ABSTRACT
Phospholipase D (PLD) is a ubiquitous enzyme that cleaves the distal phosphoester bond of phospholipids generating phosphatidic acid (PA). In plants, PA is involved in numerous cell responses triggered by stress. Similarly, in mammals, PA is also a second messenger involved in tumorigenesis. PLD is nowadays considered as a therapeutic target and blocking its activity with specific inhibitors constitutes a promising strategy to treat cancers. Starting from already described PLD inhibitors, this study aims to investigate the effect of their structural modifications on the enzyme's activity, as well as identifying new potent inhibitors of eukaryotic PLDs. Being able to purify the plant PLD from Vigna unguiculata (VuPLD), we obtained a SAXS model of its structure. We then used a fluorescence-based test suitable for high-throughput screening to review the effect of eukaryotic PLD inhibitors described in the literature. In this regard, we found that only few molecules were in fact able to inhibit VuPLD and we confirmed that vanadate is the most potent of all with an IC50 around 58 µM. Moreover, the small-scale screening of a chemical library of 3120 compounds allowed us to optimize the different screening's steps and paved the way towards the discovery of new potent inhibitors.
Subject(s)
Drug Evaluation, Preclinical , Enzyme Inhibitors/analysis , Enzyme Inhibitors/pharmacology , Phospholipase D/antagonists & inhibitors , Alcohols/pharmacology , High-Throughput Screening Assays , Humans , Hydrolysis , Phospholipase D/metabolism , Salts/pharmacology , Scattering, Small Angle , Vanadates/pharmacology , Vigna/enzymology , X-Ray DiffractionABSTRACT
Phospholipase D1 (PLD1) plays a crucial role in various inflammatory and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease. However, the role of PLD1 in the pathogenesis of RA remains unknown. Here, we first investigated the role and effects of PLD1 in collagen-induced arthritis (CIA) and found that genetic and pharmacological inhibition of PLD1 in DBA1/J mice with CIA reduced the incidence of CIA, decreased the clinical score, and abrogated disease symptoms including infiltration of leukocytes, synovial inflammation, bone erosion, and cartilage destruction. Moreover, ablation and inhibition of PLD1 suppressed the production of type II collagen-specific IgG2a autoantibody and proinflammatory cytokines, accompanied by an increase in the regulatory T (Treg) cell population and a decrease in the Th17 cell population in CIA mice. The PLD1 inhibitor also promoted differentiation of Treg cells and suppressed differentiation of Th17 cells in vitro. Furthermore, the PLD1 inhibitor attenuated pathologic bone destruction in CIA mice by suppressing osteoclastogenesis and bone resorption. Thus, our findings indicate that the targeting of PLD1 can ameliorate CIA by modulating the imbalance of Treg and Th17 cells and suppressing osteoclastogenesis, which might be a novel strategy to treat autoimmune diseases, such as RA.
Subject(s)
Arthritis, Experimental/prevention & control , Benzimidazoles/pharmacology , Osteogenesis/drug effects , Phospholipase D/antagonists & inhibitors , Piperidines/pharmacology , T-Lymphocytes, Regulatory/drug effects , Th17 Cells/drug effects , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/metabolism , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/prevention & control , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cytokines/blood , Disease Models, Animal , Knee Joint/drug effects , Knee Joint/metabolism , Knee Joint/pathology , Male , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Osteogenesis/genetics , Phospholipase D/genetics , Phospholipase D/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/immunology , Th17 Cells/metabolism , X-Ray MicrotomographyABSTRACT
N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.
