ABSTRACT
Eliglustat tartrate (Genz-112638) is a novel, orally administered agent currently in development for the treatment of lysosomal storage disorders, including type 1 Gaucher disease and Fabry disease. This glucosylceramide analogue acts as an inhibitor of glucosylceramide synthase, a Golgi complex enzyme that catalyzes the formation of glucosylceramide from ceramide and UDP-glucose and is the first step in the formation of glucocerebroside-based glycosphingolipids. Pre-clinical pharmacological studies demonstrate that the agent has a high therapeutic index, excellent oral bioavailability and limited toxicity. Phase I studies in healthy volunteers revealed limited toxicity with an excellent pharmacodynamic response, as measured by decreased plasma glucosylceramide concentrations. Phase II studies in patients with type 1 Gaucher disease have demonstrated promising clinical responses, as measured by decreases in spleen size, improvement in hemoglobin concentrations and increased platelet counts. Two randomized phase III trials testing the efficacy and safety of eliglustat tartrate are currently in progress.
ABSTRACT
We have developed a series of inhibitors of glucosylceramide synthase that are structurally based on the parent compound D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). These inhibitors provide useful tools for manipulating glycosphingolipid levels in cells and for elucidating questions associated with sphingolipid signaling. Recently, two highly active glucosylceramide synthase inhibitors, D-threo-3', 4'-ethylenedioxy-1-phenyl-2-palmitoylamino-3- pyrrolidino-1-propanol and D-threo-4'-hydroxy-1-phenyl-2-palmitoylamino-3- pyrrolidino-1-propanol, were designed, synthesized, and studied. These inhibitors markedly reduced glycosphingolipid levels in MDCK cells without any accumulation of intracellular ceramide and associated growth inhibition. Subsequently, each inhibitor was evaluated for its ability to lower glycolipid levels in virally transformed lymphoblasts from a patient with alpha-galactosidase A deficiency. Both compounds significantly reduced neutral glycosphingolipid levels in the lymphoblasts without any morphological changes and growth inhibition. Furthermore, the inhibitors were applied to a mouse knockout model of Fabry disease. Inhibitor treatment blocked accumulation of globotriaosylceramide (Gb3) in the kidney, liver and heart of mice. In contrast to another glucosylceramide synthase inhibitor, N-butyldeoxynojirimycin, this treatment was not associated with any significant change in body weight or organ weight and without immunodepletion. These results suggest that these newest PDMP homologues are promising as therapeutic agents for the treatment of glycosphingolipid storage disorders.
Subject(s)
Enzyme Inhibitors/therapeutic use , Gangliosidoses/drug therapy , Glycosphingolipids/metabolism , Morpholines/therapeutic use , Animals , Glycosphingolipids/biosynthesis , Humans , MiceABSTRACT
The CD60 antigen is expressed on a majority of T cells in autoimmune lesions, and anti-CD60 can activate T lymphocytes. CD60 has been defined as the GD3 ganglioside, and subsequently as the 9-O-acetylated form of GD3. However, other evidence suggests that anti-CD60 recognizes a glycoprotein or family of glycoproteins expressed by T lymphocytes. The current studies were undertaken to better define the identity of the CD60 antigen on both T cells and non-T cells. Treatment of intact cells with neuraminidases of various specificities confirmed that detection of the CD60 epitope depends on expression of an alpha2, 8-disialic acid carbohydrate linkage, as is found in GD3 and related gangliosides. However, the sialicacid polymer colominic acid inhibited anti-GD2 and anti-GD3, but not anti-CD60 from binding to cell surfaces. Expression of CD60 did not correlate with expression of GD3 on a variety of cell lines and T cell populations. Expression of CD60 and 9-O-acetyl-GD3 was roughly parallel on some non-T cell lines such as melanoma cells, but on T cells expression of CD60 was consistently greater. Antibodies to GD2, GD3 and 9-O-acetyl-GD3 were ineffective at inhibiting binding of anti-CD60 to CD60+ cells. Activation responses of T cells to anti-CD60 were inducible in either the presence or absence of a response to anti-GD3. A novel inhibitor of glucosyl ceramide synthesis, D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-t-P4) reduced expression of GD3 much more than CD60 on activated T lymphocytes. Following biotinylation of HUT78 T cells, anti-CD60 immunoprecipitated a 70 kDa antigen. Taken together, the present data and previous findings suggest that anti-CD60 can recognize both a modified form of the GD3 ganglioside and a carbohydrate-dependent complex epitope present on one or more glycoproteins. This glycoprotein epitope may be the more abundant and functionally significant CD60 antigen on T lymphocytes, while 9-O-acetyl-GD3 is likely to be the principal structure recognized by anti-CD60 on melanoma cells. These findings emphasize the complexity of understanding the functional roles of carbohydrate epitopes in cell activation.
