Role of membrane oxidation in controlling the activity of human group IIa secretory phospholipase A2 toward apoptotic lymphoma cells

The membranes of healthy lymphocytes normally resist hydrolysis by secretory phospholipase A₂. However, they become susceptible during the process of apoptosis. Previous experiments have demonstrated the importance of certain physical changes to the membrane during cell death such as a reduction in membrane lipid order and exposure of phosphatidylserine on the membrane surface. Nevertheless, those investigations also showed that at least one additional factor was required for rapid hydrolysis by the human group IIa phospholipase isozyme. This study was designed to test the possibility that oxidation of membrane lipids is the additional factor. Flow cytometry and confocal microscopy with a fluorescent probe of oxidative potential suggested that oxidation of the plasma membrane occurs during apoptosis stimulated by thapsigargin. When oxidative potential was high, the activity of human group IIa secretory phospholipase A₂ was enhanced 30- to 100-fold compared to that observed with conditions sufficient for maximal hydrolysis by other secretory phospholipase A₂ isoforms. Direct oxidation of cell membranes with either of two oxidizing agents also stimulated hydrolysis by secretory phospholipase A₂. Both oxidizers caused externalization of phosphatidylserine, but a change in lipid order did not always occur. These results demonstrated that membrane oxidation strongly stimulates human group IIa secretory phospholipase A₂ activity toward apoptotic cells. Interestingly, the change in membrane order, previously thought to be imperative for high rates of hydrolysis, was not required when membrane lipids were oxidized. Whether phosphatidylserine exposure is still necessary with oxidation remains unresolved since the two events could not be deconvoluted.     

Induction of distinct sets of secretory phospholipase A2 in rodents during inflammation

Although the expression of the prototypic secretory phospholipase A₂ (sPLA₂), group IIA (sPLA₂-IIA), is known to be up-regulated during inflammation, it remains uncertain if other sPLA₂ enzymes display similar or distinct profiles of induction under pathological conditions. In this study, we investigated the expression of several sPLA₂s in rodent inflammation models. In lipopolysaccharide (LPS)-treated mice, the expression of sPLA₂-V, and to a lesser extent that of sPLA₂-IID, -IIE, and -IIF, were increased, whereas that of sPLA₂-X was rather constant, in distinct tissues. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, in which the expression of sPLA₂-IID, -IIF and -V was increased, was significantly reduced by YM-26734, a competitive sPLA₂-IIA inhibitor that turned out to inhibit sPLA₂-IID, -IIE, -V and -X as well. In contrast, sPLA₂-IIA was dominant in carageenin-induced pleurisy in rats, where the accumulation of exudate fluids and leukocytes was significantly ameliorated by YM-26734. These results indicate that distinct sPLA₂s can participate in inflammatory diseases according to tissues, animal species, and types of inflammation.     

Key Role of Group V Secreted Phospholipase A2 in Th2 Cytokine and Dendritic Cell-Driven Airway Hyperresponsiveness and Remodeling

Background

Previous work has shown that disruption of the gene for group X secreted phospholipase A₂ (sPLA₂-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA₂s in the mammalian genome, the involvement of other sPLA₂s in the asthma model is possible – in particular, the group V sPLA₂ (sPLA₂-V) that like sPLA₂-X is highly active at hydrolyzing membranes of mammalian cells.

Methodology and Principal Findings

The allergen-driven asthma phenotype was significantly reduced in sPLA₂-V-deficient mice but to a lesser extent than observed previously in sPLA₂-X-deficient mice. The most striking difference observed between the sPLA₂-V and sPLA₂-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA₂-V^−/− mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V^−/− mice diminishes Th2 cytokine responses in the airways.

Conclusions

This paper illustrates the diverse roles of sPLA₂s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA₂-V as a potential new therapy to treat asthma and other allergic disorders.     

Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A₂ (GX KO). These mice have ∼80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A₂ (sPLA₂), but not a catalytically inactive mutant form of GX sPLA₂, significantly reduced steroid production 30–40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA₂ inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA₂-overexpressing Y1 cells, ruling out a role for this sPLA₂ receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was ∼2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA₂. Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA₂ antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA₂ is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.     

Effects of cholesterol on physical properties of human erythrocyte membranes: impact on susceptibility to hydrolysis by secretory phospholipase A2

The ability of secretory phospholipase A₂ (sPLA₂) to hydrolyze cell membranes is highly dependent on the physical properties of the membrane. The effects of cholesterol on these properties have been characterized in artificial bilayers and found to alter sPLA₂ activity significantly. It is hypothesized that the natural difference in cholesterol content between erythrocytes and leukocytes is in part responsible for their differing susceptibility to hydrolysis by sPLA₂. To test this hypothesis, defined amounts of cholesterol were removed from erythrocyte membranes using methyl-β-cyclodextrin. Treatment of cells with methyl-β-cyclodextrin increased the hydrolysis rate and total substrate hydrolyzed by sPLA₂. In general, this effect of cholesterol removal was more pronounced at higher temperatures. Comparison of the level of membrane order (assessed with the fluorescent probe laurdan) with hydrolysis rate revealed that sPLA₂ activity was greatly enhanced upon significant reductions in lipid order. Additional treatment of the cells with calcium ionophore further enhanced the hydrolysis rate and altered the relationship with membrane order. These data demonstrated that interactions with sPLA₂ observed in artificial bilayers apply to biological membranes. It is also proposed that the high level of cholesterol in erythrocyte membranes is a protective mechanism to guard against hydrolytic enzymes.     

Comparative efficacy of a secretory phospholipase A<Subscript>2</Subscript> inhibitor with conventional anti-inflammatory agents in a rat model of antigen-induced arthritis

Introduction  

Previously, secretory phospholipase A₂ (sPLA₂) inhibition has been used as an adjunct to conventional rheumatoid arthritis therapy in human clinical trials without significant improvement of arthritic pathology. In this study, we compared the efficacy of a potent and orally active group IIa secretory phospholipase A₂ inhibitor (sPLA₂I) to conventional anti-arthritic agents; infliximab, leflunomide and prednisolone, in a rat model of antigen-induced arthritis.     

Inhibition of secretory phospholipase A2 enzyme by bilirubin: A new role as endogenous anti-inflammatory molecule

Bilirubin is a powerful antioxidant that suppresses the inflammatory process. However its interaction with proinflammatory PLA₂ enzyme is not known. Inhibition of several secretory phospholipase A₂ (sPLA₂) enzyme activities by bilirubin was studied using ¹⁴C-oleate labeled Escherichia coli as substrate. Bilirubin inhibits purified sPLA₂ enzyme from Vipera russellii and Naja naja venom and partially purified sPLA₂ enzymes from human ascitic fluid, pleural fluid and normal serum in a dose dependent manner. IC₅₀ values calculated for these enzymes ranges from 1.75 to 10.5 μM. Inflammatory human sPLA₂ enzymes are more sensitive to inhibition by bilirubin than snake venom sPLA₂s. Inhibition of sPLA₂ activity by bilirubin is independent of calcium concentration. Increasing substrate concentration (upto 180 nmol) did not relieve the inhibition of sPLA₂ by bilirubin and it is irreversible. Bilirubin quenched the relative fluorescence intensity of sPLA₂ in a dose dependent manner in the same concentration range at which in vitro sPLA₂ inhibition was observed. In the presence of bilirubin, apparent shift in the far UV-CD spectra of sPLA₂ was observed, indicating a direct interaction with the enzyme. Inhibition of sPLA₂ induced mouse paw edema by bilirubin confirms its sPLA₂ inhibitory activity in vivo also. These findings indicate that inhibition of sPLA₂ by bilirubin is mediated by direct interaction with the enzyme and bilirubin may act as an endogenous regulator of sPLA₂ enzyme activity.     

Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase

LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A₂ (sPLA₂-IIa and sPLA₂-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA₂-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA₂-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA₂-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis.     

Inhibition of sPLA2-IIA Prevents LPS-Induced Neuroinflammation by Suppressing ERK1/2-cPLA2α Pathway in Mice Cerebral Cortex

Neuroinflammation is involved in various central nervous system (CNS) disorders, including brain infections, ischemia, trauma, stroke, and degenerative CNS diseases. In the CNS inflammation, secretory phospholipase A₂-IIA (sPLA₂-IIA) acts as a mediator, resulting in the generation of the precursors of pro-inflammatory lipid mediators, such as prostaglandins (PGs) and leukotrienes (LTs). However, the role of sPLA₂-IIA in neuroinflammation is more complicated and remains unclear yet. In the present study, we investigated the effect of sPLA₂-IIA inhibition by specific inhibitor SC-215 on the inflammation in LPS-induced mice cerebral cortex and primary astrocytes. Our results showed that the inhibition of sPLA₂-IIA alleviated the release of PGE₂ by suppressing the activation of ERK1/2, cPLA₂α, COX-2 and mPGES-1. These findings demonstrated that sPLA₂-IIA showed the potential to regulate the neuroinflammation in vivo and in vitro, indicating that sPLA₂-IIA might be a novel target for the treatment of acute neuroinflammation.     

Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host

Type-IIA secreted phospholipase A₂ (sPLA₂-IIA) has been proposed to play a role in the development of inflammatory diseases. It has been shown to release arachidonic acid, the precursor of proinflammatory eicosanoids, to hydrolyze phospholipids of pulmonary surfactant, and to bind to specific receptors located on cell surface membranes. However, the most established biological role of sPLA₂-IIA is related to its potent bactericidal property in particular toward Gram-positive bacteria. This enzyme is present in animal and human biological fluids at concentrations sufficient to kill bacteria. Human recombinant sPLA₂-IIA is able to kill Gram-positive bacteria at concentrations as low as 1.1 ng/ml. This remarkable property is due to the unique preference of sPLA₂-IIA for anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes. Much higher concentrations of sPLA₂-IIA are required for its action on host cell membranes and surfactant both of which are mainly composed by phosphatidylcholine, a poor substrate for sPLA₂-IIA. Transgenic mice over-expressing human sPLA₂-IIA are resistant to infection by Staphylococcus aureus, Escherichia coli, and Bacillus anthracis, the etiological agent of anthrax. Conversely, certain bacteria, such as B. anthracis, E. coli and Bordetella pertussis are able to inhibit sPLA₂-IIA expression by host cells, thus highlighting a mechanism by which these bacteria can subvert the host immune system. Intranasal instillation of recombinant sPLA₂-IIA protects mice from mortality caused by pulmonary anthrax. Interestingly, this protective effect was obtained even with B. anthracis strains that down-regulate the expression of endogenous sPLA₂-IIA, indicating that instilled sPLA₂-IIA can overcome the subversive action of B. anthracis. We conclude that sPLA₂-IIA is an efficient endogenous antibiotic of the host and can play a role in host defense against pathogenic bacteria. It can be used as a therapeutic agent in adjunct with current therapy to treat bacteria resistant to multiple antibiotics.     

Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases: IMPLICATIONS FOR THE REGULATION OF ADRENAL STEROIDOGENESIS*

Group X secretory phospholipase A₂ (GX sPLA₂) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA₂ is produced as a pro-enzyme (pro-GX sPLA₂) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA₂ activation have not been identified. We previously reported that GX sPLA₂ negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA₂ expression construct (FLAG-pro-GX sPLA₂), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA₂ processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA₂ processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA₂ processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA₂ to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA₂ processing and sPLA₂ activity in Y1 cells, and it significantly attenuated GX sPLA₂-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA₂ is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.     

Acidity and lipolysis by group V secreted phospholipase A2 strongly increase the binding of apoB-100-containing lipoproteins to human aortic proteoglycans

Local acidic areas characterize diffuse intimal thickening (DIT) and advanced atherosclerotic lesions. The role of acidity in the modification and extra- and intracellular accumulation of triglyceride-rich VLDL and IDL particles has not been studied before. Here, we examined the effects of acidic pH on the activity of recombinant human group V secreted phospholipase A₂ (sPLA₂-V) toward small VLDL (sVLDL), IDL, and LDL, on the binding of these apoB-100-containing lipoproteins to human aortic proteoglycans, and on their uptake by human monocyte-derived macrophages. At acidic pH, the ability of sPLA₂-V to lipolyze the apoB-100-containing lipoproteins was moderately, but significantly, increased while binding of the lipoproteins to proteoglycans increased > 60-fold and sPLA₂-V-modification further doubled the binding. Moreover, acidic pH more than doubled macrophage uptake of soluble complexes of sPLA₂-V-LDL with aortic proteoglycans. Proteoglycan-affinity chromatography at pH 7.5 and 5.5 revealed that sVLDL, IDL, and LDL consisted of populations with different proteoglycan-binding affinities, and, surprisingly, the sVLDL fractions with the highest proteoglycan-affinity contained only low amounts of apolipoproteins E and C-III. Our results suggest that in atherosclerotic lesions with acidic extracellular pH, sPLA₂-V is able to lipolyze sVLDL, IDL, and LDL, and increase their binding to proteoglycans. This is likely to provoke extracellular accumulation of lipids derived from these atherogenic lipoprotein particles and to increase the progression of the atherosclerotic lesions.     

Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge

We propose that expression of four genes encoding secretory phospholipases A₂ (sPLA₂) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA₂-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA₂ activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA₂ and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA₂s. The recombinant sPLA₂s were inhibited by sPLA₂ inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA₂ genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA₂s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA₂s showed the presence of the sPLA₂ enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA₂ genes that mediate nodulation reactions strongly supports our hypothesis that sPLA₂s are central enzymes in insect cellular immune reactions.     

Phospholipase A2 Down-Regulates the Affinity of [3H]AMPA Binding to Rat Cortical Membranes

Abstract: The effects of exogenous phospholipase A₂ on the binding of α-[³H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([³H]AMPA) to rat cortical membranes in the presence of the chaotrope potassium thiocyanate were assessed. Pretreatment of membranes with secretory phospholipase A₂ (sPLA₂) elicited a concentration-dependent decrease in specific [³H]AMPA binding due mainly to a decrease in affinity (K_D). This observation, together with protease inhibitor and western blot evidence, suggest that the sPLA₂ effect is not due to proteolysis. The sPLA₂-evoked decrease was temperature and calcium dependent. Inclusion of the specific inhibitor oleoyloxyethyl phosphocholine or preincubation of the enzyme with reducing agents to degrade its secondary structure significantly reduced the sPLA₂ inhibition. These results suggest that the effects of sPLA₂ arise from an enzymatic action rather than a competitive interaction at the AMPA binding site. However, arachidonic acid, a major metabolite of sPLA₂ action, did not cause a similar decrease in the affinity of [³H]AMPA binding. In contrast to the effects on [³H]AMPA binding, sPLA₂ caused an increase in [³H]CNQX binding, which is in accordance with the functionality of the AMPA receptor complex. These results suggest that sPLA₂ may play a role in the physiological and pathophysiological regulation of AMPA receptors.     

Refolding and purification of the human secreted group IID phospholipase A2 expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A₂ (sPLA₂s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A₂ (hsPLA₂-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA₂-IID in Escherichia coli, the DNA-coding sequence for hsPLA₂-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA₂-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A₂. The refolded recombinant hsPLA₂-IID demonstrated Ca²⁺-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA₂-IID which will advance our understanding of the structure–function relationship and biological effects of the protein.     

Secretory phospholipase A2 promotes MMP‐9‐mediated cell death by degrading type I collagen via the ERK pathway at an early stage of chondrogenesis

Background information. sPLA₂ (secretory phospholipase A₂) has been implicated in a wide range of cellular responses, including cell proliferation and ECM (extracellular matrix) remodelling. Even though ECM remodelling is an essential step for chondrogenesis, the expression and functions of sPLA₂ during chondrogenesis have not been studied. Results. In the present study, for the first time, we detect the secretion of sPLA₂ during limb development and suggest that sPLA₂ influences the proliferation and/or survival of limb mesenchymal cells. Treatment of wing bud mesenchymal cells with exogenous sPLA₂ promoted cell death by activating MMP‐9 (matrix metalloproteinase‐9) and increasing type I collagen degradation. The additive chondro‐inhibitory actions were induced by co‐treatment of mp‐BSA (p‐aminophenyl‐mannopyranoside‐BSA), a known ligand of the mannose receptor. Chondro‐inhibitory actions by sPLA₂ were prevented by functional blocking of FcRY (chicken yolk sac IgY receptor), a mannose receptor family member that is the orthologue of the mammalian PLA₂ (phospholipase A₂) receptor and by inhibition of ERK (extracellular‐signal‐regulated kinase) activity. Conclusions. Taken together, our results suggest that elevated levels of sPLA₂ secreted by wing bud mesenchymal cells promote type I collagen degradation by MMP‐9 in a manner typical of receptor‐mediated signalling and that these events lead to cell death.     

Group IID,IIE, IIF and III secreted phospholipase A2s

Among the 11 members of the secreted phospholipase A₂ (sPLA₂) family, group IID, IIE, IIF and III sPLA₂s (sPLA₂-IID, -IIE, -IIF and -III, respectively) are “new” isoforms in the history of sPLA₂ research. Relative to the better characterized sPLA₂s (sPLA₂-IB, -IIA, -V and -X), the enzymatic properties, distributions, and functions of these “new” sPLA₂s have remained obscure until recently. Our current studies using knockout and transgenic mice for a nearly full set of sPLA₂s, in combination with comprehensive lipidomics, have revealed unique and distinct roles of these “new” sPLA₂s in specific biological events. Thus, sPLA₂-IID is involved in immune suppression, sPLA₂-IIE in metabolic regulation and hair follicle homeostasis, sPLA₂-IIF in epidermal hyperplasia, and sPLA₂-III in male reproduction, anaphylaxis, colonic diseases, and possibly atherosclerosis. In this article, we overview current understanding of the properties and functions of these sPLA₂s and their underlying lipid pathways in vivo.     

Development of a Cell-Based Bioassay for Phospholipase A2-Triggered Liposomal Drug Release

The feasibility of exploiting secretory phospholipase A₂ (sPLA₂) enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA₂-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA₂-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA₂ enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA₂-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG) 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA₂ releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA₂. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA₂-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.     

Group X secretory phospholipase A2 induces potent productions of various lipid mediators in mouse peritoneal macrophages

We have previously shown the expression of group X secretory phospholipase A₂ (sPLA₂-X) in mouse splenic macrophages and its powerful potency for releasing fatty acids from various intact cell membranes. Here, we examined the potency of sPLA₂-X in the production of lipid mediators in murine peritoneal macrophages. Mouse sPLA₂-X was found to induce a marked release of fatty acids including arachidonic acid and linoleic acid, which contrasted with little, if any, release by the action of group IB and IIA sPLA₂s. In resting macrophages, sPLA₂-X elicited a modest production of prostaglandin E₂ and thromboxane A₂. After the induction of cyclooxygenase-2 (COX-2) by pretreatment with lipopolysaccharide, a dramatic increase in the production of these eicosanoids was observed in sPLA₂-X-treated macrophages, which was completely blocked by the addition of either the specific sPLA₂ inhibitor indoxam or the COX inhibitor indomethacin. In accordance with its higher hydrolyzing activity toward phosphatidylcholine, mouse sPLA₂-X induced a potent production of lysophosphatidylcholine. These findings strongly suggest that sPLA₂-X plays a critical role in the production of various lipid mediators from macrophages. These events might be relevant to the progression of various pathological states, including chronic inflammation and atherosclerosis.     

Activities and interactions among phospholipases A2 during thapsigargin-induced S49 cell death

The purpose of this study was to determine the roles of calcium-dependent phospholipase A₂ (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) in thapsigargin-induced membrane susceptibility to secretory phospholipase A₂ (sPLA₂) and programmed cell death. ³H-arachidonic acid release was observed in the presence of thapsigargin. This release was inhibited partially by an inhibitor of iPLA₂ (BEL) and completely by an inhibitor of both cPLA₂ and iPLA₂ (MAFP) suggesting that these enzymes were active during apoptosis. The process of cell death did not require the activity of either enzyme since neither inhibitor impeded the progression of apoptosis. However, both inhibitors increased the susceptibility of the membrane to sPLA₂ in the presence of thapsigargin. In the case of BEL, this effect appeared to involve direct induction of apoptosis in a sub-population of the cells independent of the action of iPLA₂. In conclusion, the results suggested that cPLA₂ and iPLA₂ are active during thapsigargin-induced apoptosis in S49 cells and that cPLA₂ tempers the tendency of the cells to become susceptible to sPLA₂ during apoptosis.