Richard Kolesnick

The Sphingomyelin Pathway in Tumor Necrosis Factor and Interleukin-1 Signaling

Richard Kolesnick and David W. Golde The Laboratories of Signal Transduction and Molecular and Cellular Hematology Memorial Sloan-Kettering Cancer Center New York, New York 10021 E:vidence suggests that tumor necrosis factor a (TNFa) and interleukin-18 (IL-18) employ the sphingomyelin path- way to effect signal transduction by their receptors. This pathway is initiated by hydrolysis of plasma membrane sphingomyelin to ceramide by the action of a sphingomye- linase. Ceramide serves as asecond messenger, stimulat- ing a serine/threonine ceramide-activated protein kinase to transduce the cytokine signal, in part through mitogen- activated protein (MAP) kinase and transcription factors such as NF-KB (Figure 1). The extent to which this signal- ing system is used in inflammation, immune responses, and apoptosis is not known, but accumulating evidence suggests that it is a commonly employed pathway that could be exploited therapeutically. The Sphingomyelin Metabolic Pathway Sphingomyelin is preferentially concentrated in the outer leaflet of the plasma membrane of most mammalian cells; it is comprised of a long chain sphingoid base backbone (predominantly sphingosine), a fatty acid, and a phospho- choline head group (Figure 2). The fatty acid in amide linkage at the second position of the sphingoid base consti- tutes ceramide. Hydrolysis of the phosphodiester bond by a sphingomyelinase to yield ceramide and phosphocho- line is the only clearly defined mechanism for sphingomye- lin degradation in mammalian cells (Kolesnick, 1991). Sphingomyelin was considered only a structural ele- ment of the plasma membrane. However, 1,2-diacylglycerol (DG), a physiologic activator of protein kinase C, stimu- lated rapid sphingomyelin degradation toceramide in GH3 rat pituitary cells (Kolesnick, 1991). Little of the generated ceramide was deacylated to sphingoid bases, potential inhibitors of protein kinase C (Hannun and Bell, 1989), prompting a search for additional derivatives of ceramide. Several investigations established the existence of a spe- cific metabolic pathway from sphingomyelin to ceramide l-phosphate (see Bajjalieh et al., 1989; Kolesnick, 1991). The sphingomyelin metabolic pathway is similar to the phosphoinositide signal transduction pathway. The cen- tral lipids in these pathways, ceramide and DG, both serve as substrates for the same bacterial DG kinase, implying they possess structural similarity. Their phosphorylated forms, ceramide l-phosphate and phosphatidic acid, were, therefore, also structurally similar. Further, neutral sphingomyelinase, the enzyme that initiates the sphingo- myelin pathway, is a phospholipase C concentrated in the plasma membrane (Kolesnick, 1991) like the enzyme ini- tiating the phosphoinositide pathway. Ceramide also ap- pears to be an ideal candidate second messenger since it readily redistributes across a membrane bilayer (Lipsky and Pagano, 1985). Since DG utilized a specific kinase, protein kinase C, for signaling, it was considered that cera- mide might stimulate a kinase. Ceramide does not activate protein kinase C. identification of Ceramidektivated Protein Kinase and Phosphatase Because natural ceramide contains long and very chain saturated or monounsaturated fatty acids and is poorly soluble in aqueous solutions, cell-permeable ana- logs were synthesized. A ceramide was constructed con- taining octanoic acid, N-octanoyl sphingosine (CS-cer), which is analogous to 1,2dioctanoylglyceroI, the hydro-

Ceramide synthase mediates daunorubicin-induced apoptosis: An alternative mechanism for generating death signals

The sphingomyelin pathway, which is initiated by sphingomyelin hydrolysis to generate the second messenger ceramide, signals apoptosis for tumor necrosis factor alpha, Fas, and ionizing radiation. In the present studies, the anticancer drug daunorubicin also stimulated ceramide elevation and apoptosis in P388 and U937 cells. Cell-permeable analogs of ceramide, but not other lipid second messengers, mimicked daunorubicin in inducing apoptosis. Daunorubicin-stimulated ceramide elevation, however, did not result from sphingomyelin hydrolysis, but rather from de novo synthesis via activation of the enzyme ceramide synthase. An obligatory role for ceramide synthase was defined, since its natural specific inhibitor, fumonisin B1, blocked daunorubicin-induced ceramide elevation and apoptosis. These studies demonstrate that ceramide synthase activity can be regulated in eukaryotes and constitute definitive evidence for a requirement for ceramide elevation in the induction of apoptosis.

