Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation

Sphingosine, a metabolite of membrane sphingolipids, regulates proliferation of quiescent Swiss 3T3 fibroblasts (Zhang, H., N. E. Buckley, K. Gibson. and S. Spiegel. 1990. J. Biol. Chem. 265:76-81). The present study provides new insights into the formation and function of a unique phospholipid, a metabolite of sphingosine, which was unequivocally identified as sphingosine-1-phosphate. The rapid increase in 32P-labeled sphingosine-1-phosphate levels induced by sphingosine was concentration dependent and correlated with its effect on DNA synthesis. Similar to the mitogenic effects of sphingosine, low concentrations of sphingosine-1-phosphate stimulated DNA synthesis and induced pronounced morphological alterations. Both sphingosine and sphingosine-1-phosphate stimulated DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester and sphingosine still elicited similar increases in sphingosine-1-phosphate levels in these cells. Although both sphingosine and sphingosine-1-phosphate acted synergistically with a wide variety of growth factors, there was no additive or synergistic effect in response to a combination of sphingosine and sphingosine-1-phosphate. Using a digital imaging system for measurement of calcium changes, we observed that both sphingosine and sphingosine-1-phosphate are potent calcium-mobilizing agonists in viable 3T3 fibroblasts. The rapid rise in cytosolic free calcium was independent of the presence of calcium in the external medium, indicating that the response is due to the mobilization of calcium from internal store. Our results suggest that sphingosine-1-phosphate may be a component of the intracellular second messenger system that is involved in calcium release and the regulation of cell growth induced by sphingosine.     

Increases in phosphatidic acid levels accompany sphingosine-stimulated proliferation of quiescent Swiss 3T3 cells

Sphingosine, a breakdown product of cellular sphingolipids, has recently been shown to stimulate DNA synthesis and act synergistically with known growth factors to induce proliferation of quiescent Swiss 3T3 fibroblasts (Hong, Z., Buckley, N. E., Gibson, K., and Spiegel, S. (1990) J. Biol. Chem. 265, 76-81). The present study demonstrates that mitogenic concentrations of sphingosine induce early increases in cytosolic phosphatidic acid, which is a potent mitogen for Swiss 3T3 cells. Structurally related analogs of sphingosine, such as N-stearoylsphingosine and other long chain aliphatic amines, did not mimic the mitogenic effect of sphingosine and did not elevate phosphatidic acid levels. Sphingosine not only stimulated [3H]thymidine incorporation with similar efficiency and kinetics as phosphatidic acid, it also induced similar morphological alterations. Both sphingosine and phosphatidic acid acted synergistically with a variety of growth factors, such as, insulin, epidermal growth factor, fibroblast growth factor, and 12-O-tetradecanoylphorbol 13-acetate. In sharp contrast, sphingosine and phosphatidic acid did not have additive or synergistic effects in either the presence or absence of other growth factors. Both sphingosine and phosphatidic acid stimulated DNA synthesis in cells made protein kinase C-deficient by prolonged treatment with phorbol ester and sphingosine still stimulated similar increases in phosphtidic acid in these cells. Furthermore, similar to the actions of phosphatidic acid on signal transduction in Swiss 3T3 cells, mitogenic concentrations of sphingosine also inhibit cAMP accumulation and trigger the hydrolysis of polyphosphoinositides. Our findings indicate that sphingosine and phosphatidic acid control cellular responses in Swiss 3T3 cells through a common pathway. In view of the prominent role of phosphatidic acid in signal transduction and cellular proliferation, our observations that sphingosine, at mitogenic concentrations, increases the level of phosphatidic acid and also mimics the effects of phosphatidic acid on signal transduction, have important implications for the mechanism of action of sphingosine.     

Signaling pathways for sphingosylphosphorylcholine-mediated mitogenesis in Swiss 3T3 fibroblasts

Sphingosylphosphorylcholine (SPC), or lysophingomyelin, a wide-spectrum growth promoting agent for a variety of cell types (Desai, N. N., and S. Spiegel. 1991. Biochem. Biophys. Res. Comm. 181: 361-366), stimulates cellular proliferation of quiescent Swiss 3T3 fibroblasts to a greater extent than other known growth factors or than the structurally related molecules, sphingosine and sphingosine-1- phosphate. SPC potentiated the mitogenic effect of an activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate, and did not compete with phorbol esters for binding to protein kinase C in intact Swiss 3T3 fibroblasts. However, downregulation of protein kinase C, by prolonged treatment with phorbol ester, reduced, but did not eliminate, the ability of SPC to stimulate DNA synthesis, indicating that SPC may act via both protein kinase C-dependent and -independent signaling pathways. SPC induced a rapid rise in intracellular free calcium ([Ca2+]i) in viable 3T3 fibroblasts determined with a digital imaging system. Although the increases in [Ca2+]i were observed even in the absence of calcium in the external medium, no increase in the levels of inositol phosphates could be detected in response to mitogenic concentrations of SPC. Furthermore, in contrast to sphingosine or sphingosine-1-phosphate, the mitogenic effect of SPC was not accompanied by increases in phosphatidic acid levels or changes in cAMP levels. SPC, but not sphingosine or sphingosine-1-phosphate, stimulates the release of arachidonic acid. Therefore, the ability of SPC to act an extremely potent mitogen may be due to activation of signaling pathway(s) distinct from those used by sphingosine or sphingosine-1- phosphate.     

Sphingosylphosphorylcholine is a remarkably potent mitogen for a variety of cell lines

The effect of spingosylphosphorylcholine on cellular proliferation was investigated in a variety of cell types. Spingosylphosphorylcholine at low concentrations greatly stimulated DNA synthesis and cell division in quiescent Swiss 3T3 fibroblasts. The increased DNA synthesis was also accompanied by pronounced morphological alterations. Spingosylphosphorylcholine was remarkably more potent than other known growth factors and also acted synergistically with insulin, epidermal growth factor, fibroblast growth factor, and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to induce cellular proliferation. Sphingosylphosphorylcholine was less effective in stimulating DNA synthesis in rapidly growing normal and transformed cells. Spingosylphosphorylcholine appears to be a new type of potent, wide-spectrum growth promoting agent.     

Sphingosine-1-phosphate, a metabolite of sphingosine, increases phosphatidic acid levels by phospholipase D activation.

Sphingosine and sphingosine-1-phosphate, metabolites of membrane sphingolipids, have recently been shown to stimulate release of calcium from internal sources and to increase proliferation of quiescent Swiss 3T3 fibroblasts (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167). The present study demonstrates that mitogenic concentrations of sphingosine induce early increases in sphingosine-1-phosphate levels which precede the increase in the potent mitogen, phosphatidic acid. Sphingosine-1-phosphate itself induces a more rapid increase in phosphatidic acid, thus suggesting that it may mediate the effects of sphingosine on phosphatidic acid accumulation. The concentration dependence for the formation of phosphatidic acid induced by sphingosine-1-phosphate correlates with its effect on DNA synthesis. Similar to sphingosine, sphingosine-1-phosphate also stimulates the activity of phospholipase D, although a significant effect is observed at a much lower concentration. However, in contrast to previous reports with sphingosine, sphingosine-1-phosphate does not inhibit the phosphatidic acid phosphohydrolase activity in cell homogenates. Thus, in addition to its effect on mobilization of calcium, sphingosine-1-phosphate can increase the level of phosphatidic acid, most likely via activation of phospholipase D. We suggest that sphingosine-1-phosphate mediates the effect of sphingosine on phosphatidic acid accumulation in Swiss 3T3 fibroblasts and may regulate cellular proliferation by affecting multiple transmembrane signaling pathways.     

