Acid sphingomyelinase mediates 50-Hz magnetic field-induced EGF receptor clustering on lipid raft

Previously, we found that exposure to a 50-Hz magnetic field (MF) could induce epidermal growth factor receptor (EGFR) clustering and phosphorylation on cell surface. In order to explore the possible mechanisms, the roles of acid sphingomyelinase (ASMase) and lipid raft in MF-induced EGFR clustering were investigated in the present study. Human amnion epithelial (FL) cells were exposed to a 50-Hz MF at 0.4?mT for different durations. Intracellular ASMase activity was detected using the Amplex® Red Sphingomyelinase Assay Kit. EGFR clustering, ASMase, and lipid rafts on cell membrane were analyzed using confocal microscopy after indirect immunofluorescence staining. Results showed that disturbing lipid rafts with nystatin could inhibit MF-induced EGFR clustering, indicating that it was dependent on intact lipid raft. Exposure of FL cells to MF significantly enhanced ASMase activity and induced ASMase translocation to membrane that co-localized with lipid rafts. Treatment with imipramine, an ASMase inhibitor, inhibited the MF-induced EGFR clustering. This inhibitory effect could be blocked by the addition of C2-ceramide in the culture medium. It suggested that ASMase mediated the 50-Hz MF-induced EGFR clustering via ceramide which was produced from hydrolyzation on lipid rafts.     

Induced endoplasmic reticulum (ER) stress and binding of over-expressed ER specific chaperone GRP78/BiP with dimerized epidermal growth factor receptor in mammalian cells exposed to low concentration of N-methyl-N'-nitro-N-nitrosoguanidine

Previously we have shown that alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) can induce the clustering of epidermal growth factor receptor (EGFR) in human amnion FL cells. However, the biological consequence of MNNG-induced clustering is different from that of epidermal growth factor (EGF)-induced clustering. In addition, MNNG strongly blocks the autophosphorylation of EGFR in response to its ligand, we speculate it might be due to the altered conformation of EGFR by MNNG alkylation, or the binding of some unknown suppressive molecules to EGFR, which could lead to the down-regulation of EGFR pathway. In this study, we further demonstrated that EGFR could not be phosphorylated by EGF in lysates prepared from MNNG-pretreated cell. In addition, it was found that the clustering of EGFR induced by low concentration (相似文献   

非律平菌素干扰甲基硝基亚硝基胍对FL细胞鞘脂代谢的影响

甲基硝基亚硝基胍（MNNG）可通过脂筏诱导细胞表面受体聚簇并激活NF-κB信号通路.本研究拟探讨脂筏干扰剂非律平菌素（filipin）对MNNG作用的影响.利用脂类组学方法分别研究了MNNG、filipin 单独处理及先用filipin再用MNNG处理情况下对人羊膜FL细胞鞘脂代谢的影响,用MALDI-TOF质谱法分析细胞鞘脂组成的变化,酶联免疫吸附法检测NF-κB通路的活化,RT-PCR法检测鞘脂代谢通路中关键酶的表达.结果表明,MNNG和filipin都可影响FL细胞鞘脂类代谢,但MNNG作用更显著.Filipin预处理可部分抑制MNNG对细胞鞘脂类代谢的影响,且能够抑制MNNG对NF-κB的活化;但filipin、MNNG单独或联合处理都不影响鞘脂代谢关键酶丝氨酸棕榈酰转移酶、酸性鞘磷脂酶和鞘磷脂合成酶在mRNA水平的表达.以上结果说明,filipin预处理会导致甲基硝基亚硝基胍引起FL细胞鞘脂代谢以及NF-κB活性的改变.而可能的机制在于,filipin破坏脂筏结构从而引起一系列信号途径的改变,而非通过改变脂类代谢关键酶的表达.     

Ceramide-rich membrane rafts mediate CD40 clustering.

Many receptor systems use receptor clustering for transmembrane signaling. In this study, we show that acid sphingomyelinase (ASM) is essential for the clustering of CD40. Stimulation of lymphocytes via CD40 ligation results in ASM translocation from intracellular stores, most likely vesicles, into distinct membrane domains on the extracellular surface of the plasma membrane. Surface ASM initiates a release of extracellularly oriented ceramide, which in turn mediates CD40 clustering in sphingolipid-rich membrane domains. ASM, ceramide, and CD40 colocalize in the cap-like structure of stimulated cells. Deficiency of ASM, destruction of sphingolipid-rich rafts, or neutralization of surface ceramide prevents CD40 clustering and CD40-initiated cell signaling. These findings indicate that the ASM-mediated release of ceramide and/or metabolites of ceramide regulate clustering of CD40, which seems to be a prerequisite for cellular activation via CD40.     

N-methyl-N'-nitro-N-nitrosoguanidine interferes with the epidermal growth factor receptor-mediated signaling pathway

Many environmental factors, such as ultraviolet (UV) and arsenic, can induce the clustering of cell surface receptors, including epidermal growth factor receptor (EGFR). This is accompanied by the phosphorylation of the receptors and the activation of ensuing cellular signal transduction pathways, which are implicated in the various cellular responses caused by the exposure to these factors. In this study, we have shown that N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, also induced the clustering of EGFR in human amnion FL cells, which was similar in morphology to that of epidermal growth factor treatment. However, MNNG treatment did not activate Ras, the downstream mediator in EGFR signaling pathway, as compared to EGF treatment. The autophosphorylation of tyrosine residues Y1068 and Y1173 at the intracellular domain of EGFR, which is related to Ras activation under EGF treatment, was also not observed by MNNG exposure. Interestingly, although MNNG did not affect the binding of EGF to EGFR, MNNG can interfere with EGF function. For instance, pre-incubating FL cells with MNNG inhibited the autophosphorylation of EGFR by EGF treatment, as well as the activation of Ras. In addition, the phosphorylation of Y845 on EGFR by EGF, which is mediated through c-Src or related kinases but not autophosphorylation, was also affected by MNNG. Therefore, MNNG may influence the tyrosine kinase activity as well as the phosphorylation of EGFR through its interaction with EGFR.     

CD95 signaling via ceramide-rich membrane rafts

Clustering seems to be employed by many receptors for transmembrane signaling. Here, we show that acid sphingomyelinase (ASM)-released ceramide is essential for clustering of CD95. In vitro and in vivo, extracellularly orientated ceramide, released upon CD95-triggered translocation of ASM to the plasma membrane outer surface, enabled clustering of CD95 in sphingolipid-rich membrane rafts and apoptosis induction. Whereas ASM deficiency, destruction of rafts, or neutralization of surface ceramide prevented CD95 clustering and apoptosis, natural ceramide only rescued ASM-deficient cells. The data suggest CD95-mediated clustering by ceramide is prerequisite for signaling and death.     

Cetuximab enhances TRAIL-induced gastric cancer cell apoptosis by promoting DISC formation in lipid rafts

TRAIL is a member of the tumor necrosis factor family that selectively induces cancer cell apoptosis. However, gastric cancer cells are insensitive to TRAIL. Our and others studies showed that the inhibition of EGFR pathway activation could increase the sensitivity of TRAIL in cancer cells. But the detailed mechanism is not fully understood. In the present study, compared with TRAIL or cetuximab (an anti-EGFR monoclonal antibody) alone, treatment with the TRAIL/cetuximab combination significantly promoted death receptor 4 (DR4) clustering as well as the translocation of both DR4 and Fas-associated death domain-containing protein (FADD) into lipid rafts. This in turn resulted in caspase-8 cleavage and the formation of the death-inducing signaling complex (DISC) in these lipid rafts. Cholesterol-depletion with methyl-β-cyclodextrin partially prevented DR4 clustering and DISC formation, and thus partially reversed apoptosis induced by the TRAIL/cetuximab dual treatment. These results indicate that cetuximab increases TRAIL-induced gastric cancer cell apoptosis at least partially through the promotion of DISC formation in lipid rafts.     

Ceramide and cell death receptor clustering

Acid sphingomyelinase (ASM) has been shown to be activated by a variety of receptor molecules and stimuli including CD95, the tumor necrosis factor receptor (TNF-R), CD40, CD28, LFA-1, CD5, during development, irradiation, heat shock, UV light or bacterial and viral infections. The central role of ASM-released ceramide in the response to those stimuli is confirmed by several genetic studies. ASM and ceramide might mediate their biological effects by the activation of several intracellular signaling molecules including cathepsin D, phospholipase A(2) or the kinase suppressor of Ras. In addition, recent fluorescence microscopy studies indicate that distinct, small membrane domains, termed rafts, are modified by ceramide to form larger domains, which serve to cluster receptor molecules. The generation of a high receptor density might be required for initiation of receptor-specific signaling and explain the function of the ASM and ceramide in multiple signaling pathways.     

Channel formation by the glycosylphosphatidylinositol-anchored protein binding toxin aerolysin is not promoted by lipid rafts

Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-beta-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin.     

A Lipidomic Study of the Effects of N-methyl-N′-nitro-N-nitrosoguanidine on Sphingomyelin Metabolism

Systems biology is a new and rapidly developing research area in which,by quantitativelydescribing the interaction among all the individual components of a cell,a systems-level understanding of abiological response can be achieved.Therefore,it requires high-throughput measurement technologies forbiological molecules,such as genomic and proteomic approaches for DNA/RNA and protein,respectively.Recently,a new concept,lipidomics,which utilizes the mass spectrometry(MS)method for lipid analysis,has been proposed.Using this lipidomic approach,the effects of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)on sphingomyelin metabolism,a major class of sphingolipids,were evaluated.Sphingomyelin moleculeswere extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS.Itwas found that MNNG induced profound changes in sphingomyelin metabolism,including the appearance ofsome new sphingomyelin species and the disappearance of some others,and the concentrations of severalsphmgomyelin species also changed.This was accompanied by the redistribution of acid sphingomyelinase(ASM),a key player in sphingomyelin metabolism.On the other hand,imipramine,an inhibitor of ASM,caused the accumulation of sphingomyelin.It also prevented some of the effects of MNNG,as well as theredistribution of ASM.Taken together,these data suggested that the lipidomic approach is highly effectivefor the systematic analysis of cellular lipids metabolism.     

A Lipidomic Study of the Effects of N-methyl-N＇-nitro-N-nitrosoguanidine on Sphingomyelin Metabolism

Systems biology is a new and rapidly developing research area in which, by quantitatively describing the interaction among all the individual components of a cell, a systems-level understanding of a biological response can be achieved. Therefore, it requires high-throughput measurement technologies for biological molecules, such as genomic and proteomic approaches for DNA/RNA and protein, respectively.Recently, a new concept, lipidomics, which utilizes the mass spectrometry （MS） method for lipid analysis,has been proposed. Using this lipidomic approach, the effects of N-methyl-N＇-nitro-N-nitrosoguanidine （MNNG） on sphingomyelin metabolism, a major class of sphingolipids, were evaluated. Sphingomyelin molecules were extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS. It was found that MNNG induced profound changes in sphingomyelin metabolism, including the appearance of some new sphingomyelin species and the disappearance of some others, and the concentrations of several sphingomyelin species also changed. This was accompanied by the redistribution of acid sphingomyelinase （ASM）, a key player in sphingomyelin metabolism. On the other hand, imipramine, an inhibitor of ASM,caused the accumulation of sphingomyelin. It also prevented some of the effects of MNNG, as well as the redistribution of ASM. Taken together, these data suggested that the lipidomic approach is highly effective for the systematic analysis of cellular lipids metabolism.     

Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.     

Sphingolipid signalling domains floating on rafts or buried in caves?

Ceramide is a novel lipid mediator involved in regulating cell growth, cell differentiation and cell death. Many studies have focused on characterizing the stimulus-induced production of ceramide and identifying putative downstream molecular targets. However, little remains known about the localization of the regulated production of ceramide through sphingomyelin metabolism in the plasma membrane. Additionally, it is unclear whether a localized increase in ceramide concentration is necessary to facilitate downstream signalling events initiated by this lipid. Recent studies have suggested that detergent-insoluble plasma membrane domains may be highly localized sites for initiating signal transduction cascades by both tyrosine kinase and sphingolipid signalling pathways. These domains are typically enriched in both sphingolipids and cholesterol and have been proposed to form highly ordered lipid rafts floating in a sea of glycerophospholipids. Alternatively, upon integration of the cholesterol binding protein caveolin, these domains may also form small cave-like structures called caveolae. Emerging evidence suggests that the enhanced sphingomyelin content of these lipid domains make them potential substrate pools for sphingomyelinases to produce a high local concentration of ceramide. The subsequent formation of ceramide microdomains in the plasma membrane may be a critical factor in regulating downstream signalling through this lipid messenger.     

Molecular mechanisms of ceramide-mediated CD95 clustering.

Receptor clustering has been suggested as a crucial mechanism to initiate receptor signaling. Here we show that ceramide in sphingolipid-rich membrane rafts mediates clustering of CD95. Neutralization of surface ceramide or inhibition of its endogenous generation prevented CD95 clustering. Furthermore, application of ceramide at the cell surface triggered clustering of active but not inactive CD95. Apoptosis was inhibited by neutralization of surface ceramide or inhibition of ceramide release in vitro and in vivo. Thus, we conclude that surface ceramide mediates CD95 clustering, which is required for initiation of apoptosis, at least in some cell types.     

Cell surface ceramide generation precedes and controls FcgammaRII clustering and phosphorylation in rafts

Despite the role of sphingolipid/cholesterol rafts as signaling platforms for Fcgamma receptor II (FcgammaRII), the mechanism governing translocation of an activated receptor toward the rafts is unknown. We show that at the onset of FcgammaRII cross-linking acid sphingomyelinase is rapidly activated. This enzyme is extruded from intracellular compartments to the cell surface, and concomitantly, exofacially oriented ceramide is produced. Both non-raft and, to a lesser extent, raft sphingomyelin pools were hydrolyzed at the onset of FcgammaRII cross-linking. The time course of ceramide production preceded the recruitment of FcgammaRII to rafts and the receptor phosphorylation. Exogenous C(16)-ceramide facilitated clustering of FcgammaRII and its association with rafts. In contrast, inhibition of acid sphingomyelinase diminished both the ceramide generation and clustering of cross-linked FcgammaRII. Under these conditions, tyrosine phosphorylation of FcgammaRII and receptor-accompanying proteins was also reduced. All the inhibitory effects were bypassed by treatment of cells with exogenous ceramide. These data provide evidence that the generation of cell surface ceramide is a prerequisite for fusion of cross-linked FcgammaRII and rafts, which triggers the receptor tyrosine phosphorylation and signaling.     

Sphingolipids and cell signaling: Involvement in apoptosis and atherogenesis

This review considers various functional aspects of cell sphingolipids (sphingomyelin, ceramides) and lysosphingolipids (sphingosine-1-phosphate (S1P) and sphingosine phosphorylcholine). Good evidence now exists that they are actively involved in numerous cell-signaling processes. The enzymes responsible for formation and interconversion of cell sphingolipids (sphingomyelinases, ceramidase, sphingosine kinase, S1P-lyase) exhibit high sensitivity to various stimulating factors. This determines the content of individual cell sphingolipids and therefore the mode of cell response. Special attention is paid to preferential localization of sphingolipids in the rigid plasma membrane domains (rafts) coupled to many signal proteins. The suggestion is discussed that ceramide signaling may be based on the modification of fine molecular interactions in lipid rafts, resulting in its clusterization inducing the signal transduction. The review also highlights involvement of sphingolipids in cell proliferation, apoptosis, and in processes implicated to atherosclerosis.     

EGF receptor exposed to oxidative stress acquires abnormal phosphorylation and aberrant activated conformation that impairs canonical dimerization

Crystallographic studies have offered understanding of how receptor tyrosine kinases from the ErbB family are regulated by their growth factor ligands. A conformational change of the EGFR (ErbB1) was shown to occur upon ligand binding, where a solely ligand-mediated mode of dimerization/activation was documented. However, this dogma of dimerization/activation was revolutionized by the discovery of constitutively active ligand-independent EGFR mutants. In addition, other ligand-independent activation mechanisms may occur. We have shown that oxidative stress (ox-stress), induced by hydrogen peroxide or cigarette smoke, activates EGFR differently than its ligand, EGF, thereby inducing aberrant phosphorylation and impaired trafficking and degradation of EGFR. Here we demonstrate that ox-stress activation of EGFR is ligand-independent, does not induce "classical" receptor dimerization and is not inhibited by the tyrosine kinase inhibitor AG1478. Thus, an unprecedented, apparently activated, state is found for EGFR under ox-stress. Furthermore, this activation mechanism is temperature-dependent, suggesting the simultaneous involvement of membrane structure. We propose that ceramide increase under ox-stress disrupts cholesterol-enriched rafts leading to EGFR re-localization into the rigid, ceramide-enriched rafts. This increase in ceramide also supports EGFR aberrant trafficking to a peri-nuclear region. Therefore, the EGFR unprecedented and activated conformation could be sustained by simultaneous alterations in membrane structure under ox-stress.     

Lipid mediators of autophagy in stress-induced premature senescence of endothelial cells

Our group (Patschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS; Am J Physiol Renal Physiol 294: F100-F109, 2008) previously observed an accumulation of gangliosides coincident with development of cell senescence and demonstrated lysosomal permeabilization in human umbilical vein endothelial cells exposed to glycated collagen I (GC). Therefore, we investigated whether the lysosome-dependent, caspase-independent or type 2-programmed cell death (autophagy) is involved in development of premature senescence of endothelial cells. The cleaved microtubule-associated protein 1 light-chain 3 (LC3), a marker of autophagosome formation, was overexpressed within 24 h of GC treatment; however, by 4-5 days, it was nearly undetectable. Early induction of autophagosomes was associated with their fusion with lysosomes, a phenomenon that later became subverted. Autophagic cell death can be triggered by the products of damaged plasma membrane, sphingolipids, and ceramide. We observed a clustering of membrane rafts shortly after exposure to GC; later, after 24 h, we observed an internalization, accompanied by an increased acid sphingomyelinase activity and accumulation of ceramide. Pharmacological inhibition of autophagy prevented development of premature senescence but did lead to the enhanced rate of apoptosis in human umbilical vein endothelial cells exposed to GC. Pharmacological induction of autophagy resulted in reciprocal changes. These observations appear to represent a mechanistic molecular cascade whereby advanced glycation end products like GC induce sphingomyelinase activity, accumulation of ceramide, clustering, and later internalization of lipid rafts.     

Glycosphingolipids and cell death

Sphingolipids have been implicated in various cellular processes including growth, cell-cell or ligand-receptor interactions, and differentiation. In addition to their importance as reservoirs of metabolites with important signaling properties, sphingolipids also help provide structural order to plasma membrane lipids and proteins within the bilayer. Glycosylated sphingolipids, and sphingomyelin in particular, are involved in the formation of lipid rafts. Although it is well accepted that ceramide, the backbone of all sphingolipids, plays a critical role in apoptosis, less is known about the biological functions of glycosphingolipids. This review summarizes current knowledge of the involvement of glycosphingolipids in cell death and in other pathological processes and diseases.