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S Spiegel

Publications and source records attributed to S Spiegel.

At least 55 records · Page 3Linked to original sources
PubMed2000

Role of ceramide in neuronal cell death and differentiation.

Ceramide is a sphingolipid metabolite that has been implicated in cellular apoptosis and differentiation. It has been shown to induce apoptosis in various mammalian cell lines and, more recently, has been implicated in neuronal apoptosis. Although the mechanisms of ceramide-induced cell death have not been fully elucidated, they appear to involve a number of signal transduction pathways, including proline-directed kinases, phosphatases, phospholipases, transcription factors, and caspases. Interestingly, ceramide also appears to promote survival and differentiation in certain neuronal systems, when applied at lower concentrations and/or at different developmental stages. Together, studies to date indicate an important multipotential role for this lipid in cell death and differentiation.

Animals↗
PubMed2000

Sphingosine 1-phosphate: a ligand for the EDG-1 family of G-protein-coupled receptors.

Ample evidence indicates that sphingosine-1-phosphate (SPP) can serve as an intracellular second messenger regulating calcium mobilization, cell growth, and survival. Moreover, the dynamic balance between levels of the sphingolipids metabolites, ceramide, and SPP, and consequent regulation of opposing signaling pathways, is an important factor that determines whether a cell survives or dies. This ceramide/SPP rheostat is an evolutionarily conserved stress regulatory mechanism influencing growth and survival of yeast. In addition, SPP also has been identified as the ligand for the G-protein-coupled receptors EDG-1, -3, -5, and -6. Binding of SPP to EDG-1 regulates chemotaxis and in vitro angiogenesis of endothelial cells, whereas EDG-5, and possibly EDG-3, are likely the cell surface receptors responsible for cell rounding and neurite retractions induced by SPP. Hence, the studies identify a family of highly specific SPP receptors that are capable of mediating different biological responses. Thus, it is suggested that SPP is a prototype for a novel class of lipid mediators that act both extracellularly as ligands for cell surface receptors and intracellularly as second messengers. Recently, sphingosine kinase was purified to homogeneity and the first mammalian sphingosine kinase, the enzyme responsible for the formation of SPP, was cloned. The studies should provide the necessary tools to develop insight into the biological roles of this important bioactive sphingolipid.

GTP-Binding Proteins↗
PubMed2000

Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation.

Sphingolipid signaling pathways have been implicated in many critical cellular events. Sphingosine-1-phosphate (SPP), a sphingolipid metabolite found in high concentrations in platelets and blood, stimulates members of the endothelial differentiation gene (Edg) family of G protein-coupled receptors and triggers diverse effects, including cell growth, survival, migration, and morphogenesis. To determine the in vivo functions of the SPP/Edg signaling pathway, we disrupted the Edg1 gene in mice. Edg1(-/-) mice exhibited embryonic hemorrhage leading to intrauterine death between E12.5 and E14.5. Vasculogenesis and angiogenesis appeared normal in the mutant embryos. However, vascular maturation was incomplete due to a deficiency of vascular smooth muscle cells/pericytes. We also show that Edg-1 mediates an SPP-induced migration response that is defective in mutant cells due to an inability to activate the small GTPase, Rac. Our data reveal Edg-1 to be the first G protein-coupled receptor required for blood vessel formation and show that sphingolipid signaling is essential during mammalian development.

Animals↗
PubMed1999

Sphingosine-1-phosphate inhibits motility of human breast cancer cells independently of cell surface receptors.

Exogenous sphingosine-1-phosphate (SPP) inhibits chemotactic motility of several transformed cell lines. We have found that SPP at high micromolar concentrations decreased chemotaxis of estrogen-independent (MDA-MB-231 and BT 549) and estrogen-dependent (MCF-7 and ZR-75-1) human breast cancer cells. Because SPP has been implicated as a lipid-signaling molecule with novel dual intra- and intercellular actions, it was of interest to determine whether the effect of SPP on chemotactic motility of human breast cancer cells is mediated intracellularly or through the recently identified endothelial differentiation gene (EDG) family of G protein-coupled SPP receptors. There was no detectable specific binding of [32P]SPP to MDA-MB-231 or MCF-7 cells; however, reverse transcription-PCR analysis revealed that both MDA-MB-231 and MCF-7 cells expressed moderate levels of EDG-3, neither expressed EDG-1, and EDG-5 mRNA was expressed in MCF-7 but not in MDA-MB-231 cells. In contrast to SPP, sphinganine-1-phosphate, which binds to and signals through SPP receptors EDG-1, EDG-3, and EDG-5, had no effect on chemotactic motility of MDA-MB-231 or MCF-7 cells. To further discriminate between intracellular and receptor-mediated actions of SPP, we used caged SPP, a photolyzable derivative of SPP that elevates intracellular levels of SPP after illumination. Caged SPP inhibited chemotactic motility of MDA-MB-231 cells only upon UV irradiation. In addition, in MCF-7 cells, overexpression of sphingosine kinase, the enzyme that produces SPP, inhibited chemotactic motility compared with vector-transfected cells and markedly increased cellular SPP levels in the absence of detectable secretion. Our results suggest that the inhibitory effect of SPP on chemotactic motility of human breast cancer cells is likely mediated through intracellular actions of SPP rather than through cell surface receptors.

Breast Neoplasms↗
PubMed1999

Sphingosine 1-phosphate stimulates cell migration through a G(i)-coupled cell surface receptor. Potential involvement in angiogenesis.

Sphingosine 1-phosphate (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low concentrations of SPP (10-100 nM) increased chemotaxis of HEK293 cells overexpressing the G protein-coupled SPP receptor EDG-1. In agreement with previous findings in human breast cancer cells (Wang, F., Nohara, K., Olivera, O., Thompson, E. W., and Spiegel, S. (1999) Exp. Cell Res. 247, 17-28), SPP, at micromolar concentrations, inhibited chemotaxis of both vector- and EDG-1-overexpressing HEK293 cells. Nanomolar concentrations of SPP also induced a marked increase in chemotaxis of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC), which express the SPP receptors EDG-1 and EDG-3, while higher concentrations of SPP were less effective. Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i)-coupled receptors, blocked SPP-induced chemotaxis. Checkerboard analysis indicated that SPP stimulates both chemotaxis and chemokinesis. Taken together, these data suggest that SPP stimulates cell migration by binding to EDG-1. Similar to SPP, sphinganine 1-phosphate (dihydro-SPP), which also binds to this family of SPP receptors, enhanced chemotaxis; whereas, another structurally related lysophospholipid, lysophosphatidic acid, did not compete with SPP for binding nor did it have significant effects on chemotaxis of endothelial cells. Furthermore, SPP increased proliferation of HUVEC and BAEC in a pertussis toxin-sensitive manner. SPP and dihydro-SPP also stimulated tube formation of BAEC grown on collagen gels (in vitro angiogenesis), and potentiated tube formation induced by basic fibroblast growth factor. Pertussis toxin treatment blocked SPP-, but not bFGF-stimulated in vitro angiogenesis. Our results suggest that SPP may play a role in angiogenesis through binding to endothelial cell G(i)-coupled SPP receptors.

Animals↗
PubMed1999

Liposomal ET-18-OCH(3) induces cytochrome c-mediated apoptosis independently of CD95 (APO-1/Fas) signaling.

ELL-12, a liposome formulation of the ether-lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH(3)), is a nonmyelosuppressive antiproliferative agent that is more effective and less toxic than the ether lipid itself in tumor model systems. We found that ELL-12 induced apoptosis in Jurkat, H9, and U-937 cells that was preceded by activation of executioner caspases. In addition, ELL-12 triggered release of cytochrome c from mitochondria to the cytoplasm before caspase-9 activation. Apoptosis, activation of caspases, and cytochrome c release were blocked by Bcl-x(L) overexpression in Jurkat T cells, suggesting a critical role for mitochondria in ELL-12-triggered cell death. Furthermore, ELL-12 had no effect on expression of CD95 ligand, and inhibition of the Fas signaling pathway with antagonistic anti-CD95 antibody did not affect apoptosis induced by ELL-12. Hence, ELL-12 could be a promising adjunct for the treatment of tumors in addition to myelosuppressive chemotherapeutic drugs and/or those that use the CD95-ligand/receptor system to trigger apoptosis.

Antineoplastic Agents↗
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↗
PubMed1999

MSE55, a Cdc42 effector protein, induces long cellular extensions in fibroblasts.

Cdc42 is a member of the Rho GTPase family that regulates multiple cellular activities, including actin polymerization, kinase-signaling activation, and cell polarization. MSE55 is a nonkinase CRIB (Cdc42/Rac interactive-binding) domain-containing molecule of unknown function. Using glutathione S-transferase-capture experiments, we show that MSE55 binds to Cdc42 in a GTP-dependent manner. MSE55 binding to Cdc42 required an intact CRIB domain, because a MSE55 CRIB domain mutant no longer interacted with Cdc42. To study the function of MSE55 we transfected either wild-type MSE55 or a MSE55 CRIB mutant into mammalian cells. In Cos-7 cells, wild-type MSE55 localized at membrane ruffles and increased membrane actin polymerization, whereas expression of the MSE55 CRIB mutant showed fewer membrane ruffles. In contrast to these results, MSE55 induced the formation of long, actin-based protrusions in NIH 3T3 cells as detected by immunofluorescence and live-cell video microscopy. MSE55-induced protrusion formation was blocked by expression of dominant-negative N17Cdc42, but not by expression of dominant-negative N17Rac. These findings indicate that MSE55 is a Cdc42 effector protein that mediates actin cytoskeleton reorganization at the plasma membrane.

Amino Acid Substitution↗
PubMed1999

Enzymatic measurement of sphingosine 1-phosphate.

Sphingosine 1-phosphate (SPP) is a sphingolipid metabolite which has novel dual actions acting as both an intracellular second messenger and a ligand for a family of G protein-coupled receptors. This paper describes a rapid enzymatic method to quantify mass levels of SPP in serum, mammalian tissues, and cultured cells. The assay utilizes an alkaline lipid extraction to selectively separate SPP from other phospholipids and sphingolipids, including sphingosine. Extracted SPP is efficiently converted to sphingosine by alkaline phosphatase treatment. Sphingosine thus formed is then quantitatively phosphorylated to [(32)P]SPP using recombinant sphingosine kinase and [gamma-(32)P]ATP. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.25 pmol to 2.5 nmol. In various rat tissues, levels of SPP varied between 0. 5 and 6 pmol/mg wet wt. The lowest levels were found in heart and testes, while brain contained the highest levels. The method was adapted easily to measure minute amounts of SPP present in various cultured cell types. The amount of SPP in cell extracts was proportional to the cell number and varied between 0.04 and 2 pmol/10(6) cells. Concurrent measurements of sphingosine levels revealed that its concentration was significantly higher than SPP in most cells and tissues. Furthermore, with this assay we were able to measure increases in intracellular SPP levels in rat pheochromocytoma PC12 cells after treatment with exogenous sphingosine or with nerve growth factor which stimulates sphingosine kinase activity.

Adenosine Triphosphate↗
PubMed1999

Differential effects of free and liposome-associated 1-O-octadecyl-2-O-methylglycerophosphocholine on protein kinase C.

Incorporation of ET-18-OCH3 into well-characterized liposomes known as ELL-12 has eliminated its gastrointestinal and hemolytic toxicity without loss of growth inhibiting activity. ET-18-OCH3, but not ELL-12, blunted the increase in membrane protein kinase C (PKC) activity induced by 12-O-tetradecanoylphorbol 13-myristate (TPA) and markedly reduced levels of PKC alpha in NIH 3T3 fibroblasts. Furthermore, prolonged treatment with ELL-12 neither inhibited TPA-induced translocations of PKC alpha and PKC delta to the particulate fraction nor caused down-regulation, and did not affect the cellular distribution of TPA-insensitive PKC zeta. In Jurkat T cells, where ELL-12 markedly induced apoptosis that was blocked by an inhibitor of caspase-3-like activities, it had no effect on PKC activity or translocation induced by TPA. Thus, it seems unlikely that PKC is involved in the therapeutic effects of ELL-12.

3T3 Cells↗
PubMed1999

Tumor necrosis factor-alpha sensitizes prostate cancer cells to gamma-irradiation-induced apoptosis.

LNCaP prostate cancer cells are highly resistant to induction of programmed cell death by y-irradiation and somewhat sensitive to the death-inducing effects of tumor necrosis factor (TNF)-alpha. Simultaneous exposure of LNCaP cells to TNF-alpha and 8 Gy of irradiation was synergistic and resulted in a 3-fold increase of apoptotic cells within 72 h compared to TNF-alpha alone. It appeared that TNF-alpha sensitized the cells to irradiation because, when cells were irradiated 24 h after exposure to TNF-alpha, increased cell death was observed. In contrast, irradiation delivered 24 h prior to TNF-alpha exposure did not result in more cell death than after TNF-alpha alone. TNF-alpha induced expression of its own mRNA, but TNF-alpha mRNA induction was neither induced nor enhanced by irradiation. Activation of the transcription factor nuclear factor kappaB can be induced by TNF-alpha and has a modulating antiapoptotic effect. But enhancement of TNF-alpha-induced cell death by irradiation did not result from altered activation of nuclear factor kappaB. TNF-alpha treatment of LNCaP cells resulted in partial activation of caspase-8 and -6 but not caspase-3. There was only minimal poly(ADP-ribose) polymerase cleavage seen in LNCaP cells after exposure to both TNF-alpha and irradiation at 72 h, a time when 60% of the cells were apoptotic. Experiments with peptide inhibitors of cysteine and serine proteases suggested that caspases were the predominant mediators of apoptosis induced by TNF-alpha alone but that serine proteases contributed significantly to cell death induced by TNF-alpha plus irradiation. TNF-alpha increased production of ceramide in LNCaP cells 48 h after exposure. Although irradiation alone had no effect on ceramide production in LNCaP cells, TNF-alpha plus irradiation induced significantly more ceramide than TNF-alpha alone. Ceramide production did not occur immediately after exposure to TNF-alpha, but rather was delayed such that ceramide levels were increased only 24 h after exposure to apoptotic stimuli. Moreover, non-toxic levels of exogenous C2-ceramide sensitized LNCaP cells to irradiation similarly to TNF-alpha, suggesting that one mechanism by which LNCaP cells were sensitized to irradiation was by increased intracellular ceramide. Hence, ceramide generation is a critical component in radiation-induced apoptosis in human prostate cancer cells. Inhibition of ceramide generation may provide a selective advantage in the development of radioresistance in prostate cancer.

Apoptosis↗
PubMed1999

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate.

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397-transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.

Breast Neoplasms↗
PubMed1999

Sphingosine 1-phosphate-induced cell rounding and neurite retraction are mediated by the G protein-coupled receptor H218.

Sphingosine 1-phosphate (SPP) is a lipid second messenger that also acts as a first messenger through the G protein-coupled receptor Edg-1. Here we show that SPP also binds to the related receptors H218 and Edg-3 with high affinity and specificity. SPP and sphinganine 1-phosphate bind to these receptors, whereas neither sphingosylphosphorylcholine nor lysophosphatidic acid compete with SPP for binding to either receptor. Transfection of HEK293 cells with H218 or edg-3, but not edg-1, induces rounded cell morphology in the presence of serum, which contains high levels of SPP. SPP treatment of cells overexpressing H218 cultured in delipidated serum causes cell rounding. A similar but less dramatic effect was observed in cells overexpressing Edg-3 but not with Edg-1. Cell rounding was correlated with apoptotic cell death, probably as a result of loss of attachment. Nerve growth factor-induced neuritogenesis in PC12 cells was inhibited by overexpression of H218 and to a lesser extent Edg-3. SPP treatment rapidly enhanced neurite retraction in PC12 cells overexpressing Edg-1, Edg-3, or H218. Thus, H218, and possibly Edg-3, may be the cell surface receptors responsible for cell rounding and neurite retraction induced by SPP. Moreover, the identification of these two additional SPP receptors indicates that a family of highly specific receptors exists that mediate different responses to SPP.

Animals↗
PubMed1999

Sphingosine 1-phosphate: a prototype of a new class of second messengers.

Sphingosine 1-phosphate (SPP) is an important sphingolipid-derived second messenger in mammalian cells that acts to promote proliferation and to inhibit apoptosis. Various growth factors increase the intracellular concentration of SPP by activating sphingosine kinase, the molecular cloning of which has revealed that it defines a new type of lipid kinase. Cell fate is influenced by the balance between the intracellular concentration of SPP and that of ceramide, a pro-apoptotic sphingolipid metabolite. The observation that a similar "rheostat" is a determinant of cell survival in yeast cells exposed to heat shock indicates that it is an evolutionarily conserved mechanism of stress regulation. SPP also acts extracellularly to inhibit cell motility and to influence cell morphology, effects that appear to be mediated by the G protein-coupled receptor EDG1. These observations indicate that SPP is the prototype of a new class of lipid mediators that exert both intracellular and extracellular actions.

Animals↗
PubMed1999

Ligand-induced trafficking of the sphingosine-1-phosphate receptor EDG-1.

The endothelial-derived G-protein-coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1-green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP-EDG-1 interaction. SPP binds to EDG-1-GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1-GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a tau1/2 of approximately 15 min. The EDG-1-GFP-containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1-GFP recycles back to the plasma membrane with a tau1/2 of approximately 30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.

Amino Acid Sequence↗
PubMed1999

Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.

Sphingosine-1-phosphate, a sphingolipid metabolite, is involved in the mitogenic response of platelet-derived growth factor (PDGF) and is formed by activation of sphingosine kinase. We examined the effect of PDGF on sphingosine kinase activation in TRMP cells expressing wild-type or various mutant betaPDGF receptors. Sphingosine kinase was stimulated by PDGF in cells expressing wild-type receptors but not in cells expressing kinase-inactive receptors (R634). Cells expressing mutated PDGF receptors with phenylalanine substitutions at five major tyrosine phosphorylation sites 740/751/771/1009/1021 (F5 mutants), which are unable to associate with PLCgamma, phosphatidylinositol 3-kinase, Ras GTPase-activating protein, or protein tyrosine phosphatase SHP-2, not only failed to increase DNA synthesis in response to PDGF but also did not activate sphingosine kinase. Moreover, mutation of tyrosine-1021 of the PDGF receptor to phenylalanine, which impairs its association with PLCgamma, abrogated PDGF-induced activation of sphingosine kinase. In contrast, PDGF was still able to stimulate sphingosine kinase in cells expressing the PDGF receptor mutated at tyrosines 740/751 and 1009, responsible for binding of phosphatidylinositol 3-kinase and SHP-2, respectively. In agreement, PDGF did not stimulate sphingosine kinase activity in F5 receptor 'add-back' mutants in which association with the Ras GTPase-activating protein, phosphatidylinositol 3-kinase, or SHP-2 was individually restored. However, a mutant PDGF receptor that was able to bind PLCgamma (tyrosine-1021), but not other signaling proteins, restored sphingosine kinase sensitivity to PDGF. These data indicate that the tyrosine residue responsible for binding of PLCgamma is required for PDGF-induced activation of sphingosine kinase. Moreover, calcium mobilization downstream of PLCgamma, but not protein kinase C activation, appears to be required for stimulation of sphingosine kinase by PDGF.-Olivera, A., Edsall, J., Poulton, S., Kazlauskas, A., Spiegel, S. Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