Subject(s)
Behavior, Animal/drug effects , Enzyme Inhibitors/chemistry , Lipid Metabolism/drug effects , Phosphatidylethanolamines/metabolism , Phospholipase D/antagonists & inhibitors , Amidohydrolases/metabolism , Animals , Blood Proteins/metabolism , Brain/metabolism , Cannabinoid Receptor Antagonists/metabolism , Cell Line, Tumor , Drug Evaluation, Preclinical , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacokinetics , Fear/drug effects , Humans , Male , Mice , Mice, Inbred C57BL , Molecular Structure , Receptors, Cannabinoid/metabolism , Signal TransductionABSTRACT
BACKGROUND: Breast cancer is the most common cancer in women. Despite high survival rates in Western countries, treatments are less effective in metastatic cases and triple-negative breast cancer (TNBC) patient survival is the shortest across breast cancer subtypes. High expression levels of stearoyl-CoA desaturase-1 (SCD1) have been reported in breast cancer. The SCD1 enzyme catalyzes the formation of oleic acid (OA), a lipid stimulating the migration of metastatic breast cancer cells. Phospholipase activity is also implicated in breast cancer metastasis, notably phospholipase D (PLD). METHODS: Kaplan-Meier survival plots generated from gene expression databases were used to analyze the involvement of SCD1 and PLD in several cancer subtypes. SCD1 enzymatic activity was modulated with a pharmaceutical inhibitor or by OA treatment (to mimic SCD1 over-activity) in three breast cancer cell lines: TNBC-derived MDA-MB-231 cells as well as non-TNBC MCF-7 and T47D cells. Cell morphology and migration properties were characterized by various complementary methods. RESULTS: Our survival analyses suggest that SCD1 and PLD2 expression in the primary tumor are both associated to metastasis-related morbid outcomes in breast cancer patients. We show that modulation of SCD1 activity is associated with the modification of TNBC cell migration properties, including changes in speed, direction and cell morphology. Cell migration properties are regulated by SCD1 activity through a PLD-mTOR/p70S6K signaling pathway. These effects are not observed in non-TNBC cell lines. CONCLUSION: Our results establish a key role for the lipid desaturase SCD1 and delineate an OA-PLD-mTOR/p70S6K signaling pathway in TNBC-derived MDA-MB-231 cell migration.
Subject(s)
Cell Movement , Stearoyl-CoA Desaturase/metabolism , Triple Negative Breast Neoplasms/pathology , Cell Line, Tumor , Datasets as Topic , Disease-Free Survival , Female , Humans , Kaplan-Meier Estimate , Neoplasm Metastasis , Oleic Acid/metabolism , Phospholipase D/antagonists & inhibitors , Phospholipase D/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Triple Negative Breast Neoplasms/mortality , Triple Negative Breast Neoplasms/surgeryABSTRACT
Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.
Subject(s)
Arthropod Proteins/antagonists & inhibitors , Brown Recluse Spider/enzymology , Drug Design , Phospholipase D/antagonists & inhibitors , Spider Venoms/antagonists & inhibitors , Animals , Arthropod Proteins/chemistry , Arthropod Proteins/genetics , Benzimidazoles/pharmacology , Brown Recluse Spider/genetics , Brown Recluse Spider/pathogenicity , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hemolysis/drug effects , Humans , Kinetics , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Necrosis , Phospholipase D/chemistry , Phospholipase D/genetics , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Piperidines/pharmacology , Rabbits , Recombinant Proteins/genetics , Skin/drug effects , Skin/pathology , Spider Bites/drug therapy , Spider Bites/enzymology , Spider Venoms/chemistry , Spider Venoms/genetics , Suramin/pharmacologyABSTRACT
Here we developed a simple, sensitive and accurate PLD detection method based on a target-controlled gating liposome (TCGL) "off-on" cascade amplified strategy and personal glucose meters (PGMs). It showed excellent sensitivity with a detection limit of 0.005 U L(-1) and well performed PLD activity analysis in breast cancer cells and inhibitor drug screening.
