Rob C. Oslund

Highly specific and broadly potent inhibitors of mammalian secreted phospholipases A2.

We report a series of inhibitors of secreted phospholipases A2 (sPLA2s) based on substituted indoles, 6,7-benzoindoles, and indolizines derived from LY315920, a well-known indole-based sPLA2 inhibitor. Using the human group X sPLA2 crystal structure, we prepared a highly potent and selective indole-based inhibitor of this enzyme. Also, we report human and mouse group IIA and IIE specific inhibitors and a substituted 6,7-benzoindole that inhibits nearly all human and mouse sPLA2s in the low nanomolar range.

Improved sensitivity mass spectrometric detection of eicosanoids by charge reversal derivatization.

Combined liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is a powerful method for the analysis of oxygenated metabolites of polyunsaturated fatty acids including eicosanoids. Here we describe the synthesis of a new derivatization reagent N-(4-aminomethylphenyl)pyridinium (AMPP) that can be coupled to eicosanoids via an amide linkage in quantitative yield. Conversion of the carboxylic acid of eicosanoids to a cationic AMPP amide improves sensitivity of detection by 10- to 20-fold compared to negative mode electrospray ionization detection of underivatized analytes. This charge reversal derivatization allows detection of cations rather than anions in the electrospray ionization mass spectrometer, which enhances sensitivity. Another factor is that AMPP amides undergo considerable collision-induced dissociation in the analyte portion rather than exclusively in the cationic tag portion, which allows isobaric derivatives to be distinguished by tandem mass spectrometry, and this further enhances sensitivity and specificity. This simple derivatization method allows prostaglandins, thromboxane B(2), leukotriene B(4), hydroxyeicosatetraenoic acid isomers, and arachidonic acid to be quantified in complex biological samples with limits of quantification in the 200-900 fg range. One can anticipate that the AMPP derivatization method can be extended to other carboxylic acid analytes for enhanced sensitivity detection.

Production of Vascular Endothelial Growth Factors from Human Lung Macrophages Induced by Group IIA and Group X Secreted Phospholipases A2

Angiogenesis and lymphangiogenesis mediated by vascular endothelial growth factors (VEGFs) are main features of chronic inflammation and tumors. Secreted phospholipases A2 (sPLA2s) are overexpressed in inflammatory lung diseases and cancer and they activate inflammatory cells by enzymatic and receptor-mediated mechanisms. We investigated the effect of sPLA2s on the production of VEGFs from human macrophages purified from the lung tissue of patients undergoing thoracic surgery. Primary macrophages express VEGF-A, VEGF-B, VEGF-C, and VEGF-D at both mRNA and protein level. Two human sPLA2s (group IIA and group X) induced the expression and release of VEGF-A and VEGF-C from macrophages. Enzymatically-inactive sPLA2s were as effective as the active enzymes in inducing VEGF production. Me-Indoxam and RO092906A, two compounds that block receptor-mediated effects of sPLA2s, inhibited group X-induced release of VEGF-A. Inhibition of the MAPK p38 by SB203580 also reduced sPLA2-induced release of VEGF-A. Supernatants of group X-activated macrophages induced an angiogenic response in chorioallantoic membranes that was inhibited by Me-Indoxam. Stimulation of macrophages with group X sPLA2 in the presence of adenosine analogs induced a synergistic increase of VEGF-A release and inhibited TNF-α production through a cooperation between A2A and A3 receptors. These results demonstrate that sPLA2s induce production of VEGF-A and VEGF-C in human macrophages by a receptor-mediated mechanism independent from sPLA2 catalytic activity. Thus, sPLA2s may play an important role in inflammatory and/or neoplastic angiogenesis and lymphangiogenesis.

Relationship between levels of secreted phospholipase A2 groups IIA and X in the airways and asthma severity

Cite this as: T. S. Hallstrand, Y. Lai, Z. Ni, R. C. Oslund, W. R. Henderson Jr, M. H. Gelb and S. E. Wenzel, Clinical & Experimental Allergy, 2011 (41) 801–810.