Subject(s)
Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , Epitopes, B-Lymphocyte/immunology , Gangliosides/immunology , Glycoproteins/immunology , N-Acetylneuraminic Acid/immunology , T-Lymphocytes/immunology , Antigens, CD/chemistry , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/chemistry , Antigens, Differentiation, T-Lymphocyte/metabolism , Carbohydrate Sequence , E-Selectin/metabolism , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/metabolism , Gangliosides/chemistry , Gangliosides/metabolism , Glucosylceramides/antagonists & inhibitors , Glycoproteins/chemistry , Humans , Jurkat Cells , Molecular Sequence Data , N-Acetylneuraminic Acid/chemistry , N-Acetylneuraminic Acid/metabolism , Propanolamines/pharmacology , Pyrrolidines/pharmacologyABSTRACT
BACKGROUND: Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism. The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. METHODS: K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days, which resulted in a marked decrease in serum and tissue K content. RESULTS: K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the V(max) of brush-border membrane (BBM) Na/Pi cotransport activity (1943 +/- 95 in control vs. 1184 +/- 99 pmol/5 sec/mg BBM protein in K deficiency, P < 0.02). Surprisingly, the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1), and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin, glucosylceramide, and ganglioside GM3 content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficient rats was the same as in control rats (1148 +/- 52 in control + PDMP vs. 1152 +/- 61 pmol/5 sec/mg BBM protein in K deficiency + PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. CONCLUSION: K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics.
Subject(s)
Carrier Proteins/metabolism , Glucosylceramides/metabolism , Kidney Tubules, Proximal/metabolism , Membrane Fluidity/physiology , Phosphates/metabolism , Potassium Deficiency/metabolism , Symporters , Animals , Carrier Proteins/genetics , G(M3) Ganglioside/metabolism , Gene Expression/physiology , Hypokalemia/metabolism , Kinetics , Male , Microvilli/metabolism , Oocytes/metabolism , Phosphorus/urine , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Receptors, Virus/genetics , Receptors, Virus/metabolism , Sodium-Phosphate Cotransporter Proteins , Sodium-Phosphate Cotransporter Proteins, Type I , Sodium-Phosphate Cotransporter Proteins, Type II , Sodium-Phosphate Cotransporter Proteins, Type III , Xenopus laevisABSTRACT
It is widely accepted that phosphorylation of the retinoblastoma (Rb) protein during the G1 phase of the mammalian division cycle is a major control element regulating passage of cells into S phase and through the division cycle. The experiments supporting G1-phase-specific Rb phosphorylation and the historical development of this idea are reviewed. By making a rigorous distinction between 'growth cessation' and the phenomena of 'cell cycle exit' or 'G1-phase arrest', the evidence for the G1-phase-specific phosphorylation of Rb protein is reinterpreted. We show that the evidence for G1-phase phosphorylation of Rb rests on few experiments and a chain of reasoning with some weak links. Evidence is reviewed that growth conditions regulate the phosphorylation of Rb. A growth-regulated control system that is independent of the cell cycle explains much of the evidence adduced to support cycle-specific phosphorylation of Rb. We propose that additional experimental evidence is needed to decide whether there is a G1-phase-specific phosphorylation of Rb protein.
