Suppressive effect of resveratrol, catechin and their conformationally constrained analogs on neutrophil extracellular trap formation by HL-60-derived neutrophils.

Neutrophil extracellular trap (NET) formation is a unique self-defense mechanism of neutrophils; however, it is also involved in many diseases, including atherosclerosis. Resveratrol and catechin are antioxidants with anti-atherosclerotic properties. Here, we examined the effects of resveratrol, catechin, and other related compounds on NET formation. HL-60-derived neutrophils were pretreated with resveratrol and other compounds before stimulation with phorbol-myristate acetate (PMA). DNA and myeloperoxidase released from neutrophils were determined. Resveratrol suppressed the DNA release from neutrophils in a dose-dependent manner. NET formation was enhanced by 1-palmitoyl-2-oxovaleroyl phosphatidylcholine (POVPC), a truncated form of oxidized phospholipid, and resveratrol suppressed NET formation induced by POVPC and PMA. Furthermore, we designed several analogs of resveratrol or catechin whose conformation was restricted by the inhibition of the free rotation of aromatic rings. The conformationally constrained analogs were more effective at inhibiting NET formation; however, their inhibitory function decreased when compound was a large, hydrophobic analog. The most potent compounds, planar catechin and resveratrol, suppressed myeloperoxidase release from activated neutrophils. In addition, these compounds suppressed DNA release from neutrophils stimulated with calcium ionophore. These results suggest that resveratrol, catechin and their analogs exert anti-NET effects, and that constraining the geometry of these compounds enhanced their inhibitory effects.

Behavior of intracellular lipid droplets during cell division in HuH7 hepatoma cells.

Intracellular lipid droplets (LDs) are ubiquitous organelles found in many cell types. During mitosis, membranous organelles, including mitochondria, are divided into small pieces and transferred to daughter cells; however, the process of LD transfer to daughter cells is not fully elucidated. Herein, we investigated the behavior of LDs during mitosis in HuH7 human hepatoma cells. While fragments of the Golgi apparatus were scattered in the cytosol during mitosis, intracellular LDs retained their size and spherical morphology as they translocated to the two daughter cells. LDs were initially distributed throughout the cell during prophase but positioned outside the spindle in metaphase, aligning at the far sides of the centrioles. A similar distribution of LDs during mitosis was observed in another hepatocarcinoma HepG2 cells. When the spindle was disrupted by nocodazole treatment or never in mitosis gene A-related kinase 2A knockdown, LDs were localized in the area outside the chromosomes, suggesting that spindle formation is not necessary for LD localization at metaphase. The amount of major LD protein perilipin 2 reduced while LDs were enriched in perilipin 3 during mitosis, indicating the potential alteration of LD protein composition. Conclusively, the behavior of LDs during mitosis is distinct from that of other organelles in hepatocytes.

High-Density Lipoprotein Suppresses Neutrophil Extracellular Traps Enhanced by Oxidized Low-Density Lipoprotein or Oxidized Phospholipids.

Neutrophil extracellular traps (NETs) are found in patients with various diseases, including cardiovascular diseases. We previously reported that copper-oxidized low-density lipoprotein (oxLDL) promotes NET formation of neutrophils, and that the resulting NETs increase the inflammatory responses of endothelial cells. In this study, we investigated the effects of high-density lipoproteins (HDL) on NET formation. HL-60-derived neutrophils were treated with phorbol 12-myristate 13-acetate (PMA) and further incubated with oxLDL and various concentrations of HDL for 2 h. NET formation was evaluated by quantifying extracellular DNA and myeloperoxidase. We found that the addition of native HDL partially decreased NET formation of neutrophils induced by oxLDL. This effect of HDL was lost when HDL was oxidized. We showed that oxidized phosphatidylcholines and lysophosphatidylcholine, which are generated in oxLDL, promoted NET formation of PMA-primed neutrophils, and NET formation by these products was completely blocked by native HDL. Furthermore, we found that an electronegative subfraction of LDL, LDL(-), which is separated from human plasma and is thought to be an in vivo oxLDL, was capable of promoting NET formation. These results suggest that plasma lipoproteins and their oxidative modifications play multiple roles in promoting NET formation, and that HDL acts as a suppressor of this response.

