA-transfected cells showed higher levels of intracellular calcium than scramble cells, while C(6)-ceramide treatment resulted in decreased intracellular calcium compared to vehicle treatment alone. Taken together, these data suggest that nSMase2 may increase DA uptake through inducing ceramide production and thereby decreasing intracellular calcium levels.

Hypoxia-induced neuronal apoptosis is mediated by de novo synthesis of ceramide through activation of serine palmitoyltransferase.

Cellular hypoxia can lead to cell death or adaptation and has important effects on development, physiology, and pathology. Here, we investigated the role and regulation of ceramide in hypoxia-induced apoptosis of SH-SY5Y neuroblastoma cells. Hypoxia increased the ceramide concentration; subsequently, we observed biochemical changes indicative of apoptosis, such as DNA fragmentation, nuclear staining, and poly ADP-ribose polymerase (PARP) cleavage. The hypoxic cell death was potently inhibited by a caspase inhibitor, zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone). l-Cycloserine, a serine palmitoyltransferase (SPT) inhibitor, and fumonisin B(1) (FB(1)), a ceramide synthase inhibitor, inhibited the hypoxia-induced increase in ceramide, indicating that the increase occurred via the de novo pathway. Hypoxia increased the activity and protein levels of SPT2, suggesting that the hypoxia-induced increase in ceramide is due to the transcriptional up-regulation of SPT2. Specific siRNA of SPT2 prevented hypoxia-induced cell death and ceramide production. However, hypoxia also increased the cellular level of glucosylceramide, which was inhibited by a glucosylceramide synthase (GCS) inhibitor and specific siRNA, but not a ceramidase inhibitor. The increase in glucosylceramide was accompanied by increases in both PARP cleavage and DNA fragmentation. Together, the current results suggest that both SPT and GCS may regulate the cellular level of ceramide, and thus may be critical enzymes for deciding the fate of the cells exposed to hypoxia.

Purification of neutral sphingomyelinase 2 from bovine brain and its calcium-dependent activation.

Ceramide is produced by sphingomyelinase (SMase) and it plays a key role in cellular responses such as apoptosis. In this study, we report the purification and characterization of neutral SMase2 (nSMase2) from bovine brain tissue. Triton X-100 extracts of bovine brain membranes were purified in nine steps, including sequential chromatography. The specific activity of purified nSMase increased 8183-fold over the brain membrane fraction. Purified nSMase showed similarities to nSMase2, which had been purified and cloned previously. Interestingly, purified nSMase2 was Ca2+-dependent and could be activated by micromolar concentrations of Ca2+ under Mg2+-free conditions. Ceramide generation was dependent upon the calcium ionophore A23187 and was observed in nSMase2-over-expressing COS-7 cells. This generation was suppressed by GW4869, an nSMase2 inhibitor, but not to fumonisin B(1), an inhibitor of the de novo ceramide synthesis pathway. The present study demonstrates the Ca2+-dependent activation of nSMase2.

D609, an inhibitor of phosphatidylcholine-specific phospholipase C, inhibits group IV cytosolic phospholipase A2.

As an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 has been widely used to explain the role of PC-PLC in various signal transduction pathways. This study shows that D609 inhibits group IV cytosolic phospholipase A2 (cPLA2), but neither secretory PLA2 nor a Ca2+ -dependent PLA2. Dixon plot analysis shows a mixed pattern of noncompetitive and uncompetitive inhibition with Ki = 86.25 microM for the cPLA2 purified from bovine spleen. D609 also time- and dose-dependently reduces the release of arachidonic acid from a Ca2+- ionophore A23187-stimulated MDCK cells. In the AA release experiment, IC50 of D609 was approximately 375 microM, suggesting that this reagent may not enter the cells easily. The present study indicates that the inhibitory effects of D609 on various cellular responses may be partially attributable to the inhibition of cPLA2.

Subcutaneous Four-Week Repeated Dose Toxicity Studies of Rice Cell-Derived Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor in Rats.

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a glycoprotein and hematopoietic growth factors that regulates the proliferation of myeloid precursor cells and activates mature granulocytes and macrophages. In a previous study, we reported that hGM-CSF could be produced in transgenic rice cell suspension culture, termed rhGM-CSF. In the present study, we examined the repeated dose toxicity of rhGM-CSF in SD rats. The repeated dose toxicity study was performed at each dose of 50 and 200 µg/kg subcutaneous administration of rhGM-CSF everyday for 28-days period. The results did not show any changes in food and water intake. There were also no significant changes in both body and organ weights between the control and the tested groups. The hematological and blood biochemical parameters were statistically not different in all groups. These results suggest that rhGM-CSF may show no repeated dose toxicity in SD rats under the conditions.

Identification of three competitive inhibitors for membrane-associated, Mg2+-dependent and neutral 60 kDa sphingomyelinase activity.

Methanol extracts of domestic plants of Korea were evaluated as a potential inhibitor of neutral pH optimum and membrane-associated 60 kDa sphingomyelinase (N-SMase) activity. In this study, we partially purified N-SMase from bovine brain membranes using ammonium sulfate. It was purified approximately 163-fold by the sequential use of DE52, Butyl-Toyopearl, DEAE-Cellulose, and Phenyl-5PW column chromatographies. The purified N-SMase activity was assayed in the presence of the plant extracts of three hundreds species. Based on the in vitro assay, three plant extracts significantly inhibited the N-SMase activity in a time- and concentration-dependent manner. To further examine the inhibitory pattern, a Dixon plot was constructed for each of the plant extracts. The extracts of Abies nephrolepis, Acer tegmentosum, and Ginkgo biloba revealed a competitive inhibition with the inhibition constant (Ki) of 11.9 microg/ mL, 9.4 microg/mL, and 12.9 microg/mL, respectively. These extracts also inhibited in a dose-dependent manner the production of ceramide induced by serum deprivation in human neuroblastoma cell line SH-SY5Y.