Subject(s)
Retinoblastoma Protein/metabolism , Animals , Cell Cycle , Cell Division , Cell Transformation, Viral , G1 Phase , Humans , Kinetics , Mammals , Phosphorylation , Simian virus 40/geneticsABSTRACT
A significant corpus of work over the last decade has firmly established an important role for sphingolipids in a variety of important biological processes. Such processes include signaling events related to cell growth, differentiation, programmed cell death, and stress responses. These processes not only involve those sphingolipids that accumulate as a result of a variety of inherited lysosomal storage disorders, but, in addition, sphingolipids associated with long-chain base metabolism. This article reviews the chemical properties, pathways, regulated metabolism, and signaling function of sphingolipids. In addition, the potential roles of sphingolipids in renal-specific processes are considered. While a variety of cellular functions have been ascribed to sphingolipids, in many cases proof of the concept has yet to be well established. Thus, a number of critical questions can be posed in interpreting these studies. Several of these questions are considered.
Subject(s)
Kidney/metabolism , Sphingolipids/chemistry , Sphingolipids/metabolism , Animals , Energy Metabolism/physiology , HumansABSTRACT
We used a potent inhibitor of glucosylceramide synthase to test whether substrate deprivation could lower globotriaosylceramide levels in alpha-galactosidase A (alpha-gal A) knockout mice, a model of Fabry disease. C57BL/6 mice treated twice daily for 3 days with D-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidi no-propanol (D-t-EtDO-P4) showed a concentration-dependent decrement in glucosylceramide levels in kidney, liver, and spleen. A single intraperitoneal injection of D-t-EtDO-P4 resulted in a 55% reduction in renal glucosylceramide, consistent with rapid renal glucosylceramide metabolism. A concentration-dependent decrement in renal and hepatic globotriaosylceramide levels was observed in alpha-Gal A(-) males treated for 4 weeks with D-t-EtDO-P4. When 8-week-old alpha-Gal A(-) males were treated for 8 weeks with 10 mg/kg twice daily, renal globotriaosylceramide fell to below starting levels, consistent with an alpha-galactosidase A-independent salvage pathway for globotriaosylceramide degradation. Complications observed with another glucosylceramide synthase inhibitor, N-butyldeoxynojirimycin, including weight loss and acellularity of lymphatic organs, were not observed with D-t-EtDO-P4. These data suggest that Fabry disease may be amenable to substrate deprivation therapy.
Subject(s)
Fabry Disease/metabolism , Trihexosylceramides/metabolism , 1-Deoxynojirimycin/analogs & derivatives , 1-Deoxynojirimycin/pharmacology , Animals , Dose-Response Relationship, Drug , Kidney/metabolism , Kidney/ultrastructure , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Propanolamines/pharmacology , Pyrrolidines/pharmacologyABSTRACT
Polymorphonuclear leukocyte (PMNL) phagocytosis mediated by FcgammaRII proceeds in concert with activation of the mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase ERK2. We hypothesized that myosin light chain kinase (MLCK) could be phosphorylated and activated by ERK, thereby linking the MAP kinase pathway to the activation of cytoskeletal components required for pseudopod formation. To explore this potential linkage, PMNLs were challenged with antibody-coated erythrocytes (EIgG). Peak MLCK activity, 3-fold increased over controls, occurred at 4 to 6 minutes, corresponding with the peak rate of target ingestion and ERK2 activity. The MLCK inhibitor ML-7 (10 micromol/L) inhibited both phagocytosis and MLCK activity to basal values, thereby providing further support for the linkage between the functional response and the requirement for MLCK activation. The MAPK kinase (MEK) inhibitor PD098059 inhibited phagocytosis, MLCK activity, and ERK2 activity by 80% to 90%. To directly link ERK activation to MLCK activation, ERK2 was immunoprecipitated from PMNLs after EIgG ingestion. The isolated ERK2 was incubated with PMNL cytosol as a source of unactivated MLCK and with MLCK substrate; under these conditions ERK2 activated MLCK, resulting in phosphorylation of the MLCK substrate or of the myosin light chain itself. Because MLCK activates myosin, we evaluated the effect of directly inhibiting myosin adenosine triphosphatase using 2,3-butanedione monoxime (BDM) and found that phagocytosis was inhibited by more than 90% but MLCK activity remained unaffected. These results are consistent with the interpretation that MEK activates ERK, ERK2 then activates MLCK, and MLCK activates myosin. MLCK activation is a critical step in the cytoskeletal changes resulting in pseudopod formation.