Acid Sphingomyelinase–Deficient Human Lymphoblasts and Mice Are Defective in Radiation-Induced Apoptosis

Stress is believed to activate sphingomyelinase to generate ceramide, which serves as a second messenger in initiating the apoptotic response. Conclusive evidence for this paradigm, however, is lacking. In the present study, we used a genetic approach to address this issue directly. We show that lymphoblasts from Niemann-Pick patients, which have an inherited deficiency of acid sphingomyelinase activity, fail to respond to ionizing radiation with ceramide generation and apoptosis. These abnormalities are reversible up on restoration of acid sphingomyelinase activity by retroviral transfer of human acid sphingomyelinase cDNA. Acid sphingomyelinase knockout mice also expressed defects in radiation-induced ceramide generation and apoptosis in vivo. Comparison with p53 knockout mice revealed that acid sphingomyelinase-mediated apoptosis and p53-mediated apoptosis are likely distinct and independent. These genetic models provide definitive evidence for the involvement of acid sphingomyelinase in one form of stress-induced apoptosis.

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.

Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy.

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with sphingosine-1-phosphate resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with sphingosine-1-phosphate. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and sphingosine-1-phosphate provides a new approach to preserve ovarian function in vivo.

Signal transduction through lipid second messengers

This review emphasizes the generation of glycerolipid and sphingolipid second messengers, and their molecular targets. The role of the phosphatidylinositol transfer protein and phospholipase D in signal transmission, and the structures of the 1, 2-diacylglycerol and calcium-binding sites of protein kinase C are discussed. Further, ceramide signaling through protein kinases and the role of cross-talk in the signaling of apoptosis and inflammation are addressed.

Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts

Pseudomonas aeruginosa infection is a serious complication in patients with cystic fibrosis and in immunocompromised individuals. Here we show that P. aeruginosa infection triggers activation of the acid sphingomyelinase and the release of ceramide in sphingolipid-rich rafts. Ceramide reorganizes these rafts into larger signaling platforms that are required to internalize P. aeruginosa, induce apoptosis and regulate the cytokine response in infected cells. Failure to generate ceramide-enriched membrane platforms in infected cells results in an unabated inflammatory response, massive release of interleukin (IL)-1 and septic death of mice. Our findings show that ceramide-enriched membrane platforms are central to the host defense against this potentially lethal pathogen.

KINASE SUPPRESSOR OF RAS IS CERAMIDE-ACTIVATED PROTEIN KINASE

A proline-directed serine/threonine ceramide-activated protein (CAP) kinase mediates transmembrane signaling through the sphingomyelin pathway. CAP kinase reportedly initiates proinflammatory TNF alpha action by phosphorylating and activating Raf-1. The present studies delineate kinase suppressor of Ras (KSR), identified genetically in Caenorhabditis elegans and Drosophila, as CAP kinase. Mouse KSR, like CAP kinase, renatures and autophosphorylates as a 100-kDa membrane-bound polypeptide. KSR overexpression constitutively activates Raf-1. TNF alpha or ceramide analogs markedly enhance KSR autophosphorylation and its ability to complex with, phosphorylate, and activate Raf-1. In vitro, low nanomolar concentrations of natural ceramide stimulate KSR to autophosphorylate, and transactivate Raf-1. Other lipid second messengers were ineffective. Moreover, Thr269 the Raf-1 site phosphorylated by CAP kinase, is also recognized by KSR. Thus, by previously established criteria, KSR appears to be CAP kinase.

FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway

Fas receptor-induced apoptosis plays critical roles in immune homeostasis. However, most of the signal transduction events distal to Fas ligation have not been elucidated. Here, we show that Ras is activated following ligation of Fas on lymphoid lines. The activation of Ras is a critical component of this apoptotic pathway, since inhibition of Ras by neutralizing antibody or a dominant-negative Ras mutant interfered with Fas-induced apoptosis. Furthermore, ligation of Fas also resulted in stimulation of the sphingomyelin signalling pathway to produce ceramides, which, in turn, are capable of inducing both Ras activation and apoptosis. This suggests that ceramides acts as second messengers in Fas signaling via Ras. Thus, ligation of the Fas molecule on lymphocyte lines induces activation of Ras via the action of ceramide, and this activation is necessary, but not sufficient, for subsequent apoptosis.

Compartmentalization of ceramide signaling: physical foundations and biological effects

J. Cell. Physiol. 184:285–300, 2000.