Sar1 Affects the Localization of Perilipin 2 to Lipid Droplets.

Lipid droplets (LDs) are intracellular organelles that are ubiquitous in many types of cells. The LD core consists of triacylglycerols (TGs) surrounded by a phospholipid monolayer and surface proteins such as perilipin 2 (PLIN2). Although TGs accumulate in the phospholipid bilayer of the endoplasmic reticulum (ER) and subsequently nascent LDs buds from ER, the mechanism by which LD proteins are transported to LD particles is not fully understood. Sar1 is a GTPase known as a regulator of coat protein complex Ⅱ (COPⅡ) vesicle budding, and its role in LD formation was investigated in this study. HuH7 human hepatoma cells were infected with adenoviral particles containing genes coding GFP fused with wild-type Sar1 (Sar1 WT) or a GTPase mutant form (Sar1 H79G). When HuH7 cells were treated with oleic acid, Sar1 WT formed a ring-like structure around the LDs. The transient expression of Sar1 did not significantly alter the levels of TG and PLIN2 in the cells. However, the localization of PLIN2 to the LDs decreased in the cells expressing Sar1 H79G. Furthermore, the effects of Sar1 on PLIN2 localization to the LDs were verified by the suppression of endogenous Sar1 using the short hairpin RNA technique. In conclusion, it was found that Sar1 has some roles in the intracellular distribution of PLIN2 to LDs in liver cells.

Regulation of Hook1-mediated endosomal sorting of clathrin-independent cargo by γ-taxilin.

In clathrin-independent endocytosis, Hook1, a microtubule- and cargo-tethering protein, participates in sorting of cargo proteins such as CD98 (encoded by SLC3A2) and CD147 (encoded by BSG) into recycling endosomes. However, the molecular mechanism that regulates Hook1-mediated endosomal sorting is not fully understood. In the present study, we found that γ-taxilin is a novel regulator of Hook1-mediated endosomal sorting. γ-Taxilin depletion promoted both CD98-positive tubular formation and CD98 recycling. Conversely, overexpression of γ-taxilin inhibited the CD98-positive tubular formation. Depletion of Hook1, or Rab10 or Rab22a (which are both involved in Hook1-mediated endosomal sorting), attenuated the effect of γ-taxilin depletion on the CD98-positive tubular formation. γ-Taxilin depletion promoted CD147-mediated spreading of HeLa cells, suggesting that γ-taxilin might be a pivotal player in various cellular functions in which Hook1-mediated cargo proteins are involved. γ-Taxilin bound to the C-terminal region of Hook1 and inhibited its interaction with CD98; the latter interaction is necessary for sorting CD98. We suggest that γ-taxilin negatively regulates the sorting of Hook1-mediated cargo proteins into recycling endosomes by interfering with the interactions between Hook1 and the cargo proteins.

Structure and Dynamics of Oxidized Lipoproteins In Vivo: Roles of High-Density Lipoprotein.

Oxidative modification of lipoproteins is implicated in the occurrence and development of atherosclerotic lesions. Earlier studies have elucidated on the mechanisms of foam cell formation and lipid accumulation in these lesions, which is mediated by scavenger receptor-mediated endocytosis of oxidized low-density lipoprotein (oxLDL). Mounting clinical evidence has supported the involvement of oxLDL in cardiovascular diseases. High-density lipoprotein (HDL) is known as anti-atherogenic; however, recent studies have shown circulating oxidized HDL (oxHDL) is related to cardiovascular diseases. A modified structure of oxLDL, which was increased in the plasma of patients with acute myocardial infarction, was characterized. It had two unique features: (1) a fraction of oxLDL accompanied oxHDL, and (2) apoA1 was heavily modified, while modification of apoB, and the accumulation of oxidized phosphatidylcholine (oxPC) and lysophosphatidylcholine (lysoPC) was less pronounced. When LDL and HDL were present at the same time, oxidized lipoproteins actively interacted with each other, and oxPC and lysoPC were transferred to another lipoprotein particle and enzymatically metabolized rapidly. This brief review provides a novel view on the dynamics of oxLDL and oxHDL in circulation.

