The role of lysosomal phospholipase A2 in the catabolism of bis(monoacylglycerol)phosphate and association with phospholipidosis.

Bis(monoacylglycerol)phosphate (BMP) is an acidic glycerophospholipid localized to late endosomes and lysosomes. However, the metabolism of BMP is poorly understood. Because many drugs that cause phospholipidosis inhibit lysosomal phospholipase A2 (LPLA2, PLA2G15, LYPLA3) activity, we investigated whether this enzyme has a role in BMPcatabolism. The incubation of recombinant human LPLA2 (hLPLA2) and liposomes containing the naturally occurring BMP (sn-(2-oleoyl-3-hydroxy)-glycerol-1-phospho-sn-1'-(2'-oleoyl-3'-hydroxy)-glycerol (S,S-(2,2',C18:1)-BMP) resulted in the deacylation of this BMP isomer. The deacylation rate was 70 times lower than that of dioleoyl phosphatidylglycerol (DOPG), an isomer and precursor of BMP. The release rates of oleic acid from DOPG and four BMP stereoisomers by LPLA2 differed. The rank order of the rates of hydrolysis were DOPG>S,S-(3,3',C18:1)-BMP>R,S-(3,1',C18:1)-BMP>R,R-(1,1',C18:1)>S,S-(2,2')-BMP. The cationic amphiphilic drug amiodarone (AMD) inhibited the deacylation of DOPG and BMP isomers by hLPLA2 in a concentration-dependent manner. Under these experimental conditions, the IC50s of amiodarone-induced inhibition of the four BMP isomers and DOPG were less than 20 µM and approximately 30 µM, respectively. BMP accumulation was observed in AMD-treated RAW 264.7 cells. The accumulated BMP was significantly reduced by exogenous treatment of cells with active recombinant hLPLA2 but not with diisopropylfluorophosphate-inactivated recombinant hLPLA2. Finally, a series of cationic amphiphilic drugs known to cause phospholipidosis were screened for inhibition of LPLA2 activity as measured by either the transacylation or fatty acid hydrolysis of BMP or phosphatidylcholine as substrates. Fifteen compounds demonstrated significant inhibition with IC50s ranging from 6.8 to 63.3 µM. These results indicate that LPLA2 degrades BMP isomers with different substrate specificities under acidic conditions and may be the key enzyme associated with BMP accumulation in drug-induced phospholipidosis.

Inhibition of lysosomal phospholipase A2 predicts drug-induced phospholipidosis.

Phospholipidosis, the excessive accumulation of phospholipids within lysosomes, is a pathological response observed following exposure to many drugs across multiple therapeutic groups. A clear mechanistic understanding of the causes and implications of this form of drug toxicity has remained elusive. We previously reported the discovery and characterization of a lysosome-specific phospholipase A2 (PLA2G15) and later reported that amiodarone, a known cause of drug-induced phospholipidosis, inhibits this enzyme. Here, we assayed a library of 163 drugs for inhibition of PLA2G15 to determine whether this phospholipase was the cellular target for therapeutics other than amiodarone that cause phospholipidosis. We observed that 144 compounds inhibited PLA2G15 activity. Thirty-six compounds not previously reported to cause phospholipidosis inhibited PLA2G15 with IC50 values less than 1 mM and were confirmed to cause phospholipidosis in an in vitro assay. Within this group, fosinopril was the most potent inhibitor (IC50 0.18 µM). Additional characterization of the inhibition of PLA2G15 by fosinopril was consistent with interference of PLA2G15 binding to liposomes. PLA2G15 inhibition was more accurate in predicting phospholipidosis compared with in silico models based on pKa and ClogP, measures of protonation, and transport-independent distribution in the lysosome, respectively. In summary, PLA2G15 is a primary target for cationic amphiphilic drugs that cause phospholipidosis, and PLA2G15 inhibition by cationic amphiphilic compounds provides a potentially robust screening platform for potential toxicity during drug development.

Structural Basis of Lysosomal Phospholipase A2 Inhibition by Zn2.