Eosinophil Cysteinyl Leukotriene Synthesis Mediated by Exogenous Secreted Phospholipase A2 Group X

Secreted phospholipase A2 group X (sPLA2-X) has recently been identified in the airways of patients with asthma and may participate in cysteinyl leukotriene (CysLT; C4, D4, and E4) synthesis. We examined CysLT synthesis and arachidonic acid (AA) and lysophospholipid release by eosinophils mediated by recombinant human sPLA2-X. We found that recombinant sPLA2-X caused marked AA release and a rapid onset of CysLT synthesis in human eosinophils that was blocked by a selective sPLA2-X inhibitor. Exogenous sPLA2-X released lysophospholipid species that arise from phospholipids enriched in AA in eosinophils, including phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine as well as plasmenyl phosphatidylcholine and phosphatidylethanolamine. CysLT synthesis mediated by sPLA2-X but not AA release could be suppressed by inhibition of cPLA2α. Exogenous sPLA2-X initiated Ser505 phosphorylation of cPLA2α, an intracellular Ca2+ flux, and translocation of cPLA2α and 5-lipoxygenase in eosinophils. Synthesis of CysLTs in response to sPLA2-X or lysophosphatidylcholine was inhibited by p38 or JNK inhibitors but not by a MEK 1/2 inhibitor. A further increase in CysLT synthesis was induced by the addition of sPLA2-X to eosinophils under conditions of N-formyl-methionyl-leucyl-phenylalanine-mediated cPLA2α activation. These results indicate that sPLA2-X participates in AA and lysophospholipid release, resulting in CysLT synthesis in eosinophils through a mechanism involving p38 and JNK MAPK, cPLA2α, and 5-lipoxygenase activation and resulting in the amplification of CysLT synthesis during cPLA2α activation. Transactivation of eosinophils by sPLA2-X may be an important mechanism leading to CysLT formation in the airways of patients with asthma.

Blockade of Human Group X Secreted Phospholipase A2 (GX-sPLA2)-induced Airway Inflammation and Hyperresponsiveness in a Mouse Asthma Model by a Selective GX-sPLA2 Inhibitor

Group X (GX) phospholipase A2, a member of a large group of secreted phospholipases A2 (sPLA2s), has recently been demonstrated to play an important in vivo role in the release of arachidonic acid and subsequent formation of eicosanoids. In a Th2 cytokine-driven mouse asthma model, deficiency of mouse GX (mGX)-sPLA2 significantly impairs development of the asthma phenotype. In this study, we generated mGX-sPLA2−/− mice with knock-in of human GX (hGX)-sPLA2 (i.e. hGX-sPLA2+/+ knock-in mice) to understand more fully the role of GX-sPLA2 in these allergic pulmonary responses and to assess the effect of pharmacological blockade of the GX-sPLA2-mediated responses. Knock-in of hGX-sPLA2 in mGX-sPLA2−/− mice restored the allergen-induced airway infiltration by inflammatory cells, including eosinophils, goblet cell metaplasia, and hyperresponsiveness to methacholine in the mGX-sPLA2-deficient mice. This knock-in mouse model enabled the use of a highly potent indole-based inhibitor of hGX-sPLA2, RO061606 (which is ineffective against mGX-sPLA2), to assess the potential utility of GX-sPLA2 blockade as a therapeutic intervention in asthma. Delivery of RO061606 via mini-osmotic pumps enabled the maintenance in vivo in the mouse asthma model of plasma inhibitor concentrations near 10 μm, markedly higher than the IC50 for inhibition of hGX-sPLA2 in vitro. RO061606 significantly decreased allergen-induced airway inflammation, mucus hypersecretion, and hyperresponsiveness in the hGX-sPLA2+/+ knock-in mouse. Thus, development of specific hGX-sPLA2 inhibitors may provide a new pharmacological opportunity for the treatment of patients with asthma.