Transfer and Enzyme-Mediated Metabolism of Oxidized Phosphatidylcholine and Lysophosphatidylcholine between Low- and High-Density Lipoproteins.

Oxidized low-density lipoprotein (oxLDL) and oxidized high-density lipoprotein (oxHDL), known as risk factors for cardiovascular disease, have been observed in plasma and atheromatous plaques. In a previous study, the content of oxidized phosphatidylcholine (oxPC) and lysophosphatidylcholine (lysoPC) species stayed constant in isolated in vivo oxLDL but increased in copper-induced oxLDL in vitro. In this study, we prepared synthetic deuterium-labeled 1-palmitoyl lysoPC and palmitoyl-glutaroyl PC (PGPC), a short chain-oxPC to elucidate the metabolic fate of oxPC and lysoPC in oxLDL in the presence of HDL. When LDL preloaded with d13-lysoPC was mixed with HDL, d13-lysoPC was recovered in both the LDL and HDL fractions equally. d13-LysoPC decreased by 50% after 4 h of incubation, while d13-PC increased in both fractions. Diacyl-PC production was abolished by an inhibitor of lecithin-cholesterol acyltransferase (LCAT). When d13-PGPC-preloaded LDL was incubated with HDL, d13-PGPC was transferred to HDL in a dose-dependent manner when both LCAT and lipoprotein-associated phospholipase A2 (Lp-PLA2) were inhibited. Lp-PLA2 in both HDL and LDL was responsible for the hydrolysis of d13-PGPC. These results suggest that short chain-oxPC and lysoPC can transfer between lipoproteins quickly and can be enzymatically converted from oxPC to lysoPC and from lysoPC to diacyl-PC in the presence of HDL.

VAMP7 Regulates Autophagosome Formation by Supporting Atg9a Functions in Pancreatic ß-Cells From Male Mice.

Dysfunctional mitochondria are observed in ß-cells of diabetic patients, which are eventually removed by autophagy. Vesicle-associated membrane protein (VAMP)7, a vesicular SNARE protein, regulates autophagosome formation to maintain mitochondrial homeostasis and control insulin secretion in pancreatic ß-cells. However, its molecular mechanism is largely unknown. In this study, we investigated the molecular mechanism of VAMP7-dependent autophagosome formation using VAMP7-deficient ß-cells and ß-cell-derived Min6 cells. VAMP7 localized in autophagy-related (Atg)9a-resident vesicles of recycling endosomes (REs), which contributed to autophagosome formation, and it interacted with Hrb, Syntaxin16, and SNAP-47. Hrb recruited VAMP7 and Atg9a from the plasma membrane to REs. Syntaxin16 and SNAP-47 mediated autophagosome formation at a step later than the proper localization of VAMP7 to Atg9a-resident vesicles. Knockdown of Hrb, Syntaxin16, and SNAP-47 resulted in defective autophagosome formation, accumulation of dysfunctional mitochondria, and impairment of glucose-stimulated insulin secretion. Our data indicate that VAMP7 and Atg9a are initially recruited to REs to organize VAMP7 and Atg9a-resident vesicles in an Hrb-dependent manner. Additionally, VAMP7 forms a SNARE complex with Syntaxin16 and SNAP-47, which may cause fusions of Atg9a-resident vesicles during autophagosome formation. Thus, VAMP7 participates in autophagosome formation by supporting Atg9a functions that contribute to maintenance of mitochondrial quality.

γ-Taxilin temporally regulates centrosome disjunction in a Nek2A-dependent manner.

