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At least 19 recordsLinked to original sources
PubMed2003

Synthesis of fluorescence-labeled sphingosine and sphingosine 1-phosphate; effective tools for sphingosine and sphingosine 1-phosphate behavior.

A fluorescence-labeled sphingosine and sphingosine 1-phosphate have been successfully synthesized from the oxazolidinone methyl ester derived from glycidol via monoalkylation and the stereoselective reduction of the resulting ketone. The labeled sphingosine was converted into its phosphate by treatment with sphingosine kinase 1 (SPHK1) from mouse, and in platelets, and it was incorporated into the Chinese Hamster Ovarian (CHO) cells. In addition, MAPK was activated by NBD-Sph-1-P through Edg-1, Sph-1-P receptor.

4-Chloro-7-nitrobenzofurazan↗
PubMed2000

Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation. A dominant-negative sphingosine kinase.

Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S1P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly(82) to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-alpha (TNFalpha), interleukin-1beta, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSK(G82D)). Basal SK activity was unaffected by expression of hSK(G82D). Expression of hSK(G82D) had no effect on TNFalpha-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSK(G82D) acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNFalpha activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation.

Amino Acid Sequence↗
PubMed2001

Stimulation of intracellular sphingosine-1-phosphate production by G-protein-coupled sphingosine-1-phosphate receptors.

Recently, a family of G-protein-coupled receptors named endothelial differentiation gene (Edg) receptor family has been identified, which are specifically activated by the two serum lipids, sphingosine-1-phosphate and lysophosphatidic acid. Sphingosine-1-phosphate can also act intracellularly to release Ca2+ from intracellular stores. Since in several cell types, G-protein-coupled lysophosphatidic acid or sphingosine-1-phosphate receptors mobilize Ca2+ in the absence of a measurable phospholipase C stimulation, it was analysed here whether intracellular sphingosine-1-phosphate production was the signalling mechanism used by extracellular sphingosine-1-phosphate for mobilization of stored Ca2+. Sphingosine-1-phosphate and the low affinity sphingosine-1-phosphate receptor agonist, sphingosylphosphorylcholine, induced a rapid, transient and nearly complete pertussis toxin-sensitive Ca2+ mobilization in human embryonic kidney (HEK-293) cells. The G-protein-coupled sphingosine-1-phosphate receptors, Edg-1, Edg-3 and Edg-5, were found to be endogenously expressed in these cells. Most interestingly, sphingosine-1-phosphate and sphingosylphosphorylcholine did not induce a measurable production of inositol-1,4,5-trisphosphate or accumulation of inositol phosphates. Instead, sphingosine-1-phosphate and sphingosylphosphorylcholine induced a rapid and transient increase in production of intracellular sphingosine-1-phosphate with a maximum of about 1.4-fold at 30 s. Stimulation of sphingosine-1-phosphate formation by sphingosine-1-phosphate and sphingosylphosphorylcholine was fully blocked by pertussis toxin, indicating that extracellular sphingosine-1-phosphate via endogenously expressed G(i)-coupled receptors induces a stimulation of intracellular sphingosine-1-phosphate production. As sphingosine-1-phosphate- and sphingosylphosphorylcholine-induced increases in intracellular Ca2+ were blunted by sphingosine kinase inhibitors, this sphingosine-1-phosphate production appears to mediate Ca2+ signalling by extracellular sphingosine-1-phosphate and sphingosylphosphorylcholine in HEK-293 cells.

Calcium Signaling↗
PubMed1996

Ca2+ mobilizing action of sphingosine in Jurkat human leukemia T cells. Evidence that sphingosine releases Ca2+ from inositol trisphosphate- and phosphatidic acid-sensitive intracellular stores through a mechanism independent of inositol trisphosphate.

Effects of sphingosine on Ca2+ mobilization in the human Jurkat T cell line were examined. Sphingosine increased the cytoplasmic Ca2+ concentration ([Ca2+]i) in a dose-dependent manner with an ED50 of around 8 microM. Sphingosine and OKT3, a CD3 monoclonal antibody, transiently increased [Ca2+]i, which declined to the resting level in the absence of extracellular Ca2+. Under the same conditions, pretreatment with sphingosine inhibited but did not abolish an increase in [Ca2+]i induced by the subsequent addition of OKT3 and vice versa. However, pretreatment with sphingosine did not affect an increase in [Ca2+]i induced by OKT3 in the presence of Ca2+. OKT3 increased IP3 formation, but sphingosine did not affect the level of IP3 by itself nor did it cause IP3 formation induced by OKT3. In permeabilized Jurkat cells, the addition of IP3 released Ca2+ from nonmitochondrial intracellular stores, but the addition of sphingosine did not. Sphingosine, stearylamine, and psychosine increased [Ca2+]i and diacylglycerol (DG) kinase activation; however, ceramide did not, whereas sphingosine 1-phosphate slightly activated DG kinase without elevation of [Ca2+]i. Pretreatment with R59022, a DG kinase inhibitor, abolished the peak but did not affect the sustained response to [Ca2+]i to sphingosine. Phosphatidic acid (PA) elevated [Ca2+]i, after which it declined to a resting level even in the presence of extracellular Ca2+. In accordance with this, PA did not stimulate 45Ca2+ uptake into cells, but sphingosine and OKT3 did. Pretreatment with PA partially inhibited a rise in [Ca2+]i induced by the subsequent addition of sphingosine and vice versa in the absence of extracellular Ca2+. Under similar conditions, pretreatment with PA affected an elevation of [Ca2+]i induced by OKT3 less, after which the subsequent addition of sphingosine did not increase [Ca2+]i. In permeabilized Jurkat cells, the addition of IP3 did not release Ca2+, but PA did in the presence of heparin. Pretreatment with thapsigargin, a microsomal Ca2+-ATPase inhibitor, abolished the rises of [Ca2+]i induced by the subsequent addition of sphingosine, OKT3, and PA in the absence of extracellular Ca2+. The present results suggest that at least two kinds of intracellular Ca2+ stores exist in Jurkat cells, both of which are IP3- and PA-sensitive, and that sphingosine mobilizes Ca2+ from both stores in an IP3-independent manner. Furthermore, the IP3- but not the PA-sensitive intracellular Ca2+ store seems to regulate Ca2+ entry induced by sphingosine.

Amines↗
PubMed2025

The Pseudomonas aeruginosa sphBC genes are important for growth in the presence of sphingosine by promoting sphingosine metabolism.

Sphingoid bases, including sphingosine, are important components of the antimicrobial barrier at epithelial surfaces where they can cause growth inhibition and killing of susceptible bacteria. Pseudomonas aeruginosa is a common opportunistic pathogen that is less susceptible to sphingosine than many Gram-negative bacteria. Here, we determined that the deletion of the sphBCD operon reduced growth in the presence of sphingosine. Using deletion mutants, complementation and growth assays in P. aeruginosa PAO1, we determined that the sphC and sphB genes, encoding a periplasmic oxidase and periplasmic cytochrome c, respectively, were important for growth on sphingosine, while sphD was dispensable under these conditions. Deletion of sphBCD in P. aeruginosa PA14, Pseudomonas protegens Pf-5 and Pseudomonas fluorescens Pf01 also showed reduced growth in the presence of sphingosine. The P. aeruginosa sphBC genes were also important for growth in the presence of two other sphingoid bases, phytosphingosine and sphinganine. In WT P. aeruginosa, sphingosine is metabolized to an unknown non-inhibitory product, as sphingosine concentrations drop in the culture. However, in the absence of sphBC, sphingosine accumulates, pointing to SphC and SphB as having a role in sphingosine metabolism. Finally, the metabolism of sphingosine by WT P. aeruginosa protected susceptible cells from full growth inhibition by sphingosine, pointing to a role for sphingosine metabolism as a public good. This work shows that the metabolism of sphingosine by P. aeruginosa presents a novel pathway by which bacteria can alter host-derived sphingolipids, but it remains an open question whether SphB and SphC act directly on sphingosine.