Group X Secretory Phospholipase A2 Negatively Regulates ABCA1 and ABCG1 Expression and Cholesterol Efflux in Macrophages

Objective—GX sPLA2 potently hydrolyzes plasma membranes to generate lysophospholipids and free fatty acids; it has been implicated in inflammatory diseases, including atherosclerosis. To identify a novel role for group X (GX) secretory phospholipase A2 (sPLA2) in modulating ATP binding casette transporter A1 (ABCA1) and ATP binding casette transporter G1 (ABCG1) expression and, therefore, macrophage cholesterol efflux. Methods and Results—The overexpression or exogenous addition of GX sPLA2 significantly reduced ABCA1 and ABCG1 expression in J774 macrophage-like cells, whereas GX sPLA2 deficiency in mouse peritoneal macrophages was associated with enhanced expression. Altered ABC transporter expression led to reduced cholesterol efflux in GX sPLA2-overexpressing J774 cells and increased efflux in GX sPLA2-deficient mouse peritoneal macrophages. Gene regulation was dependent on GX sPLA2 catalytic activity, mimicked by arachidonic acid and abrogated when liver X receptor (LXR)&agr;/&bgr; expression was suppressed, and partially reversed by the LXR agonist T0901317. Reporter assays indicated that GX sPLA2 suppresses the ability of LXR to transactivate its promoters through a mechanism involving the C-terminal portion of LXR spanning the ligand-binding domain. Conclusion—GX sPLA2 modulates gene expression in macrophages by generating lipolytic products that suppress LXR activation. GX sPLA2 may play a previously unrecognized role in atherosclerotic lipid accumulation by negatively regulating the genes critical for cellular cholesterol efflux.

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.

Group X Secreted Phospholipase A2 Proenzyme Is Matured by a Furin-like Proprotein Convertase and Releases Arachidonic Acid inside of Human HEK293 Cells

Background: Group X secreted phospholipase A2 is an enzyme produced as a proenzyme that plays an important role in arachidonic acid release. Results: Group X phospholipase A2 is matured intracellularly by a furin-like proprotein convertase and releases arachidonic acid during secretion. Conclusion: Group X phospholipase A2 can release arachidonic acid intracellularly. Significance: Group X phospholipase A2 may produce lipid mediators during secretion. Among mammalian secreted phospholipases A2 (sPLA2s), group X sPLA2 has the most potent hydrolyzing activity toward phosphatidylcholine and is involved in arachidonic acid (AA) release. Group X sPLA2 is produced as a proenzyme and contains a short propeptide of 11 amino acids ending with a dibasic motif, suggesting cleavage by proprotein convertases. Although the removal of this propeptide is clearly required for enzymatic activity, the cellular location and the protease(s) involved in proenzyme conversion are unknown. Here we have analyzed the maturation of group X sPLA2 in HEK293 cells, which have been extensively used to analyze sPLA2-induced AA release. Using recombinant mouse (PromGX) and human (ProhGX) proenzymes; HEK293 cells transfected with cDNAs coding for full-length ProhGX, PromGX, and propeptide mutants; and various permeable and non-permeable sPLA2 inhibitors and protease inhibitors, we demonstrate that group X sPLA2 is mainly converted intracellularly and releases AA before externalization from the cell. Most strikingly, the exogenous proenzyme does not elicit AA release, whereas the transfected proenzyme does elicit AA release in a way insensitive to non-permeable sPLA2 inhibitors. In transfected cells, a permeable proprotein convertase inhibitor, but not a non-permeable one, prevents group X sPLA2 maturation and partially blocks AA release. Mutations at the dibasic motif of the propeptide indicate that the last basic residue is required and sufficient for efficient maturation and AA release. All together, these results argue for the intracellular maturation of group X proenzyme in HEK293 cells by a furin-like proprotein convertase, leading to intracellular release of AA during secretion.

Expression of phospholipases A2 in primary human lung macrophages Role of cytosolic phospholipase A2-α in arachidonic acid release and platelet activating factor synthesis

Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA(2)) and secreted phospholipases A(2) (sPLA(2)) to the generation of lipid mediators in human macrophages are unclear. We investigated the expression and role of different PLA(2)s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA(2) (iPLA(2)). Two structurally-divergent inhibitors of group IV cPLA(2) completely block arachidonic acid release by macrophages in response to non-physiological (Ca(2+) ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA(2)s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA(2)s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA(2) inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA(2)-alpha, iPLA(2) and several sPLA(2)s. Cytosolic PLA(2)-alpha is the major enzyme responsible for lipid mediator production in human macrophages.