Never in mitosis A-related kinase 2A (Nek2A), a centrosomal serine/threonine kinase, is involved in mitotic progression by regulating the centrosome cycle. Particularly, Nek2A is necessary for dissolution of the intercentriole linkage between the duplicated centrosomes prior to mitosis. Nek2A activity roughly parallels its cell cycle-dependent expression levels, but the precise mechanism regulating its activity remains unclear. In this study, we found that γ-taxilin co-localized with Nek2A at the centrosome during interphase and interacted with Nek2A in yeast two-hybrid and pull-down assays and that γ-taxilin regulated centrosome disjunction in a Nek2A-dependent manner. γ-Taxilin depletion increased the number of cells with striking splitting of centrosomes. The precocious splitting of centrosomes induced by γ-taxilin depletion was attenuated by Nek2A depletion, suggesting that γ-taxilin depletion induces the Nek2A-mediated dissolution of the intercentriole linkage between the duplicated centrosomes nevertheless mitosis does not yet begin. Taken together with the result that γ-taxilin protein expression levels were decreased at the onset of mitosis, we propose that γ-taxilin participates in Nek2A-mediated centrosome disjunction as a negative regulator through its interaction with Nek2A.

ß-Taxilin participates in differentiation of C2C12 myoblasts into myotubes.

Myogenesis is required for the development of skeletal muscle. Accumulating evidence indicates that the expression of several genes are upregulated during myogenesis and these genes play pivotal roles in myogenesis. However, the molecular mechanism underlying myogenesis is not fully understood. In this study, we found that ß-taxilin, which is specifically expressed in the skeletal muscle and heart tissues, was progressively expressed during differentiation of C2C12 myoblasts into myotubes, prompting us to investigate the role of ß-taxilin in myogenesis. In C2C12 cells, knockdown of ß-taxilin impaired the fusion of myoblasts into myotubes, and decreased the diameter of myotubes. We also found that ß-taxilin interacted with dysbindin, a coiled-coil-containing protein. Knockdown of dysbindin conversely promoted the fusion of myoblasts into myotubes and increased the diameter of myotubes in C2C12 cells. Furthermore, knockdown of dysbindin attenuated the inhibitory effect of ß-taxilin depletion on myotube formation of C2C12 cells. These results demonstrate that ß-taxilin participates in myogenesis through suppressing the function of dysbindin to inhibit the differentiation of C2C12 myoblasts into myotubes.

TSG101, a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression.

BACKGROUND: Tumor susceptibility gene 101 (TSG101) was initially identified in fibroblasts as a tumor suppressor gene but subsequent studies show that TSG101 also functions as a tumor-enhancing gene in some epithelial tumor cells. Although previous studies have unraveled diverse biological functions of TSG101, the precise mechanism by which TSG101 is involved in carcinogenesis and tumor progression in a bidirectional and multifaceted manner remains unclear. METHODS: To reveal the mechanism underlying bidirectional modulation of cell invasion by TSG101, we used RNA interference to examine whether TSG101 depletion bidirectionally modulated matrix metalloproteinase (MMP)-9 expression in different cell types. RESULTS: TSG101 depletion promoted cell invasion of HT1080 cells but contrarily reduced cell invasion of HeLaS3 cells. In HT1080 cells, TSG101 depletion increased both baseline and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 secretion through enhancing MMP-9 mRNA expression, but did not affect the expression or activation of MMP-2. In contrast, TSG101 depletion decreased PMA-induced MMP-9 secretion through reducing MMP-9 mRNA expression in HeLaS3 cells. TSG101 depletion had little impact on the signaling pathways required for the activation of transcription of MMP-9 or MMP-9 mRNA stability in either cell line. CONCLUSION: TSG101 bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression in different cell types. Our results provide a mechanistic context for the role of TSG101 in cell invasion as a multifaceted gene.

Sphingomyelin Regulates the Activity of Secretory Phospholipase A2 in the Plasma Membrane.