Sphingomyelinase amplifies BMP-4-induced osteocalcin synthesis in osteoblasts: role of ceramide

We previously reported that extracellular sphingomyelinase induces sphingomyelin hydrolysis in osteoblast-like MC3T3-E1 cells and that mitogen-activated protein (MAP) kinases are involved in bone morphogenetic protein (BMP)-4-stimulated osteocalcin synthesis in these cells. In the present study, we investigated whether sphingomyelinase affects BMP-4-stimulated synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. Sphingomyelinase significantly enhanced the BMP-4-stimulated osteocalcin synthesis. Among sphingomyelin metabolites, C(2)-ceramide enhanced the BMP-4-stimulated osteocalcin synthesis while sphingosine and sphingosine 1-phosphate had little effect on the synthesis. D-erythro-MAPP, an inhibitor of ceramidase, amplified the sphingomyelinase-effect on the osteocalcin synthesis. C(2)-ceramide suppressed the BMP-4-induced phosphorylation of p44/p42 MAP kinase, while having little effect on the phosphorylation of Smad1 and p38 MAP kinase. Taken together, our results strongly suggest that extracellular sphingomyelinase enhances the BMP-stimulated osteocalcin synthesis via ceramide in osteoblasts and that the effect of ceramide is exerted at a point upstream from p44/p42 MAP kinase.     

Role of sphingosine 1-phosphate in the mitogenesis induced by oxidized low density lipoprotein in smooth muscle cells via activation of sphingomyelinase, ceramidase, and sphingosine kinase.

Oxidized LDL (oxLDL) have been implicated in diverse biological events leading to the development of atherosclerotic lesions. We previously demonstrated that the proliferation of cultured vascular smooth muscle cells (SMC) induced by oxLDL is preceded by an increase in neutral sphingomyelinase activity, sphingomyelin turnover to ceramide, and stimulation of mitogen-activated protein kinases (Augé, N., Escargueil-Blanc, I., Lajoie-Mazenc, I., Suc, I., Andrieu-Abadie, N., Pieraggi, M. T., Chatelut, M., Thiers, J. C., Jaffrézou, J. P., Laurent, G., Levade, T., Nègre-Salvayre, A., and Salvayre, R. (1998) J. Biol. Chem. 273, 12893-12900). Since ceramide can be converted to other bioactive metabolites, such as the well established mitogen sphingosine 1-phosphate (S1P), we investigated whether additional ceramide metabolites are involved in the oxLDL-induced SMC proliferation. We report here that incubation of SMC with oxLDL increased the activities of both acidic and alkaline ceramidases as well as sphingosine kinase, and elevated cellular sphingosine and S1P. Furthermore, the mitogenic effect of oxLDL was inhibited by D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and N,N-dimethylsphingosine which are inhibitors of ceramidase and sphingosine kinase, respectively. These findings suggest that S1P is a key mediator of the mitogenic effect of oxLDL. In agreement with this conclusion, exogenous addition of sphingosine stimulated the proliferation of cultured SMC, and this effect was abrogated by dimethylsphingosine but not by fumonisin B1, an inhibitor of the acylation of sphingosine to ceramide. Exogenous S1P also promoted SMC proliferation. Altogether, these results strongly suggest that the mitogenic effect of oxLDL in SMC involves the combined activation of sphingomyelinase(s), ceramidase(s), and sphingosine kinase, resulting in the turnover of sphingomyelin to a number of sphingolipid metabolites, of which at least S1P is critical for mitogenesis.     

Involvement of neutral ceramidase in ceramide metabolism at the plasma membrane and in extracellular milieu

Neutral ceramidase is a type II integral membrane protein, which is occasionally secreted into the extracellular milieu after the processing of its N-terminal anchor. We found that when overexpressed in CHOP cells, neutral ceramidase hydrolyzed cell surface ceramide, which increased in amount after the treatment of cells with bacterial sphingomyelinase, leading to an increase in the cellular level of sphingosine and sphingosine 1-phosphate. On the other hand, knockdown of the endogenous enzyme by siRNA decreased the cellular level of both sphingolipid metabolites. The treatment of cells with bovine serum albumin significantly reduced the cellular level of sphingosine, but not sphingosine 1-phosphate, generated by overexpression of the enzyme. The cellular level of sphingosine 1-phosphate increased with overexpression of the cytosolic sphingosine kinase. These results suggest that sphingosine 1-phosphate is mainly produced inside of the cell after the incorporation of sphingosine generated on the plasma membranes. The enzyme also seems to participate in the hydrolysis of serum-derived ceramide in the vascular system. Significant amounts of sphingosine as well as sphingosine 1-phosphate were generated in the cell-free conditioned medium of ceramidase transfectants, compared with mock transfectants. No increase in these metabolites was observed if serum or bacterial sphingomyelinase was omitted from the conditioned medium, suggesting that the major source of ceramide is the serum-derived sphingomyelin. A sphingosine 1-phosphate receptor, S1P(1), was internalized much faster by the treatment of S1P(1)-overexpressing cells with conditioned medium of ceramidase transfectants than that of mock transfectants. Collectively, these results clearly indicate that the enzyme is involved in the metabolism of ceramide at the plasma membrane and in the extracellular milieu, which could regulate sphingosine 1-phosphate-mediated signaling through the generation of sphingosine.     

Sphingosine stimulates cellular proliferation via a protein kinase C-independent pathway

Sphingosine, a metabolite of membrane sphingolipids, is generally considered to be cytotoxic for a variety of cell types. However, we have found that sphingosine at low concentrations stimulates DNA synthesis and acts synergistically with known growth factors to induce proliferation of quiescent Swiss 3T3 fibroblasts. Structurally related analogs of sphingosine, such as N-stearoylsphingosine and other long chain aliphatic amines, had no mitogenic effects, suggesting that sphingosine did not induce nonspecific membrane perturbations. Sphingosine, which has been proposed to be a physiological inhibitor of protein kinase C, also markedly potentiates the mitogenic effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Sphingosine still stimulates DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester. At mitogenic concentrations, sphingosine does not bind to protein kinase C as shown by its lack of effect on phorbol dibutyrate binding. Only at higher concentrations, in the cytotoxic range, was there a displacement of phorbol dibutyrate from its cellular-binding sites. In contrast to sphingosine, H-7, a known inhibitor of protein kinase C, inhibited the mitogenic response to TPA and the TPA-induced phosphorylation of the 80 kDa cellular substrate of protein kinase C. Our results suggest that sphingosine may play an important role as a positive regulator of cell growth acting in a fundamentally different, protein kinase C-independent pathway.     

Extracellular sphingomyelinase induces interleukin-6 synthesis in osteoblasts

In osteoblast-like MC3T3-E1 cells, we have recently reported that sphingosine 1-phosphate among sphingomyelin metabolites acts as a second messenger for tumor necrosis factor-alpha (TNF)-induced interleukin-6 (IL-6) synthesis. In the present study, we investigated the effect of extracellular sphingomyelinase on IL-6 synthesis in MC3T3-E1 cells. Sphingomyelinase stimulated IL-6 synthesis in a time-dependent manner for up to 24 h. This stimulative effect was dose dependent in the range between 1 and 300 mU/ml. Calphostin C, a highly and potent inhibitor of protein kinase C, enhanced sphingomyelinase-induced IL-6 synthesis. DL-Threo-dihydrosphingosine, an inhibitor of sphingosine kinase, significantly inhibited the IL-6 synthesis induced by sphingomyelinase. Sphingomyelinase markedly elicited sphingomyelin hydrolysis. In addition, the effect of a combination of sphingomyelinase and TNF on IL-6 synthesis was synergistic. These results strongly suggest that extracellular sphingomyelinase induces sphingomyelin hydrolysis in osteoblasts, resulting in IL-6 synthesis, and that protein kinase C acts as a negative controller of the IL-6 synthesis.     

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.     

Sphingolipid-mediated apoptotic signaling pathways

Various sphingolipids are being viewed as bioactive molecules and/or second messengers. Among them, ceramide (or N-acylsphingosine) and sphingosine generally behave as pro-apoptotic mediators. Indeed, ceramide mediates the death signal initiated by numerous stress agents which either stimulate its de novo synthesis or activate sphingomyelinases that release ceramide from sphingomyelin. For instance, the early generation of ceramide promoted by TNF is mediated by a neutral sphingomyelinase the activity of which is regulated by the FAN adaptor protein, thereby controlling caspase activation and the cell death programme. In addition, the activity of this neutral sphingomyelinase is negatively modulated by caveolin, a major constituent of some membrane microdomains. The enzyme sphingosine kinase also plays a crucial role in apoptosis signalling by regulating the intracellular levels of two sphingolipids having opposite effects, namely the pro-apoptotic sphingosine and the anti-apoptotic sphingosine 1-phosphate molecule. Ceramide and sphingosine metabolism therefore appears as a pivotal regulatory pathway in the determination of cell fate.     

Role of thyroid hormones in regulation of sphingomyelin metabolism in the liver.

Inhibition of thyroid gland function in rats with mercasolil sharply decreased thyroxine and triiodothyronine levels in blood serum and increased acid sphingomyelinase activity and sphingomyelin content in liver. Thyroxine injected into hypothyroid rats normalized the sphingomyelin content, reduced the free ceramide content, and further increased the acid sphingomyelinase activity in liver. Thyroxine stimulated de novo sphingomyelin synthesis. Changing the thyroid status of the rats did not influence the free sphingosine content. Thyroxine blocks the accumulation of free sphingosine in the liver during activation of sphingomyelinases.     

Bezafibrate decreases growth stimulatory action of the sphingomyelin signaling pathway in regenerating rat liver

Liver regeneration after partial hepatectomy (PH) is achieved through proliferation of hepatocytes and non-parenchymal cells. The nuclear peroxisome proliferator-activated receptor alpha (PPARalpha) is involved in regulation of lipid metabolism and proliferation of hepatic cells. The sphingomyelin signal transduction pathway is involved in the regulation of the cell cycle in eukaryotic organisms. Sphingosine-1-phosphate (S1P) and ceramide (CER)-- the intermediates of the pathway--are known to stimulate and to inhibit cellular proliferation. The aim of the present study was to investigate the effect of PPARalpha activation by bezafibrate on the sphingomyelin signaling pathway during the first 24h of liver regeneration after PH in the rat. The content of sphingomyelin, ceramide, sphingosine, sphinganine, sphingosine-1-phosphate and the activity of sphingomyelinases and ceramidases were determined at various time points after PH. It has been found that the activity of neutral Mg(2+)-dependent sphingomyelinase (nSMase) increased, whereas the activity of acidic sphingomyelinase (aSMase) decreased in the regenerating liver. Activation of PPARalpha by bezafibrate lower the activity of nSMase and increased the activity of aSMase in the regenerating rat liver. The content of ceramide was higher in bezafibrate-treated rats, whereas the content of sphingosine-1-phosphate was markedly lower as compared to the untreated rats. Therefore, it is concluded that activation of PPARalpha by bezafibrate decreases the growth-stimulatory activity of the sphingomyelin pathway in regenerating rat liver.     

Neutral sphingomyelinase: Localization in rat liver nuclei and involvement in regeneration/proliferation

We have studied the localization of neutral sphingomyelinase (N-SMase) in rat liver nuclei. The levels of neutral sphingomyelinase in regenerating liver nuclei were also assessed.We found that rat liver nuclei contain a sphingomyelinase having a pH optima of 7.2 and a kDa of 92. In intact nuclei, neutral sphingomyelinase was associated predominantly with the nuclear envelope. In regenerating/proliferating rat liver (during DNA synthesis), neutral sphingomyelinase was translocated from the nuclear envelope to the nuclear matrix. The levels of sphingomyelin in whole nuclei decreased in reverse proportion to an increase in the levels of neutral sphingomyelinase. By contrast, there was a corresponding increase in the levels of ceramide and sphingosine during cell regeneration/proliferation. Thus, endogenous nuclear neutral sphingomyelinase may play a role in the regulation of sphingomyelin levels and in relevant signal transduction reactions involving cell regeneration/proliferation. The potential significance of ceramide generation may be aimed at programmed cell death to allow the regeneration of liver mediated via target proteins such as, ceramide activated protein kinases/phospholipases or other unknown mechanisms.Abbreviations N-SMase neutral sphingomyelinase - A-SMase acid sphingomyelinase     

NAD+/NADH and/or CoQ/CoQH2 ratios from plasma membrane electron transport may determine ceramide and sphingosine-1-phosphate levels accompanying G1 arrest and apoptosis

To elucidate possible biochemical links between growth arrest from antiproliferative chemotherapeutic agents and apoptosis, our work has focused on agents (EGCg, capsaicin, cis platinum, adriamycin, anti-tumor sulfonylureas, phenoxodiol) that target tNOX. tNOX is a cancer-specific cell surface NADH oxidase (ECTO-NOX protein), that functions in cancer cells as the terminal oxidase for plasma membrane electron transport. When tNOX is active, coenzyme Q(10) (ubiquinone) of the plasma membrane is oxidized and NADH is oxidized at the cytosolic surface of the plasma membrane. However, when tNOX is inhibited and plasma membrane electron transport is diminished, both reduced coenzyme Q(10) (ubiquinol) and NADH would be expected to accumulate. To relate inhibition of plasma membrane redox to increased ceramide levels and arrest of cell proliferation in G(1) and apoptosis, we show that neutral sphingomyelinase, a major contributor to plasma membrane ceramide, is inhibited by reduced glutathione and ubiquinone. Ubiquinol is without effect or stimulates. In contrast, sphingosine kinase, which generates anti-apoptotic sphingosine-1-phosphate, is stimulated by ubiquinone but inhibited by ubiquinol and NADH. Thus, the quinone and pyridine nucleotide products of plasma membrane redox, ubiquinone and ubiquinol, as well as NAD(+) and NADH, may directly modulate in a reciprocal manner two key plasma membrane enzymes, sphingomyelinase and sphingosine kinase, potentially leading to G(1) arrest (increase in ceramide) and apoptosis (loss of sphingosine-1-phosphate). As such, the findings provide potential links between coenzyme Q(10)-mediated plasma membrane electron transport and the anticancer action of several clinically-relevant anticancer agents.     

Partial hepatectomy activates production of the pro-mitotic intermediates of the sphingomyelin signal transduction pathway in the rat liver

Sphingomyelin signal transduction pathway regulates cell cycle through a number of lipid second messengers, which stimulate cell proliferation (sphingosine-1-phosphate), initiate growth arrest or induce apoptosis (sphingosine, ceramide). To asses the functioning of sphingomyelin pathway during liver regeneration after partial hepatectomy in rat (PH) we measured the content of sphingomyelin (SM), ceramide (CER), sphingosine (SPH), sphingosine-1-phosphate (S1P), the activity of neutral Mg(2+)-dependent and acidic sphingomyelinases and ceramidases, in the remnant liver lobes during the first 24h after PH in rat. The activity of acidic ceramidase was highest at 4th hour after PH, whereas the activity of neutral ceramidases peaked at 12th hour after the operation. At these time points the activity Mg(2+)-dependent sphingomyelinase was also elevated, together with the content of SPH, S1P and the ratio of S1P to CER. The activity of acidic sphingomyelinase increased gradually from 4th to 24th hour after the operation. This was accompanied by significant increase in the content of ceramide between 4th and 24th hour and reduction in the content of S1P and S1P to CER ratio. It is concluded that partial hepatectomy induces production of the pro-mitogenic intermediates of sphingomyelin signaling pathway during the first 12h of liver regeneration in rat.     

Arachidonoyl-CoA:lysophosphatidylcholine acyltransferase activity in ras-transformed NIH 3T3 fibroblasts depends on the membrane composition.

Investigations were performed on the influence of membrane lipids on arachidonoyl-CoA:lysophosphatidylcholine acyltransferase in microsomal membranes from control and ras-transformed NIH 3T3 fibroblasts. Of all the tested phospholipids only sphingomyelin induced activation of acyltransferase in membranes from ras-transformed cells. No specific phospholipid effect on the acyltransferase was observed in microsomal membranes from control fibroblasts. Diacylglycerol was found to inhibit acyltransferase in both cell lines, whereas ceramide accumulation induced inhibition only in membranes from the transformed cells. The effects of diacylglycerol, ceramide, sphingomyelin and sphingomyelinase are discussed with respect to their putative roles in the signal transduction pathways in oncogene-expressing cells.     

A lipid analogue that inhibits sphingomyelin hydrolysis and synthesis, increases ceramide, and leads to cell death

We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 microM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells.