Activation of the sphingomyelin cycle by brefeldin A: effects of brefeldin A on differentiation and implications for a role for ceramide in regulation of protein trafficking.

The sphingomyelin (SM) cycle is an emerging pathway of signal transduction that plays a role in the control of cell growth, cell differentiation, and apoptosis. During earlier investigation of SM pools hydrolyzed in the SM cycle, we examined the effects of the fungal macrolide brefeldin A (BFA) on cellular levels of SM in HL-60 leukemia cells. We found that BFA induced up to 20-25% hydrolysis of SM. Here we show that this BFA-sensitive SM pool corresponds to the pool of SM hydrolyzed by a previously discovered activator of the SM cycle, 1,25-dihydroxyvitamin D3. BFA was also able to induce the biological end points of SM cycle activation: growth inhibition and differentiation. Reciprocally, ceramide inhibited the secretion of 35S-labeled proteins from HL-60 cells and induced a subset of effects of BFA on organelle morphology. Since a ceramide-activated protein phosphatase has been previously suggested as a direct in vitro target of ceramide action, the effects of modulators of protein kinases and phosphatases were examined. Okadaic acid enhanced protein secretion and was able to oppose the effects of both ceramide and BFA on organelle morphology. Dioctanoylglycerol and phorbol myristate acetate, known activators of protein kinase C, were also found to oppose the inhibitory actions of ceramide on secretion. These studies identify BFA as an activator of the SM cycle, with ceramide as a potential mediator of some of the effects of BFA. Additionally, taken with the effects of the PKC activators, these studies suggest that constitutive protein secretion is not a default pathway but is subject to regulation by processes of signal transduction.

Identification of a distinct pool of sphingomyelin involved in the sphingomyelin cycle.

Sphingomyelin (SM) is a membrane phosphosphingolipid that has recently been identified as a key component of the SM cycle. In this signal transduction pathway, extracellular inducers such as tumor necrosis factor alpha cause hydrolysis of membrane SM, resulting in the generation of the lipid second messenger ceramide. Only 10-20% of cellular SM appears to be involved in the SM cycle, raising the possibility of the existence of a unique "signaling" pool of SM. The existence and subcellular location of such a pool were investigated. Using bacterial sphingomyelinase from Staphylococcus aureus (bSMase), we first characterized two pools of SM, identified as an outer leaflet bSMase-sensitive pool and a distinct bSMase-resistant pool. These pools were further characterized by their differential solubility in Triton X-100 and by their kinetics of labeling. The signaling pool of SM was distinguished by the following: 1) resistance to bSMase, 2) solubility in Triton X-100, and 3) delayed labeling kinetics. In subfractionation studies, the signaling pool of SM co-fractionated with the plasma membrane. Since the SM cycle involves a cytosolic sphingomyelinase and the intracellular release of choline phosphate, this pool of SM appears to localize to the inner leaflet of the plasma membrane (or to a closely related compartment). These results identify a unique signaling pool of SM that undergoes significant hydrolysis (20-40%) in response to inducers of the SM cycle.

Identification of arachidonic acid as a mediator of sphingomyelin hydrolysis in response to tumor necrosis factor alpha.

A sphingomyelin (SM)-signaling cycle has been described in human leukemia-derived HL-60 cells (Okazaki, T., Bell, R.M., and Hannun, Y.A. (1989) J. Biol. Chem. 264, 19076-19080). Activation of the cycle by tumor necrosis factor alpha (TNF alpha) occurs rapidly, with peak levels of approximately 30% SM hydrolysis observed within 45-60 min. The mechanisms by which TNF alpha induces this SM turnover remain largely unexplored. In this study, arachidonic acid (AA) was investigated as a potential mediator of TNF alpha effects on SM turnover. In HL-60 cells, 30 nM TNF alpha stimulated the release of AA within 5-10 min. In turn, AA stimulated SM hydrolysis and concomitant ceramide generation within 20 min of addition to cells. Other fatty acids, notably oleate, mimicked the effects of AA on SM hydrolysis, but the methyl ester and alcohol analogs of fatty acids were inactive. Diacylglycerol, a candidate mediator of TNF alpha responses, AA activated a cytosolic sphingomyelinase dose dependently, with 10-100 microM AA including 3-4-fold activation, thus suggesting a direct effect of AA on sphingomyelinase. Melittin, a potent phospholipase A2 activator, induced SM hydrolysis at concentrations as low as 35 nM. However, unlike AA, melittin was unable to stimulate sphingomyelinase activation in an in vitro assay system. Finally, exogenous addition of AA also produced antiproliferative effects reminiscent of ceramide effects. Thus, a role for the phospholipase A2/AA pathway in mediating TNF alpha induction of the SM cycle is supported by multiple lines of evidence. These studies begin to elucidate a mechanism of TNF alpha signaling and identify a close relationship between glycerophospholipid and sphingolipid signaling. AA, therefore, may be pivotal to understanding the sphingomyelin-signaling cascade.

Sphingolipid breakdown products: anti-proliferative and tumor-suppressor lipids.

The sphingolipids are a family of lipids found ubiquitously in eukaryotic cell membranes. Within the last decade sphingolipids have emerged as active participants in the regulation of cell growth, differentiation, transformation, and cell-cell contact. A prototypic sphingolipid signalling pathway is the 'sphingomyelin cycle,' in which membrane sphingomyelin is hydrolyzed in response to extracellular stimuli, generating the putative second messenger ceramide. Ceramide, in turn, is thought to propagate the signal into the cell interior by the activation of a phosphatase. It is likely that other sphingolipids are components of similar signalling cycles, generating a variety of lipid messengers which participate in as yet undefined pathways. Sphingosine, for example, is a potential breakdown product of all sphingolipids, and is well-known for its pharmacologic inhibition of protein kinase C. However, it is becoming apparent that sphingosine is active in multiple signalling cascades that are independent of protein kinase C, including effects on fibroblast cell growth and the regulation of the retinoblastoma tumor suppressor protein. Similarly, lyso-sphingolipids, while comprising only a minor fraction of the cell's total sphingolipids, are turning out to have biological effects which warrant their investigation as potential signalling molecules. A distinguishing characteristic of sphingolipid breakdown products is their apparent participation in anti-proliferative pathways of cell regulation. Thus, sphingolipid breakdown products can be found to play roles in growth inhibition, induction of differentiation, and programmed cell death. In coordination with other cellular signal transduction pathways, the sphingolipid breakdown products may be the harnesses on cell growth and may also contribute to the suppression of oncogenesis.

Programmed cell death induced by ceramide.

Sphingomyelin hydrolysis and ceramide generation have been implicated in a signal transduction pathway that mediates the effects of tumor necrosis factor-alpha (TNF-alpha) and other agents on cell growth and differentiation. In many leukemic cells, TNF-alpha causes DNA fragmentation, which leads to programmed cell death (apoptosis). C2-ceramide (0.6 to 5 microM), a synthetic cell-permeable ceramide analog, induced internucleosomal DNA fragmentation, which was inhibited by zinc ion. Other amphiphilic lipids failed to induce apoptosis. The closely related C2-dihydroceramide was also ineffective, which suggests a critical role for the sphingolipid double bond. The effects of C2-ceramide on DNA fragmentation were prevented by the protein kinase C activator phorbol 12-myristate 13-acetate, which suggests the existence of two opposing intracellular pathways in the regulation of apoptosis.

Ceramide-mediated biology. Determination of structural and stereospecific requirements through the use of N-acyl-phenylaminoalcohol analogs.

Ceramide is a postulated intracellular modulator of cell growth and differentiation (Okazaki, T., Bielawska, A., Bell, R.M., and Hannun, Y. A. (1990) J. Biol. Chem. 265, 15823-15831). In order to determine the structural and stereospecific requirements for ceramide effects on HL-60 cells, N-acyl-phenylaminoalcohol analogs were synthesized and evaluated for their ability to mimic the effects of ceramide on cell proliferation and differentiation. These compounds share with ceramide a similar polar headgroup that allows the investigation of the roles of the primary and secondary hydroxyls, the hydrophobicity of the molecule, and stereospecificity. N-Myristoyl derivatives of phenylamino alcohols showed optimal activity over other chain length analogs and were able to mimic the effects of C2-ceramide on cell growth and differentiation. Neither the primary nor the secondary alcohol was necessary for activity, but the amide-linked acyl chain was required. Stereospecificity of action was demonstrated with an enantiomeric pair: D-erythro-N-myristoyl-2-amino-1-phenyl-1-propanol (C14-D-e-APP-1) and L-erythro-N-myristoyl-2-amino-1-phenyl-1-propanol (C14-L-e-APP-1). The D stereoisomer was as effective as C2-ceramide in inhibiting HL-60 cell growth and in inducing cell differentiation, whereas the L enantiomer lacked activity in both assays. These results suggest stereospecific action of ceramide and strongly support a physiologic role for ceramide as an intracellular mediator with primary roles in regulation of cell growth and differentiation.

Brefeldin A promotes hydrolysis of sphingomyelin.

The hydrolysis of sphingomyelin (SM) is a key reaction in the "sphingomyelin cycle," which plays a role in the regulation of cell proliferation and differentiation (Okazaki, T., Bell, R. M., and Hannun, Y. A. (1989) J. Biol. Chem. 264, 19076-19080). SM is produced from endoplasmic reticulum-derived ceramide and is delivered to organelle membranes in a regulated manner, presumably through the same endomembrane trafficking system used for sorting and delivery of proteins. Since brefeldin A (BFA) interferes with this endomembrane trafficking system and thus alters normal membrane and organelle distribution, we investigated the effect of BFA on SM levels in HL-60 leukemia cells. BFA caused a dose-dependent decrease of 20-25% in cellular SM levels, with effects observed at concentrations of BFA as low as 0.10 microgram/ml. BFA effects on SM levels were noted as early as 5 min and were maximal by 20 min, with no further SM hydrolysis observed up to 60 min following treatment with BFA, suggesting the presence of a fixed SM-sensitive pool. BFA did not cause SM hydrolysis at 16 degrees C, a temperature that inhibits the effects of BFA on endomembrane mixing. The very early effects and temperature dependence of BFA-induced SM hydrolysis suggest that the mechanism of hydrolysis may be closely related to endomembrane mixing. These studies are beginning to define important interrelationships between membrane trafficking and topology, SM metabolism, and cell regulation.

Modulation of cell growth and differentiation by ceramide.

Ceramide has been suggested as an intracellular modulator of cell growth and differentiation [Okazaki, T. et al. (1990) J. Biol. Chem. 265, 15823-15831]. In this study, parameters that modulate the effects of ceramide on HL-60 cell growth and differentiation were examined. A short-chain, cell-permeable analog of ceramide, C2-ceramide, induced differentiation of HL-60 human leukemia cells and inhibited HL-60 growth in a concentration-dependent manner. The potency of C2-ceramide was modulated by the starting cell density such that the concentration of C2-ceramide producing 50% inhibition of cell growth (IC50%) ranged from 2 microM (for cells suspended at 1 x 10(5) cells/ml) to 11 microM (for cells at 8 x 10(5) cells/ml). However, the IC50% showed little variation if the concentration of C2-ceramide was expressed as fmol of C2-ceramide per 10(5) cells. Therefore, the effectiveness of C2-ceramide appeared to be primarily determined by its cellular rather than molar concentration. Binding of C2-ceramide to serum proteins resulted in a 10-fold increase in the IC50%. These results demonstrate that the biologic activity of C2-ceramide is subject to surface dilution kinetics and is sensitive to the presence of lipid-binding proteins. In these properties, ceramide behaves as a prototypic lipid second messenger/intracellular mediator.