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and the activity of secretory phospholipase A2 (sPLA2 ) using two Chinese hamster ovary (CHO)-K1 cell mutants, LY-B and LY-A cells, deficient in sphingolipid synthesis. In LY-B cells, deficiency of sphingolipids enhanced the release of AA induced by bee venom sPLA2-III or human sPLA2-V. These alterations were reversed by replenishment of exogenous sphingomyelin (SM). In LY-A cells, deficiency of SM increased the release of AA induced by sPLA2. In CHO-K1 cells, decrease and increase of SM level in the plasma membrane by pharmacological methods increased and inhibited the release of AA, respectively. SM inhibited the activity of sPLA2 in vitro. Niemann-Pick disease type C (NPC) is a lysosomal storage disorder caused by mutation of either the NPC1 or NPC2 gene, and is characterized by accumulation of cholesterol and sphingolipids including SM in late endosomes/lysosomes. Increased levels of AA and sPLA2 activity are involved in various neurodegenerative diseases. In CHO cells lacking NPC1 (A101 cells), SM level was lower in the plasma membrane, while it was higher in late endosomes/lysosomes. The release of AA induced by sPLA2 was increased in A101 cells than that in parental cells (JP17 cells), which was attenuated by adding exogenous SM. In addition, sPLA2 -III-induced cytotoxicity in A101 cells was much higher than that in JP17 cells. These results suggest that SM in the plasma membrane plays important roles in regulating sPLA2 activity and the enzyme-induced cytotoxicity in A101 cells.

Trafficking of Acetyl-C16-Ceramide-NBD with Long-Term Stability and No Cytotoxicity into the Golgi Complex.

The Golgi complex plays a prominent role in the modification and sorting of lipids and proteins, and is a highly dynamic organelle that is dispersed and rearranged before and after mitosis. Several reagents including 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-C6-ceramide, a ceramide having an NBD-bound C6-N-acyl chain) and Golgi-specific proteins that emit fluorescence are used as Golgi markers. In the present study, we synthesized a new ceramide analog, acetyl-C16-ceramide-NBD (a ceramide having an acetylated C-1 hydroxyl group, C16-N-acyl chain, and NBD-bound C15-sphingosine), and showed that it preferentially accumulated in the Golgi complex without cytotoxicity for over 24 h. Pathways for cellular uptake and interorganelle trafficking of acetyl-C16-ceramide-NBD were investigated. Acetyl-C16-ceramide-NBD was transported to the Golgi complex via ceramide transport proteins. In contrast to NBD-C6-ceramide, acetyl-C16-ceramide-NBD was resistant to ceramide metabolic enzymes such as sphingomyelin synthase and glucosylceramide synthase. Because of its weaker cytotoxicity and resistance to ceramide metabolic enzymes, the localization of the Golgi complex could be observed in acetyl-C16-ceramide-NBD-labeled cells before and after mitosis.

Lactosylceramide interacts with and activates cytosolic phospholipase A2α.

Lactosylceramide (LacCer) is a member of the glycosphingolipid family and is known to be a bioactive lipid in various cell physiological processes. However, the direct targets of LacCer and cellular events mediated by LacCer are largely unknown. In this study, we examined the effect of LacCer on the release of arachidonic acid (AA) and the activity of cytosolic phospholipase A2α (cPLA2α). In CHO-W11A cells, treatment with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthase, reduced the glycosphingolipid level, and the release of AA induced by A23187 or platelet-activating factor was inhibited. The addition of LacCer reversed the PPMP effect on the stimulus-induced AA release. Exogenous LacCer stimulated the release of AA, which was decreased by treatment with an inhibitor of cPLA2α or silencing of the enzyme. Treatment of CHO-W11A cells with LacCer induced the translocation of full-length cPLA2α and its C2 domain from the cytosol to the Golgi apparatus. LacCer also induced the translocation of the D43N mutant of cPLA2α. Treatment of L929 cells with TNF-α induced LacCer generation and mediated the translocation of cPLA2α and AA release, which was attenuated by treatment with PPMP. In vitro studies were then conducted to test whether LacCer interacts directly with cPLA2α. Phosphatidylcholine vesicles containing LacCer increased cPLA2α activity. LacCer bound to cPLA2α and its C2 domain in a Ca(2+)-independent manner. Thus, we propose that LacCer is a direct activator of cPLA2α.

C2-di-ethyl-ceramide-1-phosphate as an inhibitor of group IVA cytosolic phospholipase A2.

Ceramide-1-phosphate (C1P) has been shown to bind with C2 domain in group IVA cytosolic phospholipase A(2) (cPLA(2)α, PLA2G4A) and activate the enzyme activity directly. In cells, C1P causes translocation of cPLA(2)α to perinuclear regions including the Golgi complex by interacting with C2 domain in the enzyme, and then cPLA(2)α releases arachidonic acid from substrate phospholipids in the regions. In this study, we synthesized new di-ethyl (DE) phosphate ester analogs of C1P with N-acyl chains of different lengths, and examined their effects on cPLA(2)α. A DE-C1P analog with a C2-N-acyl chain (C2-DE-C1P), but not DE-C1P analogs with longer N-acyl chain, such as C6- and C16-DE-C1P, inhibited release of arachidonic acid via cPLA(2)α activation in CHO-W11A cells expressing platelet-activating factor (PAF) receptors without changing secretory phospholipase A(2)-induced release. Treatment with C2-DE-C1P did not modify phosphorylation of extracellular signal-regulated kinase 1/2 and cPLA(2)α and increase of intracellular Ca(2+) level induced by PAF, but inhibited Ca(2+)- and PAF-induced accumulation of cPLA(2)α in the Golgi complex. Phosphatidylcholine vesicles containing C2-DE-C1P reduced cPLA(2)α activity in vitro. C2-DE-C1P disturbed the binding of the enzyme to glycerophospholipids in the lipid-protein overlay assay, and the reagent alone did not bind to the enzyme. Interestingly, C2-DE-C1P inhibited neither Ca(2+)- and PAF-induced accumulation of C2 domain of cPLA(2)α in the Golgi complex nor binding of cPLA(2)α to C16-C1P. These results suggest that C2-DE-C1P appeared to inhibit cPLA(2)α, probably by interaction with a site in the catalytic domain of the enzyme, not with the site in C2 domain responsible for native C1P.

Arachidonic acid metabolism via cytosolic phospholipase A2 α induces cytotoxicity in niemann-pick disease type C cells.

Niemann-Pick disease type C (NPC) is a neurodegenerative lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids. Here, we report that NPC1-deficient Chinese hamster ovary cells exhibit increased release of arachidonic acid (AA) and synthesis of prostaglandin E(2) compared with wild-type cells. The enhanced release of AA was inhibited by both treatment with the selective inhibitor of cytosolic phospholipase A(2) α (cPLA(2) α) and cultivation in lipoprotein-deficient medium. There was no difference in the expression of both cyclooxygenase-1 and -2 between NPC cells and wild-type cells. U18666A, a cholesterol transport-inhibiting agent commonly used to mimic NPC, also increased the release of AA in L929 mouse fibrosarcoma cells. Furthermore, U18666A-induced formation of reactive oxygen species (ROS) resulted in the induction of cell death and cell cycle delay/arrest in L929 cells. Interestingly, these responses induced by U18666A were much weaker in cPLA(2) α knockdown L929 cells. These results suggest that cPLA(2) α-AA pathway plays important roles in the cytotoxicity and the ROS formation in NPC cells.

Inhibitory effects of clinical reagents having anti-oxidative activity on transforming growth factor-ß1-induced expression of α-smooth muscle actin in human fetal lung fibroblasts.

Excessive production of transforming growth factor-ß1 (TGFß1) plays an important role in lung fibrosis, in which the differentiation of fibroblasts into myofibroblasts is a key process. Increased formation of reactive oxygen species (ROS) induced by TGFß1 is a common pathological feature of fibrosis. In the present study, probucol and lovastatin, which are therapeutics used for hyperlipidemia and proposed to act as anti-oxidants, were examined in terms of their effect on TGFß1-induced formation of ROS and expression of α-smooth muscle actin (αSMA), a myofibroblast marker, in human fetal lung fibroblasts (HFL1 cells). The effects of anti-oxidative enzymes and reagents including N-acetyl-L-cysteine, α-tocopherol, and lecithinized-superoxide dismutase (SOD) on TGFß1-induced expression of αSMA were also examined. Treatment with probucol (30 µM) and lovastatin (1 µM and 3 µM), in addition to lecithinized-SOD, significantly inhibited the TGFß1-induced formation of ROS and αSMA. Other anti-oxidants including N-acetyl-L-cysteine had marginal effects on αSMA expression under the conditions. Probucol and lovastatin, established therapeutics, may be worth trying in patients with both lung fibrosis and hypercholesterolemia.

