Daren Stephens

TLR-4 and Sustained Calcium Agonists Synergistically Produce Eicosanoids Independent of Protein Synthesis in RAW264.7 Cells

Arachidonic acid is released by phospholipase A2 and converted into hundreds of distinct bioactive mediators by a variety of cyclooxygenases (COX), lipoxygenases (LO), and cytochrome P450s. Because of the size and diversity of the eicosanoid class of signaling molecules produced, a thorough and systematic investigation of these biological processes requires the simultaneous quantitation of a large number of eicosanoids in a single analysis. We have developed a robust liquid chromatography/tandem mass spectrometry method that can identify and quantitate over 60 different eicosanoids in a single analysis, and we applied it to agonist-stimulated RAW264.7 murine macrophages. Fifteen different eicosanoids produced through COX and 5-LO were detected either intracellularly or in the media following stimulation with 16 different agonists of Toll-like receptors (TLR), G protein-coupled receptors, and purinergic receptors. No significant differences in the COX metabolite profiles were detected using the different agonists; however, we determined that only agonists creating a sustained Ca2+ influx were capable of activating the 5-LO pathway in these cells. Synergy between Ca2+ and TLR pathways was detected and discovered to be independent of NF-κB-induced protein synthesis. This demonstrates that TLR induction of protein synthesis and priming for enhanced phospholipase A2-mediated eicosanoid production work through two distinct pathways.

Systemic and Intrathecal Effects of a Novel Series of Phospholipase A2 Inhibitors on Hyperalgesia and Spinal Pge2 Release

Phospholipase A2 (PLA2) forms are expressed in spinal cord, and inhibiting spinal PLA2 induces a potent antihyperalgesia. Here, we examined the antihyperalgesic effects after systemic and i.t. delivery of four compounds constructed with a common motif consisting of a 2-oxoamide with a hydrocarbon tail and a four-carbon tether. These molecules were characterized for their ability to block group IVA calcium-dependent PLA2 (cPLA2) and group VIA calcium-independent PLA2 (iPLA2) in inhibition assays using human recombinant enzyme. The rank ordering of potency in blocking group IVA cPLA2 was AX048 (ethyl 4-[(2-oxohexadecanoyl)amino]butanoate), AX006 (4-[(2-oxohexadecanoyl)amino]butanoic acid), and AX057 (tert-butyl 4-[(2-oxohexadecanoyl)amