Biomedical subjects

S Spiegel

Publications and source records attributed to S Spiegel.

At least 91 records · Page 5Linked to original sources

PubMed1997

KSR stimulates Raf-1 activity in a kinase-independent manner.

Kinase suppressor of Ras (KSR) is an evolutionarily conserved component of Ras-dependent signaling pathways. Here, we find that murine KSR (mKSR1) translocates from the cytoplasm to the plasma membrane in the presence of activated Ras. At the membrane, mKSR1 modulates Ras signaling by enhancing Raf-1 activity in a kinase-independent manner. The activation of Raf-1 is mediated by the mKSR1 cysteine-rich CA3 domain and involves a detergent labile cofactor that is not ceramide. These findings reveal another point of regulation for Ras-mediated signal transduction and further define a noncatalytic role for mKSR1 in the multistep process of Raf-1 activation.

Animals↗

PubMed1997

Activation of sphingosine kinase in pheochromocytoma PC12 neuronal cells in response to trophic factors.

Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), dibutyryl cAMP and forskolin, known differentiating agents for pheochromocytoma PC12 cells, induced sustained activation of sphingosine kinase, the enzyme responsible for the formation of the sphingolipid second messenger, sphingosine-1-phosphate, which mediates the mitogenic effects of certain growth factors. In contrast, epidermal growth factor and insulin-like growth factor-1, which stimulate proliferation of PC12 cells, induced only small and transient increases in sphingosine kinase activity. Of the growth factors examined, NGF was the most potent activator of sphingosine kinase, inducing a 4-fold increase in Vmax. Sphingosine kinase activity induced by NGF, but not FGF, was blocked by the protein kinase inhibitor K252a when added simultaneously, with minimal effect when added after 60 min. Thus, activation of sphingosine kinase may have an important role in neural differentiation.

Animals↗

PubMed1997

Differential effects of sphingomyelinase and cell-permeable ceramide analogs on proliferation of Swiss 3T3 fibroblasts.

Metabolites of sphingomyelin, ceramide and sphingosine, have previously been implicated in cell growth regulation. Here we show that cell-permeable ceramide analogs and treatment with sphingomyelinase, which hydrolyzes sphingomyelin located on the outer leaflet of the bilayer, increase the progression of quiescent Swiss 3T3 fibroblasts through the S phase of the cell cycle leading to an increase in cell division. Although both potentiate the mitogenic effects of several growth factors [14], sphingomyelinase treatment antagonized the mitogenic effect of the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), while ceramide analogs had no effect, and sphingosine, a further metabolite of ceramide, potentiated the mitogenic effect of TPA. Concomitantly, sphingomyelinase, but not ceramide analogs, blunted the rapid increase in membrane-associated protein kinase C (PKC) activity induced by TPA without affecting the translocation of PKC alpha, delta, epsilon or zeta isoforms. Moreover, in contrast to sphingosine which activates phospholipase D (PLD) leading to an increase in phosphatidic acid levels, sphingomyelinase, but not ceramide analogs, reduced TPA-stimulated PLD activity. Our results suggest that the signaling pathways utilized by sphingomyelinase differ from those of cell-permeable ceramide analogs, and both act differently than sphingosine. The differential effects of exogenous short-chain ceramide analogs and sphingomyelinase call for caution in using these analogs as tools to study the role of ceramide in diverse cellular functions.

3T3 Cells↗

PubMed1997

Involvement of sphingosine 1-phosphate in nerve growth factor-mediated neuronal survival and differentiation.

Sphingolipid metabolites, such as ceramide and sphingosine-1-phosphate (SPP), are emerging as a new class of second messengers involved in cellular proliferation, differentiation, and apoptosis. Nerve growth factor (NGF), a neurotrophic factor for pheochromocytoma PC12 cells, induced a biphasic increase in the activity of sphingosine kinase, the enzyme that catalyzes the formation of SPP. This activation was blocked by K252a, an inhibitor of tyrosine kinase A (trkA). A rapid 1.7-fold increase was followed by a marked prolonged increase reaching a maximum of fourfold to fivefold stimulation with a concomitant increase in SPP levels and a corresponding decrease in endogenous sphingosine levels. Levels of ceramide, the precursor of sphingosine, were only slightly decreased by NGF in serum-containing medium. However, NGF decreased the elevation of ceramide induced by serum withdrawal. Treatment of PC12 cells with SPP did not induce neurite outgrowth or neurofilament expression, yet it enhanced neurofilament expression elicited by suboptimal doses of NGF. Moreover, SPP also protected PC12 cells from apoptosis induced by serum withdrawal. To further substantiate a role for SPP in the cytoprotective actions of NGF, we found that N, N-dimethylsphingosine, a competitive inhibitor of sphingosine kinase, also induced apoptosis and interfered with the survival effect of NGF. These effects were counteracted by exogenous SPP. Moreover, other structurally related compounds, such as dihydrosphingosine 1-phosphate and lysophosphatidic acid, had no significant protective effects. Our results suggest that activation of sphingosine kinase and subsequent formation of SPP may play an important role in the differentiation and survival effects induced by NGF.

Animals↗

PubMed1997

Calcium influx inhibits MT1-MMP processing and blocks MMP-2 activation.

We have previously reported that concanavalin A (ConA)-induced MMP-2 activation involves both transcriptional and non-transcriptional mechanisms. Here we examined the effects of calcium influx on MT1-MMP expression and MMP-2 activation in MDA-MB-231 cells. The calcium ionophore ionomycin caused a dose-dependent inhibition of ConA-induced MMP-2 activation, but had no effect on MT1-MMP mRNA levels. However, Western analysis revealed an accumulation of pro-MT1-MMP (63 kDa), indicating that ionomycin blocked the conversion of pro-MT1-MMP protein to the active 60 kDa form. This suggests that increased calcium levels inhibit the processing of MT1-MMP. This finding may help to elucidate the mechanism(s) which regulates MT1-MMP activation.

Blotting, Northern↗

PubMed1997

Sphingosine 1-phosphate stimulates tyrosine phosphorylation of Crk.

The proto-oncogene molecule c-Crk plays a role in growth factor-induced activation of Ras. Sphingosine 1-phosphate (SPP), a metabolite of cellular sphingolipids, has previously been shown to play a role in growth factor receptor signaling (Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560). SPP was found to strongly induce tyrosine phosphorylation of Crk, but not Shc, in NIH-3T3 parental, insulin-like growth factor-I receptor-overexpressing and Crk-overexpressing (3T3-Crk) fibroblasts. Sphingosine, a metabolic precursor of SPP, also produced a slight increase in tyrosine phosphorylation of Crk. In contrast, other sphingolipid metabolites including ceramide did not alter Crk tyrosine phosphorylation. Furthermore, Crk enhanced SPP-induced mitogenesis, as measured by SPP-stimulated [3H]thymidine incorporation in a manner proportional to the level of Crk expression in 3T3-Crk cells. This stimulation appears to be Ras-dependent, whereas SPP stimulation of MAP kinase activity is Ras-independent. These data indicate that SPP activates a tyrosine kinase that phosphorylates Crk and that Crk is a positive effector of SPP-induced mitogenesis.

3T3 Cells↗

PubMed1997

Sphingosine 1-phosphate stimulates rho-mediated tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 fibroblasts.

Sphingosine 1-phosphate (SPP), a sphingolipid second messenger implicated in the mitogenic action of platelet-derived growth factor [Olivera, A. and Spiegel, S. (1993) Nature (London) 365, 557-560], induced rapid reorganization of the actin cytoskeleton resulting in stress-fibre formation. SPP also induced transient tyrosine phosphorylation of focal adhesion kinase (p125(FAK)), a cytosolic tyrosine kinase that localizes in focal adhesions, and of the cytoskeleton-associated protein paxillin. Exoenzyme C3 transferase, which ADP-ribosylates Rho (a Ras-related small GTP binding protein) on asparagine-41 and renders it biologically inactive, inhibited both stress-fibre formation and protein tyrosine phosphorylation induced by SPP. Thus Rho may be an upstream regulator of both stress-fibre formation and tyrosine phosphorylation of p125(FAK) and paxillin. Pretreatment with PMA, an activator of protein kinase C (PKC), inhibited the stimulation of stress-fibre formation induced by 1-oleoyl-lysophosphatidic acid (LPA) but not that by SPP. Similarly, PMA also decreased LPA-induced tyrosine phosphorylation of p125(FAK) and paxillin without abrogating the response to SPP. Thus PKC is involved in LPA- but not SPP-dependent signalling. The polyanionic drug suramin, a broad-specificity inhibitor of ligand-receptor interactions, did not inhibit either the mitogenic effect of SPP or its stimulation of tyrosine phosphorylation of p125(FAK). However, suramin markedly inhibited these responses induced by LPA. These results suggest that in contrast with LPA, SPP may be acting intracellularly in Swiss 3T3 fibroblasts to stimulate tyrosine phosphorylation of p125(FAK) and paxillin and cell growth.

3T3 Cells↗

PubMed1997

Divergence in signal transduction pathways of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Involvement of sphingosine 1-phosphate in PDGF but not EGF signaling.

Platelet-derived growth factor (PDGF) and serum, but not epidermal growth factor (EGF), stimulated sphingosine kinase activity in Swiss 3T3 fibroblasts and increased intracellular concentrations of sphingosine 1-phosphate (SPP), a sphingolipid second messenger (Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560). We report herein that DL-threo-dihydrosphingosine (DHS), a competitive inhibitor of sphingosine kinase that prevents PDGF-induced SPP formation, specifically inhibited the activation of two cyclin-dependent kinases (p34(cdc2) kinase and Cdk2 kinase) induced by PDGF, but not by EGF. SPP reversed the inhibitory effects of DHS on PDGF-stimulated cyclin-dependent kinases and DNA synthesis, demonstrating that the DHS effects were mediated via inhibition of sphingosine kinase. DHS also markedly reduced PDGF-stimulated but not EGF-stimulated mitogen-activated protein kinase activity and DNA binding activity of activator protein-1. Examination of the early signaling events of PDGF action revealed that DHS did not affect PDGF-induced autophosphorylation of the growth factor receptor or phosphorylation of the SH2/SH3 adaptor protein Shc and its association with Grb2. This sphingosine kinase inhibitor did not abrogate activation of phosphatidylinositol 3-kinase by PDGF. In agreement, treatment with SPP had no effect on these responses but did, however, potently stimulate phosphorylation of Crk, another SH2/SH3 adaptor protein. Moreover, DHS inhibited PDGF-stimulated, but not EGF-stimulated, Crk phosphorylation. Thus, regulation of sphingosine kinase activity defines divergence in signal transduction pathways of PDGF and EGF receptors leading to mitogen-activated protein kinase activation.

3T3 Cells↗

PubMed1997

Sphingolipids--the enigmatic lipid class: biochemistry, physiology, and pathophysiology.

The "sphingosin" backbone of sphingolipids was so named by J. L. W. Thudichum in 1884 for its enigmatic ("Sphinx-like") properties. Although still an elusive class of lipids, research on the involvement of sphingolipids in the signal transduction pathways that mediate cell growth, differentiation, multiple cell functions, and cell death has been rapidly expanding our understanding of these compounds. In addition to the newly discovered role of ceramide as an intracellular second messenger for tumor necrosis factor-alpha, IL-1beta, and other cytokines, sphingosine, sphingosine-1-phosphate, and other sphingolipid metabolites have recently been demonstrated to modulate cellular calcium homeostasis and cell proliferation. Perturbation of sphingolipid metabolism using synthetic and naturally occurring inhibitors of key enzymes of the biosynthetic pathways is aiding the characterization of these processes; for examples, inhibition of cerebroside synthase has indicated a role for ceramide in cellular stress responses including heat shock, and inhibition of ceramide synthase (by fumonisins) has revealed the role of disruption of sphingolipid metabolism in several animal diseases. Fumonisins are currently the focus of a FDA long-term tumor study. This review summarizes recent research on (i) the role of sphingolipids as important components of the diet, (ii) the role of sphingoid base metabolites and the ceramide cycle in expression of genes regulating cell growth, differentiation, and apoptosis, (iii) the use of cerebroside synthase inhibitors as tools for understanding the role of sphingolipids as mediators of cell cycle progression, renal disease, and stress responses, and (iv) the involvement of disrupted sphingolipid metabolism in animal disease and cellular deregulation associated with exposure to inhibitors of ceramide synthase and serine palmitoyltransferase, key enzymes in de novo sphingolipid biosynthesis. These findings illustrate how an understanding of the function of sphingolipids can help solve questions in toxicology and this is undoubtedly only the beginning of this story.

Amidohydrolases↗

PubMed1997

The use of short and long PCR products for improved detection of prunus necrotic ringspot virus in woody plants.

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of prunus necrotic ringspot virus (PNRSV) in dormant peach and almond trees by the application of two different pairs of primers yielding a short and a long product, respectively. The relative amount of the short (200 base pair, bp) product was higher than the longer (785 bp) product. PNRSV was detected better in plant tissues with a low virus concentration (e.g. dormant trees) by amplification of the short PCR product, whereas the long product was product was produced at higher virus titers. Simultaneous amplification of both short and long products was demonstrated using a three-primer mixture in a single reaction tube. In this assay, amplification of either PCR product indicated the presence of PNRSV-specific sequences in the plant tissue examined, thus covering a wide range of virus concentrations in a single test. Dilution of the RNA extracted from infected plant material resulted in a steep decline in the amplification of both short and long PCR products. In contrast, serial dilutions of the intermediate cDNA template differentially affected the amplification patterns: the relative amount of the short product increased whereas that of the long product decreased. These results may explain the preferential amplification of the short PCR product observed in samples containing low virus concentrations.

DNA, Complementary↗

PubMed1997

Central motor processing in Huntington's disease. A PET study.

Repeated PET cerebral blood flow measurements using H2(15)O were performed in 13 patients with confirmed Huntington's disease and nine age-matched controls. The activation paradigm consisted of an externally triggered finger opposition task (1.5 Hz) with the dominant hand, the control condition being the auditory input. In the patients with Huntington's disease, impaired activity of the striatum and its frontal motor projection areas (rostral supplementary motor area, anterior cingulate and premotor cortex) could be demonstrated along with enhanced activity mainly in parietal areas during movement. The results suggest that the pathology of Huntington's disease causes impairment of the output part of the basal ganglia-thalamo-cortical motor circuit and may induce a compensatory recruitment of additional accessory motor pathways involving the parietal cortex.

Adult↗

PubMed1996

Fidelity of translesional synthesis past benzo[a]pyrene diol epoxide-2'-deoxyguanosine DNA adducts: marked effects of host cell, sequence context, and chirality.

We have used a site-specific approach to investigate the mutagenic potential of (+)- and (-)-trans-anti-benzo[a]pyrene diol epoxide (BPDE) DNA adducts. Oligodeoxyribonucleotides (5'TCCTCCTG1G2-CCTCTC), modified at the exocyclic amino groups of G1 or G2, were incorporated into a single-stranded shuttle vector and introduced into Escherichia coli or simian kidney (COS) cells. This experimental system permits translesional synthesis to proceed in the absence of DNA repair. The presence of (+)- or (-)-BPDE-N2-dG adducts strongly inhibited translesional synthesis in E. coli; induction of cellular SOS functions reduced this blocking effect. Vectors containing (+)-BPDE adducts at G1 or G2 generated mutation frequencies of 19% and 3%, respectively; these values were not altered significantly by induction of SOS functions. In COS cells, (+)-BPDE-modified vectors generated mutation frequencies of 13% at G1 and 45% at G2. In E. coli, the (-)-BPDE adduct generated mutation frequencies of < or = 2% at G1 and G2 and, in COS cells, 13% at G1 and 21% at G2. The predominant mutations in E. coli and COS cells were G-->T transversions targeted to the site of the lesion; however, when G2 was modified, a significant number of targeted G-->A and G-->C mutations were observed in COS cells. We conclude from this study that (+)-and (-)-BPDE-N2-dG adducts pair preferentially to dCMP and dAMP during translesional synthesis in a process that is strongly influenced by the stereochemistry of the adduct, by the bases flanking the lesion, and by host cell factors.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1996

Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.

Ceramide is an important regulatory participant of programmed cell death (apoptosis) induced by tumour-necrosis factor (TNF)-alpha and Fas ligand, members of the TNF superfamily. Conversely, sphingosine and sphingosine-1-phosphate, which are metabolites of ceramide, induce mitogenesis and have been implicated as second messengers in cellular proliferation induced by platelet-derived growth factor and serum. Here we report that sphingosine-1-phosphate prevents the appearance of the key features of apoptosis, namely intranucleosomal DNA fragmentation and morphological changes, which result from increased concentrations of ceramide. Furthermore, inhibition of ceramide-mediated apoptosis by activation of protein kinase C results from stimulation of sphingosine kinase and the concomitant increase in intracellular sphingosine-1-phosphate. Finally sphingosine-1-phosphate not only stimulates the extracellular signal-regulated kinase (ERK) pathway, it counteracts the ceramide-induced activation of stress-activated protein kinase (SAPK/JNK). Thus, the balance between the intracellular levels of ceramide and sphingosine-1-phosphate and their regulatory effects on different family members of mitogen-activated protein kinases determines the fate of the cell.

3T3 Cells↗

PubMed1996

Sphingoid bases and phospholipase D activation.

There is increased interest in physiological functions and mechanisms of action of sphingolipids metabolites, ceramide, sphingosine, and sphingosine-1-phosphate (SPP), members of a new class of lipid second messengers. This review summarizes current knowledge regarding the role of these sphingolipids metabolites in the actions of growth factors and focuses on the second messenger roles of sphingosine and its metabolite, SPP, in the regulation of cell growth. We also discuss possible interactions with intermediates of the well known glycerophospholipid cycle. Sphingosine and SPP generally provide positive mitogenic signals whereas ceramide has been reported to induce apoptosis and cell arrest in several mammalian cell lines. Stimulation of phospholipase D leading to an increase in phosphatidic acid, a positive regulator of cell growth, by sphingosine and SPP, and its inhibition by ceramide, might be related to their opposite effects on cell growth. This also indicates that sphingolipid turnover could regulate the diacylglycerol cycle. Cross-talk between sphingolipid turnover pathways and the diacylglycerol cycle increases complexity of signaling pathways leading to cellular proliferation and adds additional sites of regulation.

Animals↗

PubMed1996

Effect of acidic phospholipids on sphingosine kinase.

Sphingosine-1-phosphate (SPP) is a unique sphingolipid metabolite involved in cell growth regulation and signal transduction. SPP is formed from sphingosine in cells by the action of sphingosine kinase, an enzyme whose activity can be stimulated by growth factors. Little is known of the mechanisms by which sphingosine kinase is regulated. We found that acidic phospholipids, particularly phosphatidylserine, induced a dose-dependent increase in sphingosine kinase activity due to an increase in the apparent Vmax of the enzyme. Other acidic phospholipids, such as phosphatidylinositol, phosphatidic acid, phosphatidylinositol bisphosphate, and cardiolipin stimulated sphingosine kinase activity to a lesser extent than phosphatidylserine, whereas neutral phospholipids had no effect. Diacylglycerol, a structurally similar molecule which differs from phosphatidic acid in the absence of the phosphate group, failed to induce any changes in sphingosine kinase activity. Our results suggest that the presence of negative charges on the lipid molecules is important for the potentiation of sphingosine kinase activity, but the effect does not directly correlate with the number of negative charges. These results also support the notion that the polar group confers specificity in the stimulation of sphingosine kinase by acidic glycerophospholipids. The presence of a fatty acid chain in position 2 of the glycerol backbone was not critical since lysophosphatidylserine also stimulated sphingosine kinase, although it was somewhat less potent. Dioleoylphosphatidylserine was the most potent species, including a fourfold stimulation, whereas distearoyl phosphatidylserine was completely inactive. Thus, the degree of saturation of the fatty acid chain of the phospholipids may also play a role in the activation of sphingosine kinase.

3T3 Cells↗

PubMed1996

Involvement of sphingolipids metabolites in cellular proliferation modulated by ganglioside GM1.

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts. Recently, sphingolipids metabolites, ceramide, sphingosine and sphingosine-1-phosphate, have been implicated as second messengers in cell growth regulation and differentiation. In this paper, we examined the possibility that interaction of the B subunit with membrane GM1 leads to alterations in metabolism of glycosphingolipids and that increased levels of sphingolipids metabolites may mediate the biological effects of the B subunit. While the B subunit did not induce a change in the level of ceramide or sphingosine, the level of sphingosine-1-phosphate was rapidly and transiently increased. The B subunit also transiently activated cytosolic sphingosine kinase activity, which catalyzes the phosphorylation of the primary hydroxyl group of sphingosine to produce sphingosine-1-phosphate. To determine whether the increase in sphingosine-1-phosphate level plays a role in B subunit-induced mitogenicity, we used a competitive inhibitor of sphingosine kinase, D,L-threo-dihydrosphingosine. D,L-thereo-Dihydrosphingosine not only inhibited B subunit-induced DNA synthesis by 26%, it also reduced its ability to stimulate DNA-binding activity of the transcription factor AP-1. This sphingosine kinase inhibitor also inhibited B subunit-induced increases in the activity of cell cycle-regulated, cyclin-dependent serine/threonine kinases, cdk2 and p34cdc2. These findings suggest that sphingosine-1-phosphate may play a role in the signal transduction pathways activated by binding of the B subunit to endogenous ganglioside GM1.

3T3 Cells↗

PubMed1996

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.

Animals↗

PubMed1996

Sphingolipid metabolism and cell growth regulation.

Sphingolipids have been implicated in the regulation of cell growth, differentiation, and programmed cell death. The current paradigm for their action is that complex sphingolipids such as gangliosides interact with growth factor receptors, the extracellular matrix, and neighboring cells, whereas the backbones--sphingosine and other long-chain or "sphingoid" bases, ceramides, and sphingosine 1-phosphate--activate or inhibit protein kinases and phosphatases, ion transporters, and other regulatory machinery. Tumor necrosis factor-alpha, interleukin 1beta, and nerve growth factor, for example, induce sphingomyelin hydrolysis to ceramide. Other agonists, such as platelet-derived growth factor, trigger further hydrolysis of ceramide to sphingosine and activate sphingosine kinase to form sphingosine 1-phosphate. These metabolites either stimulate or inhibit growth and may be cytotoxic (in some cases via induction of apoptosis), depending on which products are formed (or added exogenously), the cellular levels (and possibly intracellular localization), and the cell type. In Swiss 3T3 cells, for example, sphingosine and sphingosine 1-phosphate are growth stimulatory at low concentrations via calcium mobilization from intracellular stores and activation of the mitogen-activated protein kinase (MAP kinase) pathway and transcription factors (AP-1), but are toxic at high concentrations. High levels of endogenous sphingoid bases are also produced by inhibition of ceramide synthase by fumonisins, mycotoxins produced by Fusarium moniliforme, resulting in growth stimulation or toxicity. Thus, sphingolipid metabolites appear to serve as second messengers for growth factors, cytokines, and other "physiological" agonists and, when elevated abnormally, to lead to disease.

Animals↗