Role of cytosolic phospholipase A(2)α in cell rounding and cytotoxicity induced by ceramide-1-phosphate via ceramide kinase.

Ceramide-1-phosphate (C1P), produced by ceramide kinase (CERK), is implicated in the regulation of many biological functions including cell growth and inflammation. C1P is a direct activator of group IVA cytosolic phospholipsase A(2) (PLA2G4A or cPLA(2)α). Although activation of the CERK-C1P pathway causes mitogenic and cytoprotective responses in many cells, the pathway shows cytotoxicity in several cells and the precise mechanism has not been elucidated. In the present study, we examined the effect of human CERK (hCERK) expression on cytotoxicity in two cell lines. Expression of hCERK in CHO cells caused cell rounding and lactate dehydrogenase (LDH) leakage, and co-addition of ceramide enhanced these responses. Expression of hCERK enhanced C1P formation and release of arachidonic acid in Ca(2+) ionophore-stimulated cells. Treatment with 20µM C2-C1P for 24 h caused cell rounding, and the response was significantly decreased by an inhibitor of cPLA(2)α. In L929 cells, expression of hCERK with and without ceramide caused cell rounding and LDH leakage, respectively, and the responses were significantly less in a stable clone of L929 cells lacking cPLA(2)α. These findings suggest the involvement of cPLA(2)α in CERK-C1P pathway-induced cytotoxicity.

Newly synthetic ceramide-1-phosphate analogs; their uptake, intracellular localization, and roles as an inhibitor of cytosolic phospholipase A(2)α and inducer of cell toxicity.

Ceramide-1-phosphate (C1P) regulates cellular functions including arachidonic acid (AA) metabolism and modulates cell fate. The mechanism by which C1P is taken up is unclear, and the development of lipophilic analogs may be useful for regulating C1P's actions. We synthesized new mono- and di-methyl-ester (MM and DM, respectively) analogs of C1P with N-acyl chains of different lengths, and examined their effects on AA release and cell toxicity. Short-N-acyl-DM-C1P analogs including C5- and C6-DM-C1P, but not long-N-acyl-DM-C1P analogs, inhibited the release of AA mediated by α type cytosolic phospholipase A(2) (cPLA(2)α) in Chinese hamster ovary (CHO) cells and the enzymatic activity. Short-N-acyl-DM-C1P analogs including C6-DM-C1P caused morphological changes with cell toxicity 24h after the treatment in three cells lines (CHO, L929, and RLC-18 cells), although the role of AA in the toxicity was not clear. Neither long-N-acyl-DM-C1P analogs nor MM-C1P analogs including C6-MM-C1P affected cPLA(2)α activity and cell toxicity. Similar to C6-ceramide having a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) group on a C6-N-acyl chain (NBD-C6-ceramide), NBD-C6-DM-C1P and C6-DM-C1P-NBD (with a C6-N-acyl chain and an NBD-labeled C14-alkyl chain) were accumulated in the Golgi complex, although less C6-DM-C1P-NBD than NBD-C6-DM-C1P was taken up. NBD-C6-ceramide was converted to various metabolites including NBD-C6-sphingomyelin, but both NBD-C6-DM-C1P and C6-DM-C1P-NBD were stable in cells within 2h. The short-N-acyl-DM-C1P analogs acted directly as an inhibitor of cPLA(2)α and an inducer of cell toxicity, and may be useful for the regulation of ceramide/C1P-regulated responses.

Effects of ceramide, ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2alpha; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling.

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A(2) (PLA(2)) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca(2+) binding regions in the C2 domain of alpha type cytosolic PLA(2) (cPLA(2)alpha), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA(2)alpha has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4beta-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca(2+) ionophore (A23187) -induced release of AA via cPLA(2)alpha's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca(2+) nor the phosphorylation of cPLA(2)alpha in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA(2)alpha that is independent of the Ca(2+) signal and the PKC-ERK-mediated phosphorylation signal.