Sphingosine↗
PubMed1995

Stereoselectivity of induction of the retinoblastoma gene product (pRb) dephosphorylation by D-erythro-sphingosine supports a role for pRb in growth suppression by sphingosine.

Sphingosine has been shown to inhibit cell growth in many cell lines although the mechanism of this effect remains obscure. More recently, D-erythro-sphingosine has been shown to act as an early inducer of dephosphorylation of the retinoblastoma gene product (pRb) in the lymphoblastic leukemia cell line MOLT-4 [Chao, R., Khan, W., & Hannun, Y.A. (1992) J. Biol. Chem., 267, 23459-23462]. In the current study, the role of the natural D-erythro-sphingosine in regulation of cell growth and pRb dephosphorylation was evaluated using chemically synthesized pure isomers of sphingosine. Of the four possible stereoisomers of sphingosine, D-erythro-sphingosine was most active in inducing dephosphorylation of pRb protein with an EC50% of 0.6 microM whereas its enantiomer L-erythro-sphingosine was 8-fold less potent with an EC50% of 5 microM. The dose responses for inhibition of cell growth were nearly identical to the EC50% for pRb dephosphorylation with D-erythro-sphingosine causing 50% inhibition at 0.6 microM whereas L-erythro-sphingosine was 5-6-fold less potent. All of the stereoisomers were taken up by the cells, and the greater potency of D-erythro-sphingosine was not due to differences in cellular uptake. The metabolism of D-erythro-sphingosine was also studied to evaluate the possible role of sphingosine metabolites on regulation of retinoblastoma protein. Evidence is provided against a role for ceramide or sphingosine 1-phosphate as mediators of the effects of sphingosine on pRb dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Death↗
PubMed1992

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.

3T3 Cells↗
PubMed1995

Interaction of ceramides, sphingosine, and sphingosine 1-phosphate in regulating DNA synthesis and phospholipase D activity.

C2- and C6-ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) abolished the stimulation of DNA synthesis by sphingosine 1-phosphate in rat fibroblasts. This inhibition by ceramide was partially prevented by insulin. C2-ceramide did not alter the stimulation of DNA synthesis by insulin and decreased the sphingosine-induced stimulation by only 16%. The ceramides did not significantly modify the actions of sphingosine or sphingosine 1-phosphate in decreasing cAMP concentrations. C2- and C6-ceramides blocked the activation of phospholipase D by sphingosine 1-phosphate, and this inhibition was not affected by insulin. Okadaic acid decreased the activation of phospholipase D by sphingosine 1-phosphate and did not reverse the inhibitory effect of C2-ceramide on this activation. Therefore, this effect of C2-ceramide is unlikely to involve the stimulation of phosphoprotein phosphatase activity. Sphingosine did not activate phospholipase D activity significantly after 10 min. C2-ceramide stimulated the conversion of exogenous [3H]sphingosine 1-phosphate to sphingosine and ceramide in fibroblasts. Ceramides can inhibit some effects of sphingosine 1-phosphate by stimulating its degradation via a phosphohydrolase that also hydrolyzes phosphatidate. Furthermore, C2- and C6-ceramides stimulated ceramide production from endogenous lipids, and this could propagate the intracellular signal. This work demonstrates that controlling the production of ceramide versus sphingosine and sphingosine 1-phosphate after sphingomyelinase activation could have profound effects on signal transduction.

Animals↗
PubMed2004

An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes.

Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways and high-throughput screens for agents affecting sphingosine kinase activity in vitro.

Bacterial Proteins↗
PubMed2004

D-galactosyl-beta1-1'-sphingosine and D-glucosyl-beta1-1'-sphingosine induce human natural killer cell apoptosis.

Natural killer (NK) cells perform multiple biological functions including tumor cell lysis and eradicating virally infected cells. Here, we report for the first time that D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1- 1' sphingosine damage human NK cells. We show that these cells express T-cell-associated gene-8, the receptor for glycosphingolipids. D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1-1' sphingosine induce the in vitro chemotaxis of human NK cells. Both D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1-1' sphingosine inhibit the cytotoxicity and IFN-gamma secretion by these cells. Further analysis shows that the glycosphingolipids D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1-1' sphingosine but not any other lipid examined, which include D-lactosyl-beta1-1' sphingosine, sphingosine 1-phosphate, sphingosine, lysophosphatidic acid, and phosphatidic acid, induce the apoptosis, globoid-like formation, and multinucleation in human NK cells. These results may have important implications on diseases where glycosphingolipids accumulate.

Apoptosis↗
PubMed1994

Activation of endothelial cell phospholipase D by sphingosine and sphingosine-1-phosphate.

We have investigated the activation of phospholipase D (PLD) by sphingosine and its derivatives in bovine pulmonary artery endothelial cells (BPAEC) prelabeled with [32P]orthophosphate or [32P]lyso phospholipids. Sphingosine, in a dose- and time-dependent manner, stimulated the hydrolysis of [32P]phosphatidylcholine (PC) resulting in the production of [32P]phosphatidic acid (PA), suggesting PLD activation. In the presence of ethanol (150 mM), the accumulation of [32P]phosphatidylethanol was also observed. The sphingosine-induced stimulation of PLD activity was not affected by treatment with the protein kinase C (PKC) inhibitor staurosporine or by down-regulation of PKC with TPA and was independent of extracellular Ca2+, suggesting that the PLD activation was independent of PKC and Ca2+. Chelation of intracellular Ca2+ with BAPTA actually potentiated the sphingosine-stimulated [32P]PC hydrolysis. Furthermore, the activation of PLD by sphingosine was not abolished by treatment of BPAEC with either cholera or pertussis toxin, indicating noninvolvement of toxin-sensitive G-proteins. In addition to hydrolysis of [32P]PC, sphingosine also stimulated PLD-mediated hydrolysis of [32P]phosphatidylethanolamine and [32P]phosphatidylinositol. Among the various sphingoid compounds, in addition to sphingosine, only sphingosine-1-phosphate (Sph-1-P) activated the endothelial cell PLD. The effect of sphingosine and Sph-1-P on PA phosphatase (PA Pase) activity was tested using [3H]glycerol-labeled PA. The Mg(2+)-independent and membrane-associated PA Pase activity was inhibited by sphingosine (IC50 = 200 microM) but not by Sph-1-P. This implies that sphingosine and Sph-1-P share a similar PLD-stimulating property but differ in their PA Pase inhibitory activity.

Animals↗
PubMed1998

The effects of exogenous sphingosine on Neuro2a cells are strictly related to the overall capacity of cells to metabolize sphingosine.

Neuro2a cells were exposed to different doses (1-40 nmol/10(6) cells) of [C3-3H]sphingosine and the relationship between metabolism and biological effects of sphingosine was investigated. Sphingosine appeared to be rapidly taken up and metabolized. The incorporation of sphingosine was not merely dependent on its concentration but primarily on the dose per cell of administered sphingosine. At low doses, [3H]sphingosine represented a minor portion of the cellular radioactivity, and N-acylated metabolites, particularly ceramide, largely prevailed over degradation products. Concomitantly with ceramide increase, Neuro2a differentiation took place. With increasing exogenous sphingosine/doses, the acylation process reached saturation. From this point on, [3H]sphingosine started accumulating and eventually cell toxicity occurred. In conclusion, the biological effects exerted by exogenous sphingosine on Neuro2a cells are not merely dependent on the long-chain base concentration in the culture medium, but are strictly related to the cellular dose of exogenous sphingosine and to the capacity of cells to metabolize sphingosine.

Animals↗
PubMed1999

Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival.

Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.

Animals↗
PubMed2005

Effects of exogenous sphinganine, sphingosine, and sphingosine-1-phosphate on relaxation and contraction of porcine thoracic aortic and pulmonary arterial rings.

Fumonisin mycotoxicosis in pigs causes a decrease in mean aortic pressure, an increase in mean pulmonary arterial pressure, and increases in serum concentrations of sphinganine (3.2 microM) and sphingosine (1.4 microM). To determine a causal relationship between the hemodynamic changes and sphingolipid alterations, we examined the in vitro effects of sphinganine, sphingosine, and sphingosine-1-phosphate on porcine aortic and pulmonary arterial rings. Both sphinganine and sphingosine relaxed un-contracted and phenylephrine-contracted aortic rings at > or = 10 microM and > or = 1 microM, respectively. Sphingosine (> or = 10 microM) relaxed un-contracted and phenylephrine-contracted pulmonary arterial rings, whereas sphingosine-1-phosphate (10 microM) contracted pulmonary arterial rings. Sphingosine (3 microM) also impaired the contractile response of pulmonary artery rings to 60 mM KCl. The results suggested that the systemic hypotension caused by fumonisin is mediated, in part, by increases in serum sphinganine and sphingosine concentrations, and the pulmonary hypertension is mediated, in part, by increased sphingosine-1-phosphate concentrations.

Animals↗
PubMed1999

Sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate modulate phosphatidylserine homeostasis in glioma C6 cells.

The effect of sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate on L-[U-14C]serine incorporation into phosphatidylserine and phosphatidylserine-derived phosphatidylethanolamine was investigated in intact glioma C6 cells. Sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate are potent signalling molecules which, due to their physicochemical features, may function as amphiphilic compounds. It has been found that sphingosine and sphingosylphosphorylcholine (amphiphilic cations) significantly increase [14C]phosphatidylserine synthesis and decrease the amount of 14C-labeled phosphatidylethanolamine. Sphingosine 1-phosphate (an amphiphilic anion) was without effect on phosphatidylserine synthesis but, similarly as sphingosine and sphingosylphosphorylcholine, reduced the conversion of phosphatidylserine to phosphatidylethanolamine. These results strongly suggest that sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate can modulate cellular phospholipid homeostasis by stimulation of phosphatidylserine synthesis and an interference with phosphatidylserine decarboxylase.

Brain Neoplasms↗
PubMed1993

Sphingosine and sphingosine 1-phosphate in cellular proliferation: relationship with protein kinase C and phosphatidic acid.

Previous studies from our laboratory have shown that sphingosine (Zhang et al. (1990) J. Biol. Chem. 265, 76-81) and sphingosine 1-phosphate (Zhang et al. (1991) J. Cell. Biol. 114, 155-167), metabolites of membrane sphingolipids, stimulate release of calcium from internal sources and increase proliferation of quiescent Swiss 3T3 fibroblasts acting in a fundamentally different, protein kinase C-independent pathway. The mitogenic effect of sphingosine was accompanied by an increase in the levels of phosphatidic acid (PA), a potent mitogen for a variety of cell types, that may function as an intracellular second messenger (Zhang et al. (1990) J. Biol. Chem. 265, 21309-21316). Sphingosine also induced early increases in sphingosine 1-phosphate (SPP) levels that preceded the increase in PA (Desai et al. (1992) J. Biol. Chem. 267, 23122-23128). SPP itself produced a more rapid increase in PA, thus suggesting that it may mediate the effects of sphingosine on PA accumulation. The concentration dependence for the formation of PA induced by SPP correlated with its effect on DNA synthesis. Similar to sphingosine, SPP also stimulated the activity of phospholipase D, although a significant effect was observed at a much lower concentration. However, in contrast to previous reports with sphingosine, SPP did not inhibit the PA phosphohydrolase activity in cell homogenates. Thus, in addition to its effect on mobilization of calcium, SPP can increase the level of PA, most likely via activation of phospholipase D. We suggest that SPP mediates the effect of sphingosine on PA accumulation in Swiss 3T3 fibroblasts and may regulate cellular proliferation by affecting multiple transmembrane signaling pathways.

Animals↗