Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell differentiation.

The biochemical signaling mechanisms involved in transducing the effects of tumor necrosis factor alpha (TNF alpha) and gamma-interferon (gamma-IFN) on leukemia cell differentiation are poorly defined. Recent studies established the existence of a sphingomyelin cycle that operates in response to the action of vitamin D3 on HL-60 cells and that may transduce the effects of vitamin D3 on cell differentiation (Okazaki, T., Bell, R., and Hannun, Y. (1989) J. Biol. Chem. 264, 19076-19080). The effects of TNF alpha and gamma-IFN on sphingomyelin turnover were determined, and the specificity and role of sphingomyelin hydrolysis in HL-60 human promyelocytic leukemia cells with 20% hydrolysis of sphingomyelin at 15 min, 40% hydrolysis at 30-60 min, and return to base line at 2 h. The hydrolyzed sphingomyelin (18 pmol/nmol total phospholipid) was accompanied by the concomitant generation of ceramide (11.2 pmol/nmol total phospholipid). gamma-IFN also caused reversible hydrolysis of sphingomyelin with onset at 1 h and peak effect at 2 h. This sphingomyelin cycle appeared to be specific to the monocytic pathway of HL-60 differentiation, since it was not activated by retinoic acid or dibutyryl cAMP, inducers of granulocytic differentiation, nor with phorbol myristate acetate, an inducer of macrophage-like differentiation. Addition of synthetic ceramide or bacterial sphingomyelinase induced monocytic differentiation of HL-60 cells. Cell-permeable ceramide also caused prompt down-regulation of mRNA for the c-myc protooncogene. The time course of c-myc down-regulation was consistent with the action of ceramide as the mediator of TNF alpha action. These results suggest that sphingomyelin turnover may be an important signaling mechanism transducing the actions of TNF alpha and gamma-IFN with specific function in cell differentiation.     

Curcumin induces apoptosis of multidrug-resistant human leukemia HL60 cells by complex pathways leading to ceramide accumulation

Most anti-cancer agents induce apoptosis, however, a development of multidrug resistance in cancer cells and defects in apoptosis contribute often to treatment failure. Here, the mechanism of curcumin-induced apoptosis was investigated in human leukemia HL60 cells and their HL60/VCR multidrug-resistant counterparts. In both cell lines curcumin induced a bi-phasic ceramide generation with a slow phase until 6 h followed by a more rapid one. The level of the ceramide accumulation correlated inversely with the cell viability. We found that the ceramide elevation resulted from multifarious changes of the activity of sphingolipid-modifying enzymes. In both cell lines curcumin induced relatively fast activation of neutral sphingomyelinase (nSMase), which peaked at 3 h, and was followed by inhibition of sphingomyelin synthase activity. In addition, in HL60/VCR cells the glucosylceramide synthase activity was diminished by curcumin. This process was probably due to curcumin-induced down-regulation of P-gp drug transporter, since cyclosporine A, a P-gp blocker, also inhibited the glucosylceramide synthase activity. Inhibition of nSMase activity with GW4869 or silencing of SMPD3 gene encoding nSMase2 reversed the curcumin-induced inhibition of sphingomyelin synthase without affecting the glucosylceramide synthase activity. The early ceramide generation by nSMase was indispensable for the later lipid accumulation, modulation of Bax, Bcl-2 and caspase 3 levels, and for reduction of cell viability in curcumin-treated cells, as all these events were inhibited by GW4869 or nSMase2 depletion. These data indicate that the early ceramide generation by nSMase2 induced by curcumin intensifies the later ceramide accumulation via inhibition of sphingomyelin synthase, and controls pro-apoptotic signaling.     

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.     

Daunorubicin- and Ara-C-induced interphasic apoptosis of human type II leukemia cells is caspase-8-independent

Drug-induced interphasic apoptosis in human leukemia cells is mediated through intracellular signaling pathways, of which the most proximal (initiating) event remains unclear. Indeed, both early ceramide generation and procaspase-8 cleavage have been individually identified as the initial apoptotic signaling events which precede the mitochondrial control of the apoptotic execution phase in Type II cells. In order to evaluate whether or not procaspase-8 cleavage is requisite for initial ceramide generation and rapid interphasic apoptosis, we investigated the chronological ordering of early ceramide generation and caspase-8 cleavage induced by daunorubicin (DNR) and 1-beta-D-arabinofuranosylcytosine (Ara-C) in U937 cells. We further evaluated the impact of these two drugs on initial ceramide generation and apoptosis in wild-type Jurkat cells and Jurkat clones mutated for caspase-8 and Fas-associated death domain. We show that while both DNR and Ara-C similarly induced early ceramide generation (within 5-20 min) and interphasic apoptosis in all cell models, caspase-8 cleavage was only observed farther downstream (4.5 h) and only in DNR-treated cells. Furthermore, neither DNR or Ara-C induced caspase-8 activation. These results demonstrate that caspase-8 cleavage is not requisite for the drug-induced activation of the ceramide-mediated interphasic apoptotic pathway in human Type II leukemic cells.     

De novo ceramide synthesis is responsible for the anti-tumor properties of camptothecin and doxorubicin in follicular thyroid carcinoma

Doxorubicin and camptothecin are two cytotoxic chemotherapeutic agents triggering apoptosis in various cancer cells, including thyroid carcinoma cells. Recent studies revealed a critical role of ceramide in chemotherapy and suggested that anti-cancer drugs may kill tumor cells through sphingomyelinase activation. However, in comparison to sphingomyelin hydrolysis, the relative involvement of de novo ceramide synthesis remained poorly explored and highly controversial. Here, we evidenced that both doxorubicin and camptothecin triggered ceramide accumulation in thyroid carcinoma cells. We demonstrated that ceramide increase occurred via the de novo pathway without neither acidic nor neutral sphingomyelinase contribution. Interestingly, de novo ceramide generation was responsible for the drug-induced malignant cell apoptosis through a caspase-3-dependent pathway and a decrease of thrombospondin amount. Furthermore, blocking ceramide metabolism by inhibiting glucosylceramide synthase strengthened the camptothecin and doxorubicin-dependent effects. Altogether, we evidenced that de novo ceramide synthesis mediates the anti-tumor properties of doxorubicin and camptothecin in thyroid carcinoma and suggested that glucosylation of ceramide may contribute to the drug-resistance phenotype in thyroid malignancies.     

JNK3 signaling pathway activates ceramide synthase leading to mitochondrial dysfunction

A cardinal feature of brain tissue injury in stroke is mitochondrial dysfunction leading to cell death, yet remarkably little is known about the mechanisms underlying mitochondrial injury in cerebral ischemia/reperfusion (IR). Ceramide, a naturally occurring membrane sphingolipid, functions as an important second messenger in apoptosis signaling and is generated by de novo synthesis, sphingomyelin hydrolysis, or recycling of sphingolipids. In this study, cerebral IR-induced ceramide elevation resulted from ceramide biosynthesis rather than from hydrolysis of sphingomyelin. Investigation of intracellular sites of ceramide accumulation revealed the elevation of ceramide in mitochondria because of activation of mitochondrial ceramide synthase via post-translational mechanisms. Furthermore, ceramide accumulation appears to cause mitochondrial respiratory chain damage that could be mimicked by exogenously added natural ceramide to mitochondria. The effect of ceramide on mitochondria was somewhat specific; dihydroceramide, a structure closely related to ceramide, did not inflict damage. Stimulation of ceramide biosynthesis seems to be under control of JNK3 signaling: IR-induced ceramide generation and respiratory chain damage was abolished in mitochondria of JNK3-deficient mice, which exhibited reduced infarct volume after IR. These studies suggest that the hallmark of mitochondrial injury in cerebral IR, respiratory chain dysfunction, is caused by the accumulation of ceramide via stimulation of ceramide synthase activity in mitochondria, and that JNK3 has a pivotal role in regulation of ceramide biosynthesis in cerebral IR.     

Alpha particles induce apoptosis through the sphingomyelin pathway

The sphingomyelin pathway involves the enzymatic cleavage of sphingomyelin to produce ceramide, a second messenger that serves as a key mediator in the rapid apoptotic response to various cell stressors. Low-linear energy transfer (LET) γ radiation can initiate this pathway, independent of DNA damage, via the cell membrane. Whether short-ranged, high-LET α particles, which are of interest as potent environmental carcinogens, radiotherapies and potential components of dirty bombs, can act through this mechanism to signal apoptosis is unknown. Here we show that irradiation of Jurkat cells with α particles emitted by the 22?Ac-DOTA-anti-CD3 IgG antibody construct results in dose-dependent apoptosis. This apoptosis was significantly reduced by pretreating cells with cholesterol-depleting nystatin, a reagent known to inhibit ceramide signaling by interfering with membrane raft coalescence and ceramide-rich platform generation. The effects of nystatin on α-particle-induced apoptosis were related to disruption of the ceramide pathway and not to microdosimetry alterations, because similar results were obtained after external irradiation of the cells with a broad beam of collimated α particles using a planar 2?1Am source. External irradiation allowed for more precise control of the dosimetry and geometry of the irradiation, independent of antibody binding or cell internalization kinetics. Mechanistically consistent with these findings, Jurkat cells rapidly increased membrane concentrations of ceramide after external irradiation with an average of five α-particle traversals per cell. These data indicate that α particles can activate the sphingomyelin pathway to induce apoptosis.     

Fetal asphyxia induces acute and persisting changes in the ceramide metabolism in rat brain

Fetal asphyctic preconditioning, induced by a brief episode of experimental hypoxia-ischemia, offers neuroprotection to a subsequent more severe asphyctic insult at birth. Extensive cell stress and apoptosis are important contributing factors of damage in the asphyctic neonatal brain. Because ceramide acts as a second messenger for multiple apoptotic stimuli, including hypoxia/ischemia, we sought to investigate the possible involvement of the ceramide pathway in endogenous neuroprotection induced by fetal asphyctic preconditioning. Global fetal asphyxia was induced in rats by clamping both uterine and ovarian vasculature for 30 min. Fetal asphyxia resulted in acute changes in brain ceramide/sphingomyelin metabolic enzymes, ceramide synthase 1, 2, and 5, acid sphingomyelinase, sphingosine-1-phosphate phosphatase, and the ceramide transporter. This observation correlated with an increase in neuronal apoptosis and in astrocyte number. After birth, ceramide and sphingomyelin levels remained high in fetal asphyxia brains, suggesting that a long-term regulation of the ceramide pathway may be involved in the mechanism of tolerance to a subsequent, otherwise lethal, asphyctic event.     

Apoptosis induced by capsaicin in prostate PC-3 cells involves ceramide accumulation,neutral sphingomyelinase,and JNK activation

Numerous studies have recently focused on the anticarcinogenic, antimutagenic, or chemopreventive activities of the main pungent component of red pepper, capsaicin (N-vanillyl-8-methyl-1-nonenamide). We have previously shown that, in the androgen-independent prostate cancer PC-3 cells, capsaicin inhibits cell growth and induces apoptosis through reactive oxygen species (ROS) generation [Apoptosis 11 (2006) 89–99]. In the present study, we investigated the signaling pathways involved in the antiproliferative effect of capsaicin. Here, we report that capsaicin apoptotic effect was mediated by ceramide generation which occurred by sphingomyelin hydrolysis. Using siRNA, we demonstrated that N-SMase expression is required for the effect of capsaicin on prostate cell viability. We then investigated the role of MAP kinase cascades, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, in the antiproliferative effect of capsaicin, and we confirmed that capsaicin could activate ERK and JNK but not p38 MAPK. Pharmacological inhibition of JNK kinase, as well as inhibition of ROS by the reducing agent N-acetylcysteine, prevented ceramide accumulation and capsaicin-induced cell death. However, inhibition of ceramide accumulation by the SMase inhibitor D609 did not modify JNK activation. These data reveal JNK as an upstream regulator of ceramide production. Capsaicin-promoted activation of ERK was prevented with all the inhibitors tested. We conclude that capsaicin induces apoptosis in PC-3 cells via ROS generation, JNK activation, ceramide accumulation, and second, ERK activation.     

Involvement of sphingomyelinases in TNF signaling pathways

Sphingomyelin (N-acylsphingosin-1-phosphorylcholine) is a phospholipid preferentially found in the plasma membrane of mammalian cells. Signaling through the sphingomyelin pathway is associated with generation of ceramide, which acts as a second messenger in activating a variety of cellular functions. Ceramide belongs to the group of sphingosine-based lipid second messenger molecules that are critically involved in the regulation of signal transduction of diverse cell surface membrane receptors. The emerging picture suggests that coupling of ceramide to specific signaling cascades is both stimulus- and cell type-specific and depends on the subcellular topology of its production. Following membrane receptor triggering, neutral and acid isoforms of sphingomyelinases are rapidly activated generating ceramide through sphingomyelin hydrolysis. Here the molecular mechanisms of TNF-induced activation of sphingomyelinases and the functional consequences of ceramide generation will be discussed.     

4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2

We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (ΔΨm), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.     

Ceramide accumulation is independent of camptothecin-induced apoptosis in prostate cancer LNCaP cells

We have investigated to determine the source of ceramide produced during the genotoxic apoptosis induced by the anti-cancer drug, camptothecin (CPT), in human prostate cancer LNCaP cells by measuring the activities of acid and neutral sphingomyelinases (SMase) and by using fumonisinB(1) (FB(1)), the inhibitor of ceramide synthase involving de novo synthesis of ceramide. In contrast to time-dependent elevation of intracellular ceramide level after CPT-treatment, the activities of both SMases were not increased but rather decreased. Instead, pretreatment for 3 h with FB(1) (100 microM), an inhibitor of ceramide synthase, almost completely abrogated ceramide accumulation observed in cells exposed to CPT for 18 h. These results indicate that ceramide is produced via de novo pathway but not via sphingomyelin hydrolysis pathway. Furthermore, it is to be noted that the pretreatment with FB(1) did not affect the CPT-induced apoptosis as assessed by DNA ladder formation, Hoechst 33342 staining, flow cytometry, and mitochondrial potential thereby leading us to propose that ceramide accumulation is independent of apoptosis in this system.     

L-carnitine prevents doxorubicin-induced apoptosis of cardiac myocytes: role of inhibition of ceramide generation.

Besides the well-documented effect of the chemotherapeutic drug doxorubicin on free radical generation, the exact signaling mechanisms by which it causes cardiac damage remain largely unknown and are of fundamental importance in understanding anthracycline cardiotoxicity. In this study, we describe that a 1 h treatment of isolated adult rat cardiac myocytes with doxorubicin (0.5 microM) induced DNA fragmentation associated with the classical morphological features of apoptosis observed after 7 days of culture. The doxorubicin toxicity was preceded by an increase in intracellular ceramide levels with a concurrent decrease in sphingomyelin. Anthracycline-induced ceramide accumulation resulted from the activation of a sphingomyelinase assayed under acidic conditions, an effect related to an increase in V(max). Pretreatment of cardiac myocytes with L-carnitine (200 microgram/ml), a compound known for its protective effect on cardiac metabolic injuries, was found to dose-dependently inhibit the doxorubicin-induced sphingomyelin hydrolysis and ceramide generation as well as subsequent cell death. However, L-carnitine did not protect cardiac myocytes from apoptosis induced by exogenous cell-permeant ceramide. L-carnitine pretreatment did not affect the sphingomyelinase basal activity but abolished the doxorubicin-induced increase in V(max). Moreover, in vitro studies conducted on cell extracts or with purified acid sphingomyelinase demonstrated that L-carnitine exerted a dose-dependent, sphingomyelinase inhibitory effect (through V(max) reduction). Taken together, these findings show that by inhibiting a (perhaps novel) drug-activated acid sphingomyelinase and ceramide generation, L-carnitine can prevent doxorubicin-induced apoptosis of cardiac myocytes.     

The role of sphingomyelin and sphingomyelin synthases in cell death,proliferation and migration—from cell and animal models to human disorders

Sphingomyelin constitutes membrane microdomains such as lipid raft, caveolae, and clathrin-coated pits and implicates in the regulation of trans-membrane signaling. On the other hand, sphingomyelin emerges as an important molecule to generate bioactive sphingolipids through ceramide. Sphingomyelin synthase is an enzyme that generates sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide. Although ceramide has a well-known role as a lipid mediator to regulate cell death and survival, the only known biological role of sphingomyelin regulated by sphingomyelin synthases was limited to being a source of bioactive lipids. Here, we describe the basic characters of sphingomyelin synthases and discuss additional roles for sphingomyelin and sphingomyelin synthase in biological functions including cell migration, apoptosis, autophagy, and cell survival/proliferation as well as in human disorders such as cancer and cardiovascular disorders. It is expected that a better understanding of the role of sphingomyelin regulated by sphingomyelin synthase will shed light on new mechanisms in cell biology, physiology and pathology. In the future, novel therapeutic procedures for currently incurable diseases could be developed through modifying the function of not only sphingolipids, such as sphingomyelin and ceramide, but also of their regulatory enzymes. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Different vitamin D analogues induce sphingomyelin hydrolysis and apoptosis in the human keratinocyte cell line HaCaT.

Sphingomyelin hydrolysis seems to be a ubiquitous pathway generating ceramide, an important cell response modifier. Upon agonist-stimulation this pathway is linked to biological responses as inhibition of proliferation, promotion of differentiation and induction of apoptosis. One of the agonists described is 1alpha,25-dihydroxyvitamin D3. Recently, we could demonstrate the existence of sphingomyelin hydrolysis in human primary keratinocytes as well as in the human keratinocyte cell line HaCaT after treatment with 1alpha,25-dihydroxyvitamin D3. In the present study we tested four vitamin D analogues on HaCaT keratinocytes for their ability to inhibit cell proliferation and to induce sphingomyelin hydrolysis. These analogues, calcipotriol, EB 1213, GS 1500 and tacalcitol inhibit cell growth after 48 hrs. of incubation and trigger the hydrolysis of sphingomyelin. Moreover, all analogues tested induce apoptotic cell death in HaCaT keratinocytes after 24 hrs. of incubation. This study indicates that sphingomyelin hydrolysis, subsequently leading to the elevation of cellular ceramide levels, may represent an important signal transduction pathway for 1alpha,25-dihydroxyvitamin D3 and its analogues in human keratinocytes. Possible differences of the mechanism underlying vitamin D-induced sphingomyelin hydrolysis has to be studied in more detail and may contribute to the antipsoriatic action of these analogues.     

Ceramide stimulates a cytosolic protein phosphatase.

A sphingomyelin cycle has been identified whereby the action of certain extracellular agents results in reversible sphingomyelin hydrolysis and the concomitant generation of ceramide. Moreover, a cell-permeable ceramide, C2-ceramide (N-acetylsphingosine), is a potent modulator of cell proliferation and differentiation. We report herein that C2-ceramide, C6-ceramide, and natural ceramides activate a cytosolic serine/threonine protein phosphatase in a dose-dependent manner. Initial activation is observed at concentrations of ceramide as low as 0.1 microM with peak response occurring at 5-10 microM. However, other closely related sphingolipids, sphingosine and sphingomyelin, were largely inactive. Ceramide-stimulated phosphatase was inhibited by okadaic acid, an inhibitor of protein phosphatases, with an IC50 of 0.1-1 nM, depending on the concentration of ceramide. Ceramide-stimulated phosphatase was insensitive to Mg2+ and Mn2+ cations. Using sequential anion exchange chromatography, ceramide-stimulated phosphatase activity could be resolved from ceramide-nonresponsive phosphatases. The activity of partially purified enzyme was stimulated 3.5-fold by ceramide. The identification of a phosphatase as a molecular target for the action of ceramide defines a novel intracellular signaling pathway with potential roles in the regulation of cell proliferation and differentiation.     

Sphingomyelin metabolites in vascular cell signaling and atherogenesis

The atherosclerotic lesion most probably develops through a number of cellular events which implicate all vascular cell types and include synthesis of extracellular proteins, cell proliferation, differentiation and death. Sphingolipids and sphingolipid metabolizing enzymes may play important roles in atherogenesis, not only because of lipoprotein alterations but also by mediating a number of cellular events which are believed to be crucial in the development of the vascular lesions such as proliferation or cell death. Exogenous sphingolipids may mediate various biological effects such as apoptosis, mitogenesis or differentiation depending on the cell type. Moreover, several molecules present in the atherogenic lesion, such as oxidized LDL, growth factors or cytokines, which activate intracellular signaling pathways leading to vascular cell modifications, can stimulate sphingomyelin hydrolysis and generation of ceramide (and other metabolites as sphingosine-1-phosphate). Here we review the potential implication of the sphingomyelin/ceramide cycle in vascular cell signaling related to atherosclerosis, and more generally the role of sphingolipids in the events observed during the atherosclerotic process as cell differentiation, migration, adhesion, retraction, proliferation and death.     

Differential Effects of Ceramide and Sphingosine 1-Phosphate on ERM Phosphorylation: PROBING SPHINGOLIPID SIGNALING AT THE OUTER PLASMA MEMBRANE*

ERM proteins are regulated by phosphorylation of the most C-terminal threonine residue, switching them from an activated to an inactivated form. However, little is known about the control of this regulation. Previous work in our group demonstrated that secretion of acid sphingomyelinase acts upstream of ERM dephosphorylation, suggesting the involvement of sphingomyelin (SM) hydrolysis in ERM regulation. To define the role of specific lipids, we employed recombinant bacterial sphingomyelinase (bSMase) as a direct probe of SM metabolism at the plasma membrane. bSMase induced a rapid dose- and time-dependent decrease in ERM dephosphorylation. ERM dephosphorylation was driven by ceramide generation and not by sphingomyelin depletion, as shown using recombinant sphingomyelinase D. The generation of ceramide at the plasma membrane was sufficient for ERM regulation, and no intracellular SM hydrolysis was required, as was visualized using Venus-tagged lysenin probe, which specifically binds SM. Interestingly, hydrolysis of plasma membrane bSMase-induced ceramide using bacterial ceramidase caused ERM hyperphosphorylation and formation of cell surface protrusions. The effects of plasma membrane ceramide hydrolysis were due to sphingosine 1-phosphate formation, as ERM phosphorylation was blocked by an inhibitor of sphingosine kinase and induced by sphingosine 1-phosphate. Taken together, these results demonstrate a new regulatory mechanism of ERM phosphorylation by sphingolipids with opposing actions of ceramide and sphingosine 1-phosphate. The approach also defines a tool kit to probe sphingolipid signaling at the plasma membrane.