Subject(s)
Mitogen-Activated Protein Kinase Kinases/metabolism , Myosin-Light-Chain Kinase/metabolism , Neutrophils/immunology , Phagocytosis , Azepines/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Erythrocytes/immunology , Flavonoids/pharmacology , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Myosin-Light-Chain Kinase/antagonists & inhibitors , Myosins/antagonists & inhibitors , Naphthalenes/pharmacology , Neutrophils/enzymology , Phagocytosis/drug effects , PhosphorylationABSTRACT
BACKGROUND: Fabry disease is an inherited X-linked disorder resulting in the loss of activity of the lysosomal hydrolase alpha-galactosidase A and causing the clinical manifestations of renal failure, cerebral vascular disease, and myocardial infarction. The phenotypic expression of this disorder is manifest by the accumulation of glycosphingolipids containing alpha-galactosyl linkages, most prominently globotriaosylceramide. METHODS: Based on quantitative structure activity studies, we recently reported two newly designed glucosylceramide synthase inhibitors based on 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4). These inhibitors, 4'-hydroxy-P4 and ethylenedioxy-P4, were evaluated for their ability to deplete globotriaosylceramide and other glucosylceramide-based lipids in Fabry lymphocytes and were compared with N-butyldeoxynojirimycin, another reported glucosylceramide synthase inhibitor. RESULTS: Concentrations as low as 10 nmol/L of 4'-hydroxy-P4 and ethylenedioxy-P4 resulted in 70 and 80% depletion, respectively, of globotriaosylceramide, with maximal depletion occurring at three days of treatment. There was no impairment of cell growth. In contrast, N-butyldeoxynojirimycin only minimally lowered globotriaosylceramide levels, even at concentrations as high as 10 micromol/L. Globotriaosylceramide depletion was confirmed by the loss of binding of FITC-conjugated verotoxin B subunit to the lymphoblasts. CONCLUSIONS: These findings suggest that selective glucosylceramide synthase inhibitors are highly effective in the depletion of globotriaosylceramide from Fabry cell lines. We suggest that these compounds have potential therapeutic utility in the treatment of Fabry disease.
Subject(s)
B-Lymphocytes/enzymology , Enzyme Inhibitors/pharmacology , Fabry Disease/metabolism , Glucosyltransferases/antagonists & inhibitors , Neutral Glycosphingolipids/metabolism , Propanolamines/pharmacology , Pyrrolidines/pharmacology , Trihexosylceramides/biosynthesis , 1-Deoxynojirimycin/analogs & derivatives , 1-Deoxynojirimycin/pharmacology , B-Lymphocytes/cytology , Bacterial Toxins , Cell Line, Transformed , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Fabry Disease/drug therapy , Fabry Disease/immunology , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Genetic Vectors , Glucosyltransferases/metabolism , Glycosphingolipids/analysis , Herpesvirus 4, Human , Humans , Neutral Glycosphingolipids/analysis , Propanolamines/chemistry , Pyrrolidines/chemistry , Shiga Toxin 1 , alpha-Galactosidase/metabolismABSTRACT
Glycosphingolipids have been proposed to be critical components of clustered lipids within cell membranes that serve as rafts for the attachment and sorting of proteins to the cell membrane. Density gradient centrifugation was used to isolate and to ascertain the lipid composition of caveolin-enriched membranes. These membranes demonstrated a significant enrichment of sphingolipids and cholesterol containing up to 20 and 30%, respectively, of the cellular glucosylceramide and lactosylceramide. A specific inhibitor of glucosylceramide synthase, d-threo-1-phenyl-2-palmitoyl-3-pyrrolidino-propanol, was used to test the hypothesis that glycosphingolipids are required for the sorting of proteins to caveolae. When NIH 3T3 cells were depleted of their glucosylceramide based glycosphingolipid mass, the caveolar structure remained intact as determined by electron microscopy and confocal microscopy. The caveolar proteins caveolin and annexin II sorted normally to caveolae, as determined by immunoblotting and confocal microscopy. When the GPI-linked protein B61 was inducibly expressed in these cells, sorting to caveolar membranes occurred normally, even in the presence of glucosylceramide depletion. These observations suggest that protein sorting to caveolae in fibroblasts occurs independently of glycosphingolipid synthesis.
Subject(s)
Caveolins , Cell Membrane/metabolism , Glycosphingolipids/metabolism , Membrane Lipids/metabolism , Membrane Proteins/metabolism , 3T3 Cells , Animals , Annexin A2/metabolism , Caveolin 1 , Enzyme Inhibitors/pharmacology , Glucosyltransferases/antagonists & inhibitors , Membrane Lipids/chemistry , Mice , Microscopy, Confocal , Propanolamines/pharmacology , Pyrrolidines/pharmacologyABSTRACT
Glucosylceramide synthesis is a key step in the formation of most mammalian glycosphingolipids. The expanding number of cellular functions that may be glycosphinolipid dependent and the identification of this glucosylceramide synthase as a potential therapeutic target for several sphingolipid storage disorders necessitate the availability of a reliable assay for glucosylceramide synthase. Coupled with the recent sequencing of this enzyme, the liposome-based assay utilizing a single extraction step should aid in the understanding of this critical early pathway in glycosphingolipid formation.
Subject(s)
Glucosyltransferases/analysis , Glucosyltransferases/metabolism , Cations, Divalent , Ceramides/metabolism , Enzyme Induction , Glucosyltransferases/antagonists & inhibitors , Humans , Hydrogen-Ion Concentration , Liposomes/metabolism , Sphingolipidoses/therapy , Sphingolipids/chemical synthesis , Sphingosine/analogs & derivatives , Sphingosine/chemical synthesis , Substrate Specificity , Uridine Diphosphate Glucose/metabolismSubject(s)
Acyltransferases/isolation & purification , Ceramides/metabolism , Sphingolipids/biosynthesis , Acylation , Acyltransferases/metabolism , Animals , Arachidonic Acid/metabolism , Brain/enzymology , Cattle , Cells, Cultured , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Phospholipases A2 , Sphingosine/analogs & derivatives , Sphingosine/metabolismSubject(s)
Glucosylceramides/biosynthesis , Glucosyltransferases/antagonists & inhibitors , Morpholines/chemistry , Cell Division/drug effects , Ceramides/biosynthesis , Drug Carriers , Enzyme Inhibitors/pharmacology , Glycosphingolipids/biosynthesis , Morpholines/chemical synthesis , Propanolamines/chemical synthesis , Pyrrolidines/chemical synthesis , Sphingolipids/biosynthesis , Sphingolipids/chemical synthesis , StereoisomerismABSTRACT
We investigated the requirement for Syk activation to initiate downstream signaling events during polymorphonuclear leukocyte (PMN) phagocytosis of Ab-coated erythrocytes (EIgG). When PMN were challenged with EIgG, Syk phosphorylation increased in a time-dependent manner, paralleling the response of PMN phagocytosis. Pretreatment of PMN with piceatannol, a Syk-selective inhibitor, blocked EIgG phagocytosis and Syk phosphorylation. We found that piceatannol inhibited protein kinase Cdelta (PKCdelta) and Raf-1 translocation from cytosol to plasma membrane by >90%. Extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly blocked. We also investigated phosphatidylinositide 3-kinase (PI 3-kinase) activity and Syk phosphorylation using piceatannol, wortmannin, and LY294002, inhibitors of PI 3-kinase. The phosphorylation of Syk preceded the activation of PI 3-kinase. Both wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol inhibited Syk. In contrast to piceatannol, wortmannin did not inhibit PKCdelta and Raf-1 translocation. To elucidate signaling downstream of Syk activation, we assessed whether the cell-permeable diacylglycerol analogue didecanoylglycerol could normalize PMN phagocytosis, PKCdelta and Raf-1 translocation, and ERK1 and ERK2 phosphorylation inhibited by piceatannol. The addition of didecanoylglycerol to the Syk-inhibited phagocytosing PMN normalized all three without a concomitant effect on PI 3-kinase activity and Syk phosphorylation. We conclude that Syk activation following Fcgamma receptor engagement initiates downstream signaling events leading to mitogen-activated protein kinase activation independent of PI 3-kinase activation.
Subject(s)
Enzyme Precursors/metabolism , Neutrophils/enzymology , Neutrophils/immunology , Phagocytosis/immunology , Protein-Tyrosine Kinases/metabolism , Signal Transduction/immunology , Androstadienes/pharmacology , Animals , Biological Transport/drug effects , Biological Transport/immunology , Cell Membrane/enzymology , Cell Membrane/immunology , Cell Migration Inhibition , Diglycerides/pharmacology , Enzyme Activation/drug effects , Enzyme Activation/immunology , Enzyme Inhibitors/pharmacology , Enzyme Precursors/antagonists & inhibitors , Erythrocytes/immunology , Humans , Immunoglobulin G/blood , Intracellular Signaling Peptides and Proteins , Isoenzymes/metabolism , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Neutrophils/drug effects , Neutrophils/metabolism , Opsonin Proteins/blood , Phagocytosis/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Phosphotyrosine/metabolism , Protein Kinase C/metabolism , Protein Kinase C-delta , Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, IgG/physiology , Sheep , Stilbenes/pharmacology , Syk Kinase , WortmanninABSTRACT
Previous work has led to the identification of inhibitors of glucosylceramide synthase, the enzyme catalyzing the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. These inhibitors have two identified sites of action: the inhibition of glucosylceramide synthase, resulting in the depletion of cellular glycosphingolipids, and the inhibition of 1-O-acylceramide synthase, resulting in the elevation of cell ceramide levels. A new series of glucosylceramide synthase inhibitors based on substitutions in the phenyl ring of a parent compound, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), was made. For substitutions of single functional groups, the potency of these inhibitors in blocking glucosylceramide synthase was primarily dependent upon the hydrophobic and electronic properties of the substituents. An exponential relationship was found between the IC50 of each inhibitor and the sum of derived hydrophobic (pi) and electronic (sigma) parameters. This relationship demonstrated that substitutions that increased the electron-donating characteristics and decreased the lipophilic characteristics of the homologues enhanced the potency of these compounds in blocking glucosylceramide formation. A novel compound was subsequently designed and observed to be even more active in blocking glucosylceramide formation. This compound, D-threo-4'-hydroxy-P4, inhibited glucosylceramide synthase at an IC50 of 90 nM. In addition, a series of dioxane substitutions was designed and tested. These included 3',4'-methylenedioxyphenyl-, 3',4'-ethylenedioxyphenyl-, and 3'4'-trimethylenedioxyphenyl-substituted homologues. D-threo-3', 4'-Ethylenedioxy-P4-inhibited glucosylceramide synthase was comparably active to the p-hydroxy homologue. 4'-Hydroxy-P4 and ethylenedioxy-P4 blocked glucosylceramide synthase activity at concentrations that had little effect on 1-O-acylceramide synthase activity. These novel inhibitors resulted in the inhibition of glycosphingolipid synthesis in cultured cells at concentrations that did not significantly raise intracellular ceramide levels or inhibit cell growth.
