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M Liscovitch

Publications and source records attributed to M Liscovitch.

At least 37 records · Page 2Linked to original sources
PubMed1995

Role of phospholipase D in laminin-induced production of gelatinase A (MMP-2) in metastatic cells.

Metastatic spread depends critically upon the invasiveness of tumor cells, i.e. their ability to breach basement membranes by elaborating and secreting specific proteolytic enzymes such as gelatinase A (MMP-2). Laminin is a major constituent of the extracellular matrix that can trigger production of MMP-2 in metastatic cells, but not in non-metastatic cells. The present study was designed to examine the role of phospholipase D (PLD) and its product, phosphatidic acid, in the intracellular signal transduction mechanisms that mediate induction of MMP-2 by laminin. Here we show that stimulation of tumor cells with laminin results in a time- and dose-dependent activation of PLD. Laminin-induced production of MMP-2 is attenuated by 1-butanol, a competitive substrate of PLD that reduces PLD-catalyzed production of PA. Moreover, phosphatidic acid itself can induce production of MMP-2 in metastatic tumor cells. MMP-2 can also be induced by exposing the cells to exogenous bacterial PLD. Elevated cellular phosphatidic acid induces MMP-2 in metastatic ras-transformed 3T3 fibroblasts but, like laminin, fails to do so in normal cells. These data indicate that laminin-induced activation of PLD and consequent generation of phosphatidic acid are involved in a signal propagation pathway leading to induction of MMP-2 and enhanced invasiveness of metastatic tumor cells.

3T3 Cells↗
PubMed1994

Novel function of phosphatidylinositol 4,5-bisphosphate as a cofactor for brain membrane phospholipase D.

The activation of phospholipase D (PLD) is a receptor-mediated event that has been implicated in signal transduction and membrane traffic in eukaryotic cells. Little is known about the biochemical and molecular properties of signal-activated PLDs, and none has been isolated. Here we report that phosphatidylinositol 4,5-bisphosphate (PIP2) potently stimulates brain membrane PLD activity in vitro in a highly specific manner. PIP2 increases 10-fold the maximal activity of a partially purified PLD with an EC50 of < 0.5 mol %. Other acidic phospholipids, including phosphatidylinositol 4-phosphate, phosphatidylinositol, phosphatidylserine, and phosphatidic acid, are completely or nearly ineffective. Neomycin, a high affinity ligand of PIP2, inhibits membrane-bound PLD but has no effect on the activity of a detergent-solubilized or partially purified enzyme. The addition of PIP2 restores the sensitivity of partially purified PLD to neomycin inhibition, indicating that neomycin blocks membrane PLD activity by binding to endogenous PIP2. These results define a novel function of PIP2 as a cofactor for brain membrane PLD and suggest that PIP2 synthesis and hydrolysis could be important determinants in regulating PLD action in signal transduction and membrane transport.

Animals↗
PubMed1994

Modulation by sphingolipids of calcium signals evoked by epidermal growth factor.

Receptor-activated breakdown of complex sphingolipids has been proposed as a mechanism for generating sphingoid base-containing putative second messenger molecules whose actions may modulate responses to extracellular signals. In human epidermoid carcinoma A431 cells, sphingosine (1-10 microM) by itself had no effect on intracellular free calcium concentrations ([Ca2+]i), yet within seconds, markedly enhanced the epidermal growth factor (EGF)-evoked Ca2+ influx (by up to 2-fold), but failed to alter Ca2+ release from the intracellular stores. Ca2+ signals evoked by serum were not affected by sphingosine. The response to sphingosine was dose-dependent and saturable, exhibiting an EC50 of 2.3 microM. In contrast, a ceramide, N-acetylsphingosine (10 microM), sphingosine 1-phosphate (10 microM), and sphingosylphosphorylcholine (10 microM) inhibited EGF-evoked elevations in [Ca2+]i. The latter two compounds by themselves transiently increased [Ca2+]i. N-Octanoylsphingosine, N,N-dimethylsphingosine, sphingomyelin, and stearylamine were inactive. The potentiation of calcium signals by sphingosine occurred at all concentrations of EGF tested (0.15-15 nM) and did not alter the EGF receptor protein kinase activity as determined by antiphosphotyrosine immunoblotting. Antiphosphoserine immunoblotting revealed that sphingosine (10 microM for 3 min) increased the phosphoserine content of two proteins with approximate molecular masses of 40 and 70 kDa. Serine hyperphosphorylation of the 40-kDa protein was also observed in cells treated with EGF alone, whereas the intensity of the 70-kDa band was highest in cells treated with both sphingosine and EGF. The modulation of growth factor receptor-regulated signaling, including changes in [Ca2+]i, may constitute a mechanism by which elevations in cellular levels of specific sphingolipids, which occur transiently upon activation of certain receptors and chronically in sphingolipid storage diseases, exert their physiological and pathophysiological effects.

Calcium↗
PubMed1994

Formation of endogenous free sphingoid bases in cells induced by changing medium conditions.

Sphingoid bases are precursors and breakdown products of sphingolipids and may function as second messengers. Here we have tested the hypothesis that sphingoid bases are produced in cells in response to external stimuli. Using a high-performance liquid chromatography system, the pattern and the amounts of free sphingoid bases in various cell types (i.e., NIH-3T3, A431, NG108-15) were determined. The predominant sphingoid base in these mammalian cells was identified as C-18 sphingosine, followed by C-18 sphinganine (dihydrosphingosine). In all cells examined, the levels of endogenous sphingoid bases can be rapidly elevated by replacing cell-conditioned medium with Hepes-buffered saline or with fresh medium, causing a dramatic increase (up to 9.5-fold) in sphingosine levels within 60 min; sphinganine levels were raised to a lesser extent (up to 4.5-fold). Addition of ammonium ions inhibited the generation of sphingoid bases. These results suggest that the machinery for metabolizing sphingoid bases can be stimulated rapidly, although the exact nature of the stimulus remains obscure. Nevertheless, the ability to control sphingosine formation in cells by changing medium conditions provides a powerful tool for investigations of the physiological roles of endogenous sphingosine.

3T3 Cells↗
PubMed1994

Role of phospholipase-D and phosphatidic acid in mediating gonadotropin-releasing hormone-induced inhibition of preantral granulosa cell differentiation.

The activation of phospholipase-D (PLD) was previously implicated in mediating the differentiative action of GnRH on preovulatory granulosa cells. The activation of PLD and the action of its product, phosphatidic acid (PA), were further studied in preantral granulosa cells, where GnRH exerts an antidifferentiative effect. A GnRH receptor agonist (GnRH-A) activated PLD in the cells, causing a sustained elevation of phosphatidylethanol and a transient increase in cellular PA levels. PLD was also activated by 12-O-tetradecanoylphorbol-13-acetate (TPA). Both GnRH-A and TPA inhibited FSH-induced production of progesterone, a marker of granulosa cell differentiation. D,L-Propranolol, which elevates cellular PA by inhibiting its degradation, mimicked the antidifferentiative action of GnRH-A and TPA in a dose-dependent manner. Addition of PA similarly inhibited FSH-induced progesterone production in a dose-dependent manner. The effect of forskolin, which mimics the steroidogenic effect of FSH by elevating intracellular levels of cAMP, could also be suppressed by GnRH-A and PA. FSH- and cAMP-induced differentiation of preantral granulosa cells is characterized by cell rounding and breakdown of actin filament bundles. This effect is inhibited by GnRH-A and TPA as well as PA. It is concluded that activation of PLD and the resultant production of PA could mediate the antidifferentiative action of GnRH in preantral granulosa cells. Moreover, GnRH-induced PLD-generated signals counteract the FSH-induced cAMP-dependent signals that modulate the organization of the actin cytoskeleton characteristic of steroidogenic cells.

Actins↗
PubMed1993

Phospholipase D-catalyzed hydrolysis of phosphatidylcholine provides the choline precursor for acetylcholine synthesis in a human neuronal cell line.

To identify the metabolic pathway that generates choline (Cho) for acetylcholine (AcCho) from its storage pool in membrane phosphatidylcholine (PtdCho), human neuronal cells (LA-N-2) were radioisotopically labeled with 1-O-hexadecyl-2-hydroxy-sn-glycero(3)phospho[14C]choline. The compound was efficiently taken up by the cells and metabolically labelled PtdCho, Cho, AcCho, and phosphocholine pools. In pulse-chase experiments, the specific radioactivities of the metabolites of 1-O-hexadecyl-2-hydroxy-sn-glycero(3)-phospho[14C]choline indicated that it was rapidly acylated to Ptd-Cho and then hydrolyzed first to free Cho and not to phosphocholine or glycerophosphocholine. This Cho was subsequently converted to AcCho and to phosphocholine. In the absence of exogenous Cho, at least 15% of the total cellular AcCho pool was synthesized by this pathway in 1 h. The data demonstrate that the liberation of the free Cho precursor for AcCho synthesis from PtdCho can be accomplished in a one-step process, indicating the involvement of a phospholipase D-type enzyme. In the presence of hemicholinium-3, which inhibits Cho transport, the amount of intracellular [14C]Cho metabolites that accumulated during the chase period was higher than in control cells, indicating that PtdCho hydrolysis liberated Cho directly into the cytoplasm. These data show that cholinergic cells are characterized by an intracellular pathway, catalyzed by a phospholipase D, that generates Cho for AcCho synthesis from PtdCho. Abnormalities in the regulation of this pathway may contribute to selective vulnerability of cholinergic neurons in certain neurodegenerative diseases, e.g., Alzheimer disease.

Acetylcholine↗
PubMed1993

Distinct mechanisms of phospholipase D activation and attenuation utilized by different mitogens in NIH-3T3 fibroblasts.

The activation of phospholipase D (PLD) by platelet-derived growth factor (PDGF), prostaglandin F2 alpha and 12-O-tetradecanoylphorbol 13-acetate (TPA) was studied in NIH-3T3 fibroblasts. PLD activation was determined by measuring the production of both [3H]phosphatidic acid and [3H]phosphatidylpropanol (products of the PLD-catalyzed hydrolysis and transphosphatidylation reactions, respectively), in cells that were metabolically pre-labeled with [3H]oleic acid. All mitogens caused a rapid (within 2 min) activation of PLD. Activation of PLD by prostaglandin F2 alpha and PDGF was transient and declined to near basal levels by 15 min and 55 min, respectively. In contrast, TPA-induced activation of PLD was sustained for at least 60 min of incubation. A combination of maximally effective concentrations of PDGF and TPA stimulated PLD activity in a non-additive manner, while the effect of prostaglandin F2 alpha was additional to that of either PDGF or TPA. The protein kinase inhibitor staurosporine inhibited PLD activation by PDGF or TPA with almost identical dose/response curves. In contrast, staurosporine potentiated prostaglandin-F2 alpha-induced PLD activation. The specific protein kinase C inhibitor GF109203X (a bisindolylmaleimide) inhibited PLD activation by prostaglandin F2 alpha and PDGF at concentrations higher than those required for inhibition of PLD activation induced by TPA. Depletion of cellular protein kinase C abolished PLD activation by all three mitogens without affecting in vitro activity of membrane-bound PLD. The distinct kinetics of PLD activation and its differential susceptibility to protein kinase inhibitors suggest the existence of agonist-specific activation and/or inactivation mechanisms. The results indicate also that protein kinase C participates in the mechanism of PLD activation via PDGF, while the effect of prostaglandin F2 alpha involves a pathway independent of protein kinase C.

3T3 Cells↗
PubMed1993

Up-regulation of phospholipase D activity induced by overexpression of protein kinase C-alpha. Studies in intact Swiss/3T3 cells and in detergent-solubilized membranes in vitro.

The role of protein kinase C in the mechanism of phospholipase D activation by platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate was studied in Swiss/3T3 fibroblasts that overexpress protein kinase C-alpha. Production of [3H]phosphatidylpropanol (specific product of the phospholipase D-catalyzed transphosphatidylation reaction) was determined in cells which were prelabeled with [3H]oleic acid. Accumulation of [3H]phosphatidylpropanol in response to platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate was 2-3-fold greater in protein kinase C-alpha-overexpressing SF1.4 cells compared with the vector control cells, SC1. Basal [3H] phosphatidylpropanol production also was 2-fold higher in SF1.4 cells than in SC1 cells. Hence, -fold stimulation of basal phospholipase D activity by platelet-derived growth factor and 12-O-tetradecanoyl-phorbol-13-acetate was comparable in the two cell lines and was not significantly altered by the overexpression of protein kinase C-alpha. Similarly, overexpression of protein kinase C-alpha did not affect either the kinetics of phospholipase D activation nor its dependence on platelet-derived growth factor or 12-O-tetradecanoylphorbol-13-acetate concentration. In vitro assay of phospholipase D activity in membranes isolated from the cells, utilizing exogenous [3H]phosphatidylcholine as a substrate, revealed nearly 2-fold higher phospholipase D activity in SF1.4 cell membranes. Kinetic analysis of detergent-solubilized phospholipase D activity indicated that the apparent Vmax and Km of phospholipase D derived from SF1.4 and SF3.2 (protein kinase C-alpha-overexpressing) cells are significantly higher than those of phospholipase D from control cells. These results indicate that in Swiss/3T3 cells overexpression of protein kinase C-alpha elevates basal and agonist-stimulated phospholipase D activity in intact cells as well as phospholipase D activity in vitro. These data are consistent with the hypothesis that overexpression of protein kinase C-alpha up-regulates phospholipase D, leading to a constitutive higher level of enzyme activity. Thus, protein kinase C-alpha may play a role in regulating phospholipase D expression.

3T3 Cells↗
PubMed1993

Phospholipase D-mediated hydrolysis of phosphatidylcholine: role in cell signalling.

Studies carried out in many laboratories have demonstrated the activation of phospholipase D (PLD) by a variety of receptor agonists and in many cell types. The signal-dependent formation of phosphatidic acid (PA), by PLD-catalyzed hydrolysis of phosphatidylcholine (PC), may represent a novel and ubiquitous signal transduction pathway in mammalian cells. The mode(s) of coupling between agonist receptors and PLD activation are not well understood. Studies utilizing NIH-3T3 fibroblasts indicated that PLD activation by different mitogens involves distinct mechanisms. Protein kinase C (PKC) seems to play a role both as a mediator and as a modulator of PLD activation. The role of PKC was further examined in Swiss/3T3-derived fibroblasts which stably overexpress PKC-alpha. In these cells, both basal and agonist-stimulated PLD activity are higher than in control cells. In vitro analysis of PLD activity in detergent-solubilized cell membranes, utilizing exogenous C6-NBD-PC as fluorescent substrate, showed nearly 2-fold higher activity in membranes from cells that overexpress PKC-alpha. These results suggest that PKC-alpha may play a role in regulating PLD expression. The PLD product PA was identified as a precursor of 'late phase' diacylglycerol which, at least in some cases, was temporally correlated and causally related to the sustained activation of PKC. However, PA may itself act as an intracellular messenger in its own right, although immediate targets for its action have not yet been identified. Activation of phosphoinositide-phospholipase C, PLD and phospholipase A2 seems to comprise a signaling cascade which is typically utilized by most (if not all) Ca(2+)-mobilizing agonists.

3T3 Cells↗
PubMed1992

Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.

We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1992

Bimodal distribution of phosphatidic acid phosphohydrolase in NG108-15 cells. Modulation by the amphiphilic lipids oleic acid and sphingosine.

The properties and bimodal distribution of phosphatidic acid phosphohydrolase (PAP) were investigated in neuroblastoma X glioma hybrid NG108-15 cells. Two PAP activities distinguished by their differential sensitivity to Mg2+ and Triton X-100 were identified in the cytosolic and microsomal fractions. A digitonin permeabilization method was employed to study the basal distribution of the cytosolic PAP and its redistribution upon cell exposure to amphiphilic lipids. Under conditions which release 100% of the cytosolic marker enzyme lactate dehydrogenase, only 60% of total cellular PAP activity was released into the medium through the digitonin-induced membrane pores, suggesting that about 40% of the total are membrane associated. Elevated plasma-membrane levels of phosphatidic acid, accomplished by incubating cells with Streptomyces chromofuscus phospholipase D, did not affect the distribution of cytosolic PAP. In contrast, oleic acid induced a marked concentration-dependent redistribution of the cytosolic enzyme to the particulate fraction. PAP redistribution was completely abolished in the presence of the sphingoid base sphingosine, previously shown to inhibit PAP activity in vitro (Lavie, Y., Piterman, O. & Liscovitch, M. (1990) FEBS Lett. 277, 7-10). Thus, the distribution of cytosolic PAP is reciprocally regulated by a long-chain (fatty) acid and a long-chain (sphingoid) base which are breakdown products of phospholipids and sphingolipids, respectively. These effects might influence PAP function in glycerolipid metabolism and signal transduction under physiological and pathophysiological conditions.

Cations, Divalent↗
PubMed1992

Signal transduction mechanisms involved in carbachol-induced aldosterone secretion from bovine adrenal glomerulosa cells.

In cultured bovine adrenal glomerulosa cells, diacylglycerol content remains elevated for up to 75 min following the removal of angiotensin II. This maintained increase could provide a mechanism by which angiotensin II pretreatment may prime cells to secrete aldosterone in response to the calcium channel agonist Bay K 8644. In the present study we find that carbachol failed both to produce this persistent diacylglycerol elevation and to exert a priming effect. In addition, because carbachol was also a less potent activator of phospholipase D than angiotensin II, our results implicate phospholipase D in the maintained increase in diacylglycerol content observed following stimulation with and removal of angiotensin II. Carbachol also elicited changes in the radiolabeled levels of both myristate- and arachidonate-containing diacylglycerol. However, the rapid decline in diacylglycerol content following carbachol removal resembled the rapid fall in arachidonate-diacylglycerol; we therefore proposed that the diacylglycerol species generated with carbachol stimulation contains predominantly arachidonic acid. In summary, our results suggest that prolonged elevations in diacylglycerol content following removal of hormones such as angiotensin II, as well as the identity of the diacylglycerol species itself, may be important in the regulation of cellular responses.

Aldosterone↗
PubMed1992

Crosstalk among multiple signal-activated phospholipases.

Transduction of extracellular signals across the plasma membrane often involves activation of several phospholipases that generate multiple, sometimes interconvertible, lipid-derived messengers. Coordination and integration of these signal-activated phospholipases may require crosstalk between both the messengers and target protein constituents of these pathways.

Animals↗
PubMed1991

Delayed activation of phospholipase D by gonadotropin-releasing hormone in a clonal pituitary gonadotrope cell line (alpha T3-1).

Stimulation of cultured pituitary cells from a gonadotrope lineage (alpha T3-1) by the gonadotropin-releasing hormone agonist analog [D-Trp6]GnRH (GnRH-A) resulted in a manifold increase in accumulation of phosphatidylethanol, a specific product of phospholipase D phosphatidyl transferase activity when ethanol is the phosphatidyl group acceptor. Levels of the natural lipid product of phospholipase D, phosphatidic acid, were increased 2-3-fold. Activation of phospholipase D by GnRH-A was dose- and time-dependent and was blocked by a GnRH receptor antagonist [D-pClPhe2,D-Trp3.6]GnRH. GnRH-A stimulated phospholipase D activity after a lag of 1-2 min. We conclude that in alpha T3-1 gonadotropes GnRH receptor occupancy results in delayed activation of phospholipase D which could participate in late phases of gonadotrope regulation by the neurohormone.

Animals↗
PubMed1991

Inhibition of neural phospholipase D activity by aminoglycoside antibiotics.

The effects of aminoglycoside antibiotics on phospholipase D (PLD) activity were investigated in permeabilized NG108-15 cells and in isolated rat brain membranes. Neomycin inhibited guanosine 5'-[gamma-thio]triphosphate-stimulated PLD activity in digitonin-permeabilized NG108-15 cells in a concentration-dependent manner (50% inhibition at 100 microM). Neomycin similarly inhibited PLD activity present in rat brain membranes and assayed in vitro with [3H]phosphatidylcholine as substrate (50% inhibition at 65 microM). Other aminoglycosides tested (kanamycin, geneticin and streptomycin) were nearly equipotent inhibitors of rat brain PLD. These results indicate that aminoglycoside antibiotics inhibit phosphatidylcholine-PLD activity with comparable and sometimes greater potency than their well known inhibition of phosphoinositide-phospholipase C. The possibility that PLD inhibition could mediate some of the toxic side effects of aminoglycosides is suggested.

Aminoglycosides↗