Lysosomal phospholipase A2 (LPLA2/PLA2G15) is a key enzyme involved in lipid homeostasis and is characterized by both phospholipase A2 and transacylase activity and by an acidic pH optimum. Divalent cations such as Ca2+ and Mg2+ have previously been shown to have little effect on the activity of LPLA2, but the discovery of a novel crystal form of LPLA2 with Zn2+ bound in the active site suggested a role for this divalent cation in regulating enzyme activity. In this complex, the cation directly coordinates the serine and histidine of the α/ß-hydrolase triad and stabilizes a closed conformation. This closed conformation is characterized by an inward shift of the lid loop, which extends over the active site and effectively blocks access to one of its lipid acyl chain binding tracks. Therefore, we hypothesized that Zn2+ would inhibit LPLA2 activity at a neutral but not acidic pH because histidine would be positively charged at lower pH. Indeed, Zn2+ was found to inhibit the esterase activity of LPLA2 in a noncompetitive manner exclusively at a neutral pH (between 6.5 and 8.0). Because lysosomes are reservoirs of Zn2+ in cells, the pH optimum of LPLA2 might allow it to catalyze acyl transfer unimpeded within the organelle. We conjecture that Zn2+ inhibition of LPLA2 at higher pH maintains a lower activity of the esterase in environments where its activity is not typically required.

Determinants of pH profile and acyl chain selectivity in lysosomal phospholipase A2.

Lysosomal phospholipase A2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1-O-acyl-N-acetylsphingosine to measure the hydrolysis of sn-1 versus sn-2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain.

Inhibitors of Glucosylceramide Synthase.

Glucosylceramide synthase can be targeted by high affinity small molecular weight inhibitors for the study of glycosphingolipid metabolism and function or for the treatment of glycosphingolipid storage disorders, including Gaucher and Fabry disease. This work is exemplified by the discovery and development of eliglustat tartrate, the first stand-alone small chemical entity approved for the treatment of Gaucher disease type 1. The development of inhibitors of glucosylceramide synthase that have utility for either research or clinical purposes begins with a testing funnel for screening candidate inhibitors for activity against this enzyme and for activity in lowering the content of glucosylceramide in intact cells. Two common assays for glucosylceramide synthase, one enzyme based and another cell based, are the focus of this chapter.

Modulation of hepatic transcription factor EB activity during cold exposure uncovers direct regulation of bis(monoacylglycero)phosphate lipids by Pla2g15.

Cold exposure is an environmental stress that elicits a rapid metabolic shift in endotherms and is required for survival. The liver provides metabolic flexibility through its ability to rewire lipid metabolism to respond to an increased demand in energy for thermogenesis. We leveraged cold exposure to identify novel lipids contributing to energy homeostasis and found that lysosomal bis(monoacylglycero)phosphate (BMP) lipids were significantly increased in the liver during acute cold exposure. BMP lipid changes occurred independently of lysosomal abundance but were dependent on the lysosomal transcriptional regulator transcription factor EB (TFEB). Knockdown of TFEB in hepatocytes decreased BMP lipid levels. Through molecular biology and biochemical assays, we found that TFEB regulates lipid catabolism during cold exposure and that TFEB knockdown mice were cold intolerant. To identify how TFEB regulates BMP lipid levels, we used a combinatorial approach to identify TFEB target Pla2g15 , a lysosomal phospholipase, as capable of degrading BMP lipids in in vitro liposome assays. Knockdown of Pla2g15 in hepatocytes led to a decrease in BMP lipid species. Together, our studies uncover a required role of TFEB in mediating lipid liver remodeling during cold exposure and identified Pla2g15 as an enzyme that regulates BMP lipid catabolism.

Ceramide-1-phosphate in phagocytosis and calcium homeostasis.

Sphingolipids are well established sources of important signaling molecules. For example, ceramide (Cer) has been described as a potent inhibitor of cell growth and inducer of apoptosis. In contrast, ceramide-1-phosphate (C1P) has been reported to have mitogenic properties and to inhibit apoptosis. Our understanding of the distinct biological roles of C1P in the regulation of DNA synthesis, inflammation, membrane fusion and intracellular Ca2+ increase has rapidly expanded. C1P is a bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). This chapter specifically focuses on the role of C1P in phagocytosis and Ca2+ homeostasis. Studies of the metabolism of C1P during phagocytosis, may lead to a better understanding of its role in signaling. Potentially, the inhibition of CERK and C1P formation may be a therapeutic target for inflammation.

Optimization of Eliglustat-Based Glucosylceramide Synthase Inhibitors as Substrate Reduction Therapy for Gaucher Disease Type 3.

There remain no approved therapies for rare but devastating neuronopathic glyocosphingolipid storage diseases, such as Sandhoff, Tay-Sachs, and Gaucher disease type 3. We previously reported initial optimization of the scaffold of eliglustat, an approved therapy for the peripheral symptoms of Gaucher disease type 1, to afford 2, which effected modest reductions in brain glucosylceramide (GlcCer) in normal mice at 60 mg/kg. The relatively poor pharmacokinetic properties and high Pgp-mediated efflux of 2 prompted further optimization of the scaffold. With a general objective of reducing topological polar surface area, and guided by multiple metabolite identification studies, we were successful at identifying 17 (CCG-222628), which achieves remarkably greater brain exposure in mice than 2. After demonstrating an over 60-fold improvement in potency over 2 at reducing brain GlcCer in normal mice, we compared 17 with Sanofi clinical candidate venglustat (Genz-682452) in the CBE mouse model of Gaucher disease type 3. At doses of 10 mg/kg, 17 and venglustat effected comparable reductions in both brain GlcCer and glucosylsphingosine. Importantly, 17 achieved these equivalent pharmacodynamic effects at significantly lower brain exposure than venglustat.

The role of sphingosine-1-phosphate and ceramide-1-phosphate in calcium homeostasis.

During the last several years, sphingolipids have been identified as a source of important signaling molecules. Particularly, the understanding of the distinct biological roles of ceramide, sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P) and lyso-sphingomyelin in the regulation of cell growth, death, senescence, adhesion, migration, inflammation, angiogenesis and intracellular trafficking has rapidly expanded. Additional studies have elucidated the biological roles of sphingolipids in maintaining a homeostatic environment in cells, as well as in regulating numerous cellular responses to environmental stimuli. This review focuses on the role of S1P and C1P in maintaining Ca2+ homeostasis. By studying changes in the metabolism of S1P and C1P in pathological conditions, it is hoped that altered sphingolipid-metabolizing enzymes and their metabolites can be used as therapeutic targets.

Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase.

Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high-resolution crystal structures of human LPLA2 and a low-resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/ß hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome.

Ceramide-dependent PP2A regulation of TNFalpha-induced IL-8 production in respiratory epithelial cells.

IL-8 is a key mediator in the pathophysiology of acute lung injury. TNFalpha stimulates IL-8 production in respiratory epithelial cells by activating both the NF-kappaB and MAP kinase pathways. The precise mechanism by which these pathways are downregulated to terminate IL-8 production remains unclear. We studied the regulatory role of the serine/threonine phosphatase, PP2A, on the signaling pathways involved in IL-8 production from respiratory epithelial cells. Inhibition of PP2A using okadaic acid or gene knockdown using siRNA resulted in an augmentation of TNFalpha-induced IL-8 production. We also found that PP2A inhibition resulted in prolonged activation of JNK, p38, and ERK resulting in both increased transcriptional activation of the IL-8 promoter and posttranscriptional stabilization of IL-8 mRNA. Because TNFalpha had been shown to activate ceramide accumulation, and separate studies had linked ceramide with activation of PP2A, we hypothesized the pathway of TNFalpha-inducing ceramide to activate PP2A comprised an endogenous regulatory pathway. Inhibition of the immediate sphingomyelinase-dependent pathway as well as the de novo synthesis pathway of ceramide production reduced serine/threonine phosphatase activity and augmented IL-8 production. These data suggest that ceramide plays a role in activating PP2A to terminate ongoing IL-8 production. In summary, our data suggest that in respiratory epithelium, TNFalpha induces ceramide accumulation, resulting in subsequent activation of PP2A, which targets those kinases responsible for transcriptional activation of IL-8.

Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells.

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcgammaRIIA or both FcgammaRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcgammaRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcgammaRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcgammaRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.

Tristetraprolin (TTP)-14-3-3 complex formation protects TTP from dephosphorylation by protein phosphatase 2a and stabilizes tumor necrosis factor-alpha mRNA.

Tumor necrosis factor (TNF)-alpha is a major cytokine produced by alveolar macrophages in response to pathogen-associated molecular patterns such as lipopolysaccharide. TNF-alpha secretion is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation occurs by modulation of TNF-alpha mRNA stability via the binding of tristetraprolin (TTP) to the adenosine/uridine-rich elements found in the 3'-untranslated region of the TNF-alpha transcript. Phosphorylation plays important roles in modulating mRNA stability, because activation of p38 MAPK by lipopolysaccharide stabilizes TNF-alpha mRNA. We hypothesized that the protein phosphatase 2A (PP2A) regulates this signaling pathway. Our results show that inhibition of PP2A by okadaic acid or small interference RNA significantly enhanced the stability of TNF-alpha mRNA. This result was associated with increased phosphorylation of p38 MAPK and MAPK-activated kinase 2 (MK-2). PP2A inhibition increased TTP phosphorylation and enhanced complex formation with chaperone protein 14-3-3. TTP physically interacted with PP2A in transfected mammalian cells. A functional consequence of TTP-14-3-3 complex formation appeared to be protection of TTP from dephosphorylation by inhibition of the binding of PP2A to phosphorylated TTP. Mutation of the MK-2 phosphorylation sites of TTP did not influence TNF-alpha adenosine/uridine-rich element binding and did not alter the increased TNF-alpha 3'-untranslated region-dependent luciferase activity induced by PP2A-small interference RNA silencing. Our data indicate that, although phosphorylation stabilizes TNF-alpha mRNA, PP2A regulates the mRNA stability by modulating the phosphorylation state of members of the p38/MK-2/TTP pathway.

Phagocytic signaling molecules in lipid rafts of COS-1 cells transfected with FcgammaRIIA.

COS-1 cells bearing FcgammaRIIA were used as a model to demonstrate co-localization of several enzymes previously shown to regulate neutrophil phagocytosis. In COS-1 cells, phospholipase D (PLD) in the membrane fraction was activated during phagocytosis. PLD was found almost exclusively in lipid rafts, along with RhoA and ARF1. Protein kinase C-delta (PKCdelta) and Raf-1 translocated to lipid rafts. In neutrophils, ceramide levels increase during phagocytosis, indicating that FcgammaRIIA engagement initiates ceramide generation. Applying this model, we transfected COS-1 cells with FcgammaRIIA that had been mutated in the ITAM region, rendering them unable to ingest particles. When the mutant receptors were engaged, ceramide was generated and MAPK was activated normally, thus these processes did not require actual ingestion of particles. These results indicate that signaling proteins for phagocytosis are either constitutively present in, or are recruited to, lipid rafts where they are readily available to activate one another.

Ceramide 1-phosphate, a mediator of phagocytosis.

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the FcgammaRIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERK-transfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERK-transfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.

Enhanced phagocytosis through inhibition of de novo ceramide synthesis.

Fcgamma receptors are important mediators of the binding of IgG to and induction of phagocytosis in neutrophils. COS-1 cells provide a potentially useful model for studying these receptors because transfection with the FcgammaRIIA renders these cells phagocytic. During FcgammaRIIA-mediated phagocytosis in COS-1 cells, endogenous ceramide levels increased 52% by 20 min (p < 0.01). Phospholipase D activity increased by 62% (p < 0.01). Correspondingly, the phagocytic index increased by 3.7-fold by 20 min. Two inhibitors of ceramide formation were used to assess the consequences of reduced ceramide generation. l-Cycloserine, an inhibitor that blocks serine palmitoyltransferase activity, lowered both sphingosine and ceramide levels. Under these conditions, the phagocytic index increased 100% in the presence of 2 mm l-cycloserine. The formation of ceramide resulting from the N-acylation of dihydrosphingosine or sphingosine by ceramide synthase is inhibited by the fungal toxin fumonisin B(1). When cells were treated with 5-50 microm fumonisin B(1), the cellular level of ceramide decreased in a concentration-dependent manner, while simultaneously the phagocytic index increased by 52%. Concomitantly, three indirect measures of FcgammaRIIA activity were altered with the fall in ceramide levels. Syk phosphorylation, phospholipase D activity, and mitogen-activated protein (MAP) kinase phosphorylation were increased at 30 min. When Syk phosphorylation was blocked with piceatannol and cells were similarly challenged, phosphatidylinositol 3-kinase activation was blocked, but no changes in either ceramide accumulation or MAP kinase activation were observed. Ceramide formation and MAP kinase activation are therefore not dependent on Syk kinase activity in this system. These results indicate that COS-1 cells provide a useful model for the recapitulation of sphingolipid signaling in the study of phagocytosis. Ceramide formed by de novo synthesis may represent an important mechanism in the regulation of phagocytosis.

Granulocyte colony-stimulating factor primes NADPH oxidase in neutrophils through translocation of cytochrome b(558) by gelatinase-granule release.

Granulocyte colony-stimulating factor (GCSF) primes reduced neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in response to formyl peptide but does not increase oxidase activity when used alone. Both oxidase activity and degranulation require phospholipase D (PLD) activation, and exogenous C(2)-ceramide inhibits both functions through inhibition of PLD activity. We extended these observations to investigate neutrophil responses to GCSF. GCSF at a dosage of 30 to 100 ng/mL, a concentration range that primes superoxide release, stimulated a 60% to 100% increase in gelatinase release from tertiary granules but did not stimulate lactoferrin release from secondary granules. A 75% to 100% dose-dependent increase in PLD activity in GCSF-treated neutrophils was also observed. Gelatinase release and PLD activity were inhibited by 10 micromol/L C(2)-ceramide. The increase in gelatinase release in response to priming concentrations of GCSF suggests that tertiary granules contribute a component of the NADPH oxidase to the plasma membrane. Neutrophils treated with 50 ng/mL GCSF were found to contain 20% more cytochrome b(558) in the plasma membrane fraction than unstimulated cells, consistent with degranulation of only tertiary granules. Correspondingly, in the presence of 10 micromol/L C(2)-ceramide, cytochrome b(558) content in the plasma membrane did not increase after neutrophil activation. In contrast, GCSF did not lead to p47phox translocation to the plasma membrane or phosphorylation. Because phosphorylation and translocation of p47phox are required for oxidase activity, these findings account for the inability of GCSF alone to generate the respiratory burst. We conclude that translocation of cytochrome b(558) was responsible for GCSF priming of NADPH oxidase in neutrophils.

Regulation of polymorphonuclear leukocyte degranulation and oxidant production by ceramide through inhibition of phospholipase D.

Exogenous C(2)-ceramide has been shown to inhibit polymorphonuclear leukocyte (PMN) phagocytosis through inhibition of phospholipase D (PLD) and downstream events, including activation of extracellular signal-regulated kinases 1 and 2, leading to the hyphothesis that the sphingomyelinase pathway is involved in termination of phagocytosis. Here it is postulated that increased PLD activity generating phosphatidic acid and diacylglycerol (DAG) is essential for superoxide release and degranulation and that ceramide, previously shown to be generated during PMN activation, inhibits PLD activation, thereby leading to inhibition of PMN function. When PMNs were primed with granulocyte colony-stimulating factor (G-CSF) and then activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C(2)-ceramide (10 microM) completely inhibited release of superoxide, lactoferrin, and gelatinase; the DAG analog sn-1,2-didecanoylglycerol (DiC10) (10 microM) restored oxidase activation and degranulation in the ceramide-treated cells. Similarly, C(2)-ceramide inhibited oxidase activity and degranulation of PMNs treated with cytochalasin B followed by FMLP, and DiC10 restored function. In contrast, C(2)-ceramide did not inhibit phosphorylation of p47phox or p38 mitogen-activated protein kinase, or translocation of p47phox, PLD-containing organelles, adenosine diphosphate-ribosylation factor 1, RhoA, protein kinase C (PKC)-beta or PKC-alpha to the plasma membrane in G-CSF or cytochalasin B-treated, FMLP-activated PMNs. PLD activity increased by 3-fold in G-CSF-primed PMNs stimulated by FMLP and by 30-fold in cytochalasin B-treated PMNs stimulated by FMLP. Both PLD activities were completely inhibited by 10 microM C(2)-ceramide. In conclusion, superoxide, gelatinase, and lactoferrin release require activation of the PLD pathway in primed PMNs and cytochalasin B-treated PMNs. Ceramide may affect protein interactions with PLD in the plasma membrane, thereby attenuating PMN activation.

Ceramide inhibition of phospholipase D and its relationship to RhoA and ARF1 translocation in GTP gamma S-stimulated polymorphonuclear leukocytes.

Phospholipase D (PLD) regulates the polymorphonuclear leukocyte (PMN) functions of phagocytosis, degranulation, and oxidant production. Ceramide inhibition of PLD suppresses PMN function. In streptolysin O-permeabilized PMNs, PLD was directly activated by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulation of adenosine diphosphate (ADP)-ribosylation factor (ARF) and Rho, stimulating release of lactoferrin from specific granules of permeabilized PMNs; PLD activation and degranulation were inhibited by C2-ceramide but not dihydro-C2-ceramide. To investigate the mechanism of ceramide's inhibitory effect on PLD, we used a cell-free system to examine PLD activity and translocation from cytosol to plasma membrane of ARF, protein kinase C (PKC)alpha and beta, and RhoA, all of which can activate PLD. GTP gamma S-activated cytosol stimulated PLD activity and translocation of ARF, PKC alpha and beta, and RhoA when recombined with cell membranes. Prior incubation of PMNs with 10 microM C2-ceramide inhibited PLD activity and RhoA translocation, but not ARF1, ARF6, PKC alpha, or PKC beta translocation. However, in intact PMNs stimulated with N-formyl-1-methionyl-1-leucyl-1-phenylalamine (FMLP) or permeabilized PMNs stimulated with GTP gamma S, C2-ceramide did not inhibit RhoA translocation. Exogenous RhoA did not restore ceramide-inhibited PLD activity but bound to membranes despite ceramide treatment. These observations suggest that, although ceramide may affect RhoA in some systems, ceramide inhibits PLD through another mechanism, perhaps related to the ability of ceramide to inhibit phosphatidylinositol-bisphosphate (PIP2) interaction with PLD.