Subject(s)
Enzyme Inhibitors/pharmacology , Glucosyltransferases/antagonists & inhibitors , Morpholines/pharmacology , Cells, Cultured , Chromatography , Dose-Response Relationship, DrugABSTRACT
In the present study, we investigated the mechanism by which sphingosine and its analogues, dihydrosphingosine and phytosphingosine, inhibit polymorphonuclear leukocyte (PMN) phagocytosis of IgG-opsonized erythrocytes (EIgG) and inhibit ERK1 and ERK2 phosphorylation. We used antibodies that recognized the phosphorylated forms of ERK1 (p44) and ERK2 (p42) (extracellular signal-regulated protein kinases 1 and 2). Sphingoid bases inhibited ERK1 and ERK2 activation and phagocytosis of EIgG in a concentration-dependent manner. Incubation with glycine, N,N'-[1, 2-ethanediylbis(oxy-2, 1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-bis[ (acetylox y)methyl]ester (BAPTA,AM), an intracellular chelator of calcium, failed to block either phagocytosis or ERK1 and ERK2 phosphorylation, consistent with the absence of a role for a calcium-dependent protein kinase C (PKC) in ERK1 and ERK2 phosphorylations. Western blotting demonstrated that sphingosine inhibited the translocation of Raf-1 and PKCdelta from PMN cytosol to the plasma membrane during phagocytosis. These data are consistent with the interpretation that sphingosine regulates ERK1 and ERK2 phosphorylation through inhibition of PKCdelta, and this in turn leads to inhibition of Raf-1 translocation to the plasma membrane. Consistent with this interpretation, the sphingosine-mediated inhibition of phagocytosis, ERK2 activation, and PKCdelta translocation to the plasma membrane could be abrogated with a cell-permeable diacylglycerol analog. The increase in the diacylglycerol mass correlated with the translocation of PKCdelta and Raf-1 to the plasma membrane by 3 minutes after the initiation of phagocytosis. Additionally, the diacylglycerol analog enhanced phagocytosis by initiating activation of PKCdelta and its translocation to the plasma membrane. Because PMN generate sufficient levels of sphingosine by 30 minutes during phagocytosis of EIgG to inhibit phagocytosis, it appears that sphingosine can serve as an endogenous regulator of EIgG-mediated phagocytosis by downregulating ERK activation.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Mitogen-Activated Protein Kinases , Neutrophils/physiology , Phagocytosis/drug effects , Sphingosine/pharmacology , Biological Transport/drug effects , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Membrane/metabolism , Diglycerides/pharmacology , Erythrocytes , Humans , Immunoglobulin G , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Opsonin Proteins , Phosphorylation , Protein Kinase C/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Receptors, Fc/physiologyABSTRACT
Phosphorylation of the retinoblastoma protein (Rb) during the G1-phase of the mammalian cell division cycle is currently believed to be a controlling element regulating the passage of cells into S-phase. We find, however, that the suspension-grown cell lines U937, L1210, and MOLT-4 contain exclusively hyperphosphorylated Rb. Furthermore, when adherent NIH3T3 cells are grown at very low densities to avoid overgrowth and contact inhibition, they also contain only hyperphosphorylated Rb. NIH3T3 cells exhibit hypophosphorylation when the cells are grown at moderate to high cell densities. We propose that cultures of adherent cells such as NIH3T3, when grown to moderate cell densities, are made up of two populations of cells: (a) cells that are relatively isolated and therefore growing exponentially without contact inhibition, and (b) cells that are growth-inhibited by local cell density or contact inhibition. The common observation in adherent cell lines, that Rb is both hyper- and hypophosphorylated in the G1-phase and only hyperphosphorylated in the S- and G2-phases, is explained by the effects of cell density and contact inhibition. Thus, phosphorylation-dephosphorylation of Rb protein during the G1 phase is not a necessary process during the NIH3T3, L1210, MOLT-4, and U937 division cycles. We propose that phosphorylation-dephosphorylation of Rb is independent of the division cycle and is primarily determined by growth conditions throughout the division cycle.
Subject(s)
Cell Cycle/physiology , Retinoblastoma Protein/metabolism , 3T3 Cells , Animals , Cell Count , Cell Line , G1 Phase/physiology , Humans , Leukemia L1210/metabolism , Leukemia L1210/pathology , Mice , Phosphorylation , U937 CellsABSTRACT
Ceramide, a product of agonist-stimulated sphingomyelinase activation, is known to be generated during the phagocytosis of antibody-coated erythrocytes by polymorphonuclear leukocytes. Agonist-stimulated formation of ceramide-1-phosphate is now shown to occur in 32PO4-labeled neutrophils. Ceramide-1-phosphate is formed by a calcium-dependent ceramide kinase, found predominately in the neutrophil plasma membrane. The neutrophil kinase is specific for ceramide because, in contrast to the bacterial diglyceride kinase, ceramide is not phosphorylated under conditions specific for diglyceride phosphorylation. Conversely, 1,2-diacylglycerol does not serve as substrate for the neutrophil ceramide kinase. Ceramide kinase activation occurs in a time-dependent fashion, reaching peak activity 10 min after formyl peptide stimulation and challenge with antibody-coated erythrocytes. The lipid kinase activity is optimal at pH 6.8. Because the formation of the phagolysosome is a critical event in phagocytosis, the effect of ceramide-1-phosphate in promoting the fusion of liposomes was determined. Both the addition of increasing concentrations of sphingomyelinase D and ceramide-1-phosphate promoted liposomal fusion. In summary, ceramide-1-phosphate is formed during phagocytosis through activation of ceramide kinase. Ceramide-1-phosphate may promote phagolysosome formation.
Subject(s)
Ceramides/biosynthesis , Liposomes/metabolism , Membrane Fusion , Neutrophils/metabolism , Phagocytosis , Calcium/metabolism , Enzyme Activation , Humans , In Vitro Techniques , Neutrophils/enzymology , Neutrophils/immunology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Subcellular Fractions/enzymology , Subcellular Fractions/metabolismABSTRACT
The sphingomyelin cycle, which plays an important role in regulation of cell growth, differentiation, and apoptosis, involves the formation of ceramide by the action of a membrane-associated, Mg2+-dependent, neutral sphingomyelinase and/or a lysosomal acid sphingomyelinase. In human polymorphonuclear leukocytes (PMNs), ceramide production correlates with and plays a role in the regulation of functional responses such as oxidant release and Fcgamma receptor-mediated phagocytosis. To increase our understanding of the sphingomyelin cycle in human PMNs, the cellular location of neutral and acid sphingomyelinases was investigated in resting, formylmethionylleucylphenylalanine (FMLP)-activated, and FMLP-activated PMNs engaged in phagocytosis. In resting PMNs, a Mg2+-dependent, neutral sphingomyelinase was the predominant activity and was localized to the plasma membrane fractions along with the majority of ceramide. Upon FMLP-activation, there was a 1. 9-fold increase in this neutral, Mg2+-dependent sphingomyelinase activity, which increased to 2.7-fold subsequent to phagocytosis of IgG opsonized targets. This increase in sphingomyelinase activity was restricted to the plasma membrane fractions, which were also the site of increased ceramide levels. Phospholipase D (PLD) activity, which is a target of ceramide action and is required for phagocytosis, was also found primarily in the plasma membrane fractions of FMLP-activated and phagocytosing PMNs. Our findings indicate that in human PMNs engaged in phagocytosis, the sphingomyelin cycle is restricted to the plasma membrane where intracellular targets of ceramide action, such as PLD, are localized.