Subject(s)
Blood Glucose Self-Monitoring/instrumentation , Breast Neoplasms/enzymology , Enzyme Assays/instrumentation , Phospholipase D/analysis , Amylose/metabolism , Biosensing Techniques/instrumentation , Cell Line, Tumor , Drug Evaluation, Preclinical/instrumentation , Female , Glucan 1,4-alpha-Glucosidase/metabolism , Glucose/analysis , Glucose/metabolism , Humans , Limit of Detection , Liposomes/metabolism , Liposomes/ultrastructure , Phospholipase D/antagonists & inhibitors , Phospholipase D/metabolismABSTRACT
Long-term memory (LTM) of fear stores activity dependent modifications that include changes in amygdala signaling. Previously, we identified an enhanced probability of release of glutamate mediated signaling to be important in rat fear potentiated startle (FPS), a well-established translational behavioral measure of fear. Here, we investigated short- and long-term synaptic plasticity in FPS involving metabotropic glutamate receptors (mGluRs) and associated downstream proteomic changes in the thalamic-lateral amygdala pathway (Th-LA). Aldolase A, an inhibitor of phospholipase D (PLD), expression was reduced, concurrent with significantly elevated PLD protein expression. Blocking the PLD-mGluR signaling significantly reduced PLD activity. While transmitter release probability increased in FPS, PLD-mGluR agonist and antagonist actions were occluded. In the unpaired group (UNP), blocking the PLD-mGluR increased while activating the receptor decreased transmitter release probability, consistent with decreased synaptic potentials during tetanic stimulation. FPS Post-tetanic potentiation (PTP) immediately following long-term potentiation (LTP) induction was significantly increased. Blocking PLD-mGluR signaling prevented PTP and reduced cumulative PTP probability but not LTP maintenance in both groups. These effects are similar to those mediated through mGluR7, which is co-immunoprecipitated with PLD in FPS. Lastly, blocking mGluR-PLD in the rat amygdala was sufficient to prevent behavioral expression of fear memory. Thus, our study in the Th-LA pathway provides the first evidence for PLD as an important target of mGluR signaling in amygdala fear-associated memory. Importantly, the PLD-mGluR provides a novel therapeutic target for treating maladaptive fear memories in posttraumatic stress and anxiety disorders.
Subject(s)
Amygdala/physiology , Fear/physiology , Long-Term Potentiation , Phospholipase D/physiology , Receptors, Metabotropic Glutamate/physiology , Reflex, Startle/physiology , Amygdala/enzymology , Animals , Conditioning, Classical/drug effects , Conditioning, Classical/physiology , Cyclopropanes/pharmacology , Electric Stimulation , Excitatory Postsynaptic Potentials/drug effects , Fear/drug effects , Fructose-Bisphosphate Aldolase/metabolism , Glycine/analogs & derivatives , Glycine/pharmacology , Long-Term Potentiation/drug effects , Male , Memory, Long-Term/drug effects , Memory, Long-Term/physiology , Neural Pathways/drug effects , Neural Pathways/physiology , Phospholipase D/antagonists & inhibitors , Phospholipase D/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Reflex, Startle/drug effects , Thalamus/physiologyABSTRACT
In eukaryotic cells, phosphatidic acid (PA) and diacylglycerol (DAG), are at the origin of all membrane glycerolipids. Their interconversion is achieved by dephosphorylation of PA and phosphorylation of DAG: they form therefore a metabolic hub. PA and DAG are also known to be versatile signaling molecules. Two independent pharmacological screenings conducted on plant and human targets, led to the discovery of a new family of compounds acting on enzymes binding to either PA or DAG, in biological contexts that seemed initially independent. On the one hand, in plants, monogalactosyldiacylglycerol synthases (MGDG synthases or MGD) are responsible for the synthesis of MGDG, which is the most profuse lipid of photosynthetic membranes, and thus essential for metabolism and development. MGD use DAG as substrate. On the other hand, in mammals, phospholipases D (PLD), that produce PA, are involved in a variety of signaling cascades that control a broad spectrum of cellular functions, and play a role in the development of cancers. The two independent pharmacological screenings described in this review aimed to identify inhibitory molecules of either MGD of the plant model Arabidopsis, or human PLD. In both cases, the obtained molecules are piperidinyl-benzimidazolone derivatives, thereby allowing to propose this family of molecules as a novel source of inspiration for the search of compounds interfering with glycerolipid metabolism, that could be useful for other biological and therapeutics contexts.
Subject(s)
Drug Discovery/methods , Drug Evaluation, Preclinical , Enzyme Inhibitors/isolation & purification , Glycerides/antagonists & inhibitors , Glycerides/metabolism , Lipid Metabolism/drug effects , Galactosyltransferases/antagonists & inhibitors , Humans , Inventions , Phospholipase D/antagonists & inhibitors , PlantsABSTRACT
Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based short hairpin RNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, P<0.05) and their onset delayed when compared with control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (5-fluoro-2-indolyl des-chlorohalopemide and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide). These inhibitors led to significant (>70%, P<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid, Wiscott-Aldrich Syndrome protein, growth receptor-bound protein 2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows for the first time that PLD2 has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target.
Subject(s)
Breast Neoplasms/pathology , Phospholipase D/metabolism , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Female , Humans , Lung Neoplasms/secondary , MCF-7 Cells , Mice , Mice, SCID , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Transplantation , Phosphatidic Acids/biosynthesis , Phospholipase D/antagonists & inhibitors , Phospholipase D/genetics , Protein Isoforms/metabolism , RNA Interference , RNA, Small Interfering , Signal TransductionABSTRACT
The impact of polyunsaturated fatty acid (PUFA) supplementation on phospholipase D (PLD) trafficking and activity in mast cells was investigated. The enrichment of mast cells with different PUFA including α-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) revealed a PUFA-mediated modulation of the mastoparan-stimulated PLD trafficking and activity. All PUFA examined, except AA, prevented the migration of the PLD1 to the plasma membrane. For PLD2 no PUFA effects on trafficking could be observed. Moreover, PUFA supplementation resulted in an increase of mastoparan-stimulated total PLD activity, which correlated with the number of double bonds of the supplemented fatty acids. To investigate, which PLD isoform was affected by PUFA, stimulated mast cells were supplemented with DHA or AA in the presence of specific PLD-isoform inhibitors. It was found that both DHA and AA diminished the inhibition of PLD activity in the presence of a PLD1 inhibitor. By contrast, only AA diminished the inhibition of PLD activity in the presence of a PLD2 inhibitor. Thus, PUFA modulate the trafficking and activity of PLD isoforms in mast cells differently. This may, in part, account for the immunomodulatory effect of unsaturated fatty acids and contributes to our understanding of the modulation of mast cell activity by PUFA.
Subject(s)
Fatty Acids, Unsaturated/pharmacology , Mast Cells/enzymology , Phospholipase D/metabolism , Animals , Dogs , Enzyme Inhibitors/pharmacology , Green Fluorescent Proteins/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Mast Cells/drug effects , Mice , Phospholipase D/antagonists & inhibitors , Protein Transport/drug effectsABSTRACT
Shiga toxin (Stx) causes diarrhea-associated hemolytic uremic syndrome by damaging renal microvascular endothelium. The pentameric B subunits of Stx types 1 and 2 (Stx1B and Stx2B) are sufficient to stimulate acute VWF secretion from endothelial cells, but Stx1B and Stx2B exert distinct effects on Ca(2+) and cAMP pathways. Therefore, we investigated other signaling components in StxB-induced VWF exocytosis. Incubation of HUVECs with StxB transiently increased phospholipase D (PLD) activity. Inhibition of PLD activity or shRNA-mediated PLD1 knockdown abolished StxB-induced VWF secretion. In addition, treatment with StxB triggered actin polymerization, enhanced endothelial monolayer permeability, and activated RhoA. PLD activation and VWF secretion induced by Stx1B were abolished on protein kinase Cα (PKCα) inhibition or gene silencing but were only moderately reduced by Rho or Rho kinase inhibitors. Conversely, PLD activation and VWF exocytosis induced by Stx2B were reduced by Rho/Rho kinase inhibitors and dominant-negative RhoA, whereas attenuation of PKCα did not affect either process. Another PLD1 activator, ADP-ribosylation factor 6, was involved in VWF secretion induced by Stx1B or Stx2B, but not histamine. These data indicate that Stx1B and Stx2B induce acute VWF secretion in a PLD1-dependent manner but do so by differentially modulating PKCα, RhoA, and ADP-ribosylation factor 6.
Subject(s)
Phospholipase D/physiology , Shiga Toxins/pharmacology , von Willebrand Factor/metabolism , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Cells, Cultured , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Enzyme Activation/physiology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Permeability/drug effects , Phospholipase D/antagonists & inhibitors , Phospholipase D/genetics , Phospholipase D/metabolism , Protein Kinase C-alpha/metabolism , Protein Subunits/metabolism , Protein Subunits/pharmacology , RNA, Small Interfering/pharmacology , Shiga Toxin 2/pharmacology , Shiga Toxins/chemistry , Time Factors , rhoA GTP-Binding Protein/metabolismABSTRACT
Hexanal, an inhibitor of phospholipase D, has been successfully applied for the pre- and post-harvest treatment of fruits, vegetables and flowers. Changes in gene expression induced by hexanal and the ethylene antagonist 1-MCP, were analyzed by microarray using TOM2 tomato oligo-array containing approximately 12 000 unigenes. Mature green tomato fruits were treated with 1-MCP and hexanal, RNA isolated after 10 days of storage, and labeled cDNA synthesized for microarray analysis. A large variation in gene expression profile was observed in 1-MCP-treated fruits. Genes for ethylene biosynthetic pathway enzymes such as ACC- synthase/oxidase, ethylene receptor and ethylene response factors were heavily down-regulated in 1-MCP-treated fruits. In addition, genes for key enzymes involved in cell wall degradation and carotenoid development pathways were down-regulated. Hexanal treatment significantly down-regulated ACC-synthase, and to a lesser extent, other components of ethylene signal transduction. By contrast to MCP-treated fruits, hexanal-treated fruits gradually ripened and showed higher levels of lycopene and ß-carotene. GC-MS analysis of volatiles showed a higher level of major volatile components in hexanal-treated fruits. Similarities in the modulation of gene expression by hexanal and 1-MCP suggest that hexanal, in addition to being a PLD inhibitor, may also act as a weak ethylene inhibitor.
Subject(s)
Aldehydes/pharmacology , Ethylenes/antagonists & inhibitors , Fruit/drug effects , Gene Expression Regulation, Plant/drug effects , Genes, Plant , Lyases/pharmacology , Solanum lycopersicum/metabolism , Carotenoids/genetics , Carotenoids/metabolism , Cell Wall/metabolism , DNA, Complementary/biosynthesis , Down-Regulation , Enzymes/genetics , Enzymes/metabolism , Fruit/growth & development , Fruit/metabolism , Gene Expression/drug effects , Gene Expression Profiling/methods , Lyases/antagonists & inhibitors , Lycopene , Solanum lycopersicum/genetics , Solanum lycopersicum/growth & development , Oligonucleotide Array Sequence Analysis , Phospholipase D/antagonists & inhibitors , RNA/isolation & purification , Signal Transduction/drug effects , Volatile Organic Compounds/metabolismABSTRACT
Walnuts are a rich source of essential fatty acids, including the polyunsaturated fatty acids alpha-linolenic acid and linoleic acid. Essential fatty acids have been shown to modulate a number of cellular processes in the brain, including the activation state of microglia. Microglial activation can result in the generation of cytotoxic intermediates and is associated with a variety of age-related and neurodegenerative conditions. In vitro, microglial activation can be induced with the bacterial cell wall component lipopolysaccharide (LPS). In the present study, we generated a methanolic extract of English walnuts (Juglans regia) and examined the effects of walnut extract exposure on LPS-induced activation in BV-2 microglial cells. When cells were treated with walnut extract prior to LPS stimulation, production of nitric oxide and expression of inducible nitric oxide synthase were attenuated. Walnut extract also induced a decrease in tumor necrosis-alpha (TNFalpha) production. We further found that walnut extract induced internalization of the LPS receptor, toll-like receptor 4, and that the anti-inflammatory effects of walnut were dependent on functional activation of phospholipase D2. These studies represent the first to describe the anti-inflammatory effects of walnuts in microglia, which could lead to nutritional interventions in the prevention and treatment of neurodegeneration.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Juglans , Lipopolysaccharides/pharmacology , Microglia/drug effects , Phospholipase D/metabolism , Plant Extracts/pharmacology , Toll-Like Receptor 4/drug effects , 1-Butanol/pharmacology , Animals , Anti-Inflammatory Agents/chemistry , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme Inhibitors/pharmacology , Fatty Acids/analysis , Mice , Microglia/enzymology , Microglia/immunology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolism , Nuts , Phospholipase D/antagonists & inhibitors , Plant Extracts/chemistry , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Enzymes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases alpha and beta (DAGLalpha and beta) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLalpha to screen new synthetic substances as DAGLalpha inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLalpha and MAGL activities were assessed by using sn-1-[14C]-oleoyl-2-arachidonoyl-glycerol and 2-[3H]-arachidonoylglycerol as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat) (IC50 approximately 60 nM), the most potent inhibitors of DAGLalpha were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC50 = 500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC50 = 160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat N-acylphosphatidyl-ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB1 and CB2 receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl-O-p-nitro-phenyl oleylphosphonate] inhibited both DAGLalpha and MAGL with similar potencies (IC50 = 0.8-0.1 and 3.7-3.2 microM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.
Subject(s)
Cannabinoid Receptor Modulators/antagonists & inhibitors , Cannabinoid Receptor Modulators/biosynthesis , Endocannabinoids , Amidohydrolases/antagonists & inhibitors , Animals , COS Cells , Chlorocebus aethiops , Drug Design , Drug Evaluation, Preclinical , Humans , In Vitro Techniques , Lactones/pharmacology , Lipase/antagonists & inhibitors , Lipoprotein Lipase/antagonists & inhibitors , Lipoprotein Lipase/genetics , Liver/enzymology , Molecular Structure , Monoacylglycerol Lipases/antagonists & inhibitors , Monoacylglycerol Lipases/genetics , Oleic Acids/chemical synthesis , Oleic Acids/chemistry , Oleic Acids/pharmacology , Orlistat , Phospholipase D/antagonists & inhibitors , Rats , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/geneticsABSTRACT
Envenomation by the brown recluse spider (Loxosceles reclusa) may cause local dermonecrosis and, rarely, coagulopathies, kidney failure and death. A venom phospholipase, SMaseD (sphingomyelinase D), is responsible for the pathological manifestations of envenomation. Recently, the recombinant SMaseD from Loxosceles laeta was demonstrated to hydrolyse LPC (lysophosphatidylcholine) to produce LPA (lysophosphatidic acid) and choline. Therefore activation of LPA signalling pathways may be involved in some manifestations of Loxosceles envenomation. To begin investigating this idea, we cloned a full-length cDNA encoding L. reclusa SMaseD. The 305 amino acid sequence of the L. reclusa enzyme is 87, 85 and 60% identical with those of L. arizonica, L. intermedia and L. laeta respectively. The recombinant enzyme expressed in bacteria had broad substrate specificity. The lysophospholipids LPC, LPI (18:1-1-oleyol lysophosphatidylinositol), LPS, LPG (18:1-1-oleoyl-lysophosphatidylglycerol), LBPA (18:1-1-oleoyl-lysobisphosphatidic acid) (all with various acyl chains), lyso-platelet-activating factor (C16:0), cyclic phosphatidic acid and sphingomyelin were hydrolysed, whereas sphingosylphosphorylcholine, PC (phosphatidylcholine; C22:6, C20:4 and C6:0), oxidized PCs and PAF (platelet-activating factor; C16:0) were not hydrolysed. The PAF analogue, edelfosine, inhibited enzyme activity. Recombinant enzyme plus LPC (C18:1) induced the migration of A2058 melanoma cells, and this activity was blocked by the LPA receptor antagonist, VPC32183. The recombinant spider enzyme was haemolytic, but this activity was absent from catalytically inactive H37N (His37-->Asn) and H73N mutants. Our results demonstrate that Loxosceles phospholipase D hydrolyses a wider range of lysophospholipids than previously supposed, and thus the term 'SMaseD' is too limited in describing this enzyme.
Subject(s)
Lysophospholipids/metabolism , Phospholipase D/metabolism , Phosphoric Diester Hydrolases/metabolism , Spider Venoms/metabolism , Spiders/enzymology , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/chemistry , Gene Expression Regulation, Enzymologic , Molecular Sequence Data , Mutation , Phospholipase D/antagonists & inhibitors , Phospholipase D/chemistry , Phospholipid Ethers , Point Mutation , Sequence Alignment , Sequence Homology, Amino Acid , Spiders/metabolism , Substrate SpecificityABSTRACT
The Toxic Oil Syndrome (TOS) was a toxic epidemic disease, related to the consumption of rapeseed oil denatured with aniline that affected more than 20,000 people in Spain and resulted in more than 330 deaths after its sudden appearance in 1981. It has been reported that the fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP) have shown a strong association with TOS. These PAP-esters could be absorbed and metabolized in a similar way than phospholipids. This is of interest because some products of phospholipid metabolism are important mediators in downstream pathways involved in the regulation of different nuclear factors. In particular, phospholipase D activity is involved in the activation of c-fos. Thus, we have investigated the effect of different PAP-esters in the induction of c-fos in lung fibroblasts. Results indicate that PAP-esters rapidly induced the expression of c-fos in a dose-dependent manner. In addition, both butanol and propranolol prevent this induction pointing to the involvement of phospholipase D in this activation. These results suggest that deregulation of some nuclear factors such as AP-1 could be involved in the pathogenesis of TOS.
Subject(s)
Genes, fos/drug effects , Plant Oils/toxicity , Propylene Glycols/toxicity , Proto-Oncogene Proteins c-fos/biosynthesis , RNA, Messenger/biosynthesis , Anilides/metabolism , Anilides/toxicity , Animals , Blotting, Western , Butanols/pharmacology , Enzyme Inhibitors/pharmacology , Fatty Acids, Monounsaturated , Fibroblasts/drug effects , Fibroblasts/metabolism , Foodborne Diseases/etiology , Gene Expression Regulation/drug effects , Male , Phospholipase D/antagonists & inhibitors , Phospholipase D/metabolism , Plant Oils/chemistry , Propranolol/pharmacology , Propylene Glycols/chemistry , Proto-Oncogene Proteins c-fos/genetics , RNA, Messenger/genetics , Rapeseed Oil , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The creosote bush (Larrea tridentata) is a xerophytic evergreen C3 shrub thriving in vast arid areas of North America. As the first step toward understanding the molecular mechanisms controlling the drought tolerance of this desert plant, we have isolated a dozen genes encoding transcription factors, including LtWRKY21 that encodes a protein of 314 amino acid residues. Transient expression studies with the GFP-LtWRKY21 fusion construct indicate that the LtWRKY21 protein is localized in the nucleus and is able to activate the promoter of an abscisic acid (ABA)-inducible gene, HVA22, in a dosage-dependent manner. The transactivating activity of LtWRKY21 relies on the C-terminal sequence containing the WRKY domain and a N-terminal motif that is essential for the repression activity of some regulators in ethylene signaling. LtWRKY21 interacts synergistically with ABA and transcriptional activators VP1 and ABI5 to control the expression of the HVA22 promoter. Co-expression of VP1, ABI5, and LtWRKY21 leads to a much higher expression of the HVA22 promoter than does the ABA treatment alone. In contrast, the Lt-WRKY21-mediated transactivation is inhibited by two known negative regulators of ABA signaling: 1-butanol, an inhibitor of phospholipase D, and abi1-1, a dominant negative mutant protein phosphatase. Interestingly, abi1-1 does not block the synergistic effect of LtWRKY21, VP1, and ABI5 co-expression, indicating that LtWRKY21, VP1, and ABI5 may form a complex that functions downstream of ABI1 to control ABA-regulated expression of genes.
Subject(s)
Abscisic Acid/metabolism , DNA-Binding Proteins/physiology , Gene Expression Regulation, Plant , Plant Proteins/physiology , Plants/metabolism , Transcription Factors/physiology , 1-Butanol/pharmacology , Amino Acid Motifs , Amino Acid Sequence , Blotting, Northern , Cell Nucleus/metabolism , DNA/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Ethylenes/chemistry , Gene Library , Genes, Dominant , Genes, Plant , Genes, Reporter , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Models, Genetic , Molecular Sequence Data , Mutation , Phospholipase D/antagonists & inhibitors , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , RNA/chemistry , Recombinant Fusion Proteins/chemistry , Sequence Homology, Amino Acid , Signal Transduction , Transcriptional ActivationABSTRACT
The role of phospholipase (PL) D in secretion was examined in RBL-2H3 mast cells which contain both PLD1 and 2. The effects of pharmacologic stimulants and inhibitors of Ca(2+)/calmodulin-dependent kinase II, protein kinase C, and protein kinase A suggested that all three kinases synergistically stimulate PLD and, when associated with a calcium signal, secretion as well to indicate a possible linkage between these two events. Overexpression of either PLD1 or 2 markedly enhanced the activation of PLD by pharmacologic stimulants as well as antigen and both isoforms thus appear co-ordinately regulated. As the expressed PLD1 was associated with secretory granules and PLD2 with the plasma membrane, the two isoforms may serve distinct but complementary functions in secretion.
Subject(s)
Mast Cells/enzymology , Phospholipase D/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Antigens/immunology , Carbachol/pharmacology , Cholera Toxin/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/pharmacology , Mast Cells/immunology , Mast Cells/metabolism , Phospholipase D/antagonists & inhibitors , Rats , Tetradecanoylphorbol Acetate/pharmacology , Thapsigargin/pharmacology , Tumor Cells, CulturedABSTRACT
CT-2584, an anticancer agent that inhibits phospholipid signaling, is under development by Cell Therapeutics Inc (CTI) as a potential treatment for various types of cancer. Phase II trials are underway for the treatment of prostate cancer and soft-tissue sarcoma [306617], [324290]. According to CIBC World Markets, completion of enrolment for these trials was expected in the fourth quarter of 2000. Furthermore, the initiation of phase II/III trials in combination with taxotere for the treatment of prostate cancer was anticipated in the second half of 2000, as were phase I/II trials in combination with cisplatin for the treatment of other cancers, including lung cancer [396582]. Results of a phase II study in patients with soft-tissue sarcomas evaluating pharmacokinetics, tolerance and therapeutic activity were presented at the 2000 American Society of Clinical Oncology (ASCO) meeting [367283]. Further data are expected to be presented at the ASCO meeting in May 2001 [396582]. Cell Therapeutics is seeking development and commercialization partners for CT-2584 [386398].
Subject(s)
Antineoplastic Agents/pharmacology , Drugs, Investigational , Xanthines/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , Fatigue/chemically induced , Hematuria/chemically induced , Humans , Male , Molecular Structure , Phosphatidic Acids/metabolism , Phosphatidylcholines/metabolism , Phospholipase D/antagonists & inhibitors , Prostatic Neoplasms/drug therapy , Sarcoma/drug therapy , Tumor Cells, Cultured , Xanthines/chemistry , Xanthines/pharmacokinetics , Xanthines/therapeutic useABSTRACT
Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, beta-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of beta-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified beta-actin potently inhibited both phosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2 activities in a concentration-dependent manner (IC50 = 5 nm). In a previous paper, we reported that alpha-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversible manner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo, J.-S., Du, G., Frohman, M. A., Suh, P.-G., and Ryu, S. H. (2000) J. Biol. Chem. 275, 21295-21301). In vitro binding analyses showed that beta-actin could displace alpha-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of beta-actin on PLD2. We also observed that beta-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivo interaction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, beta-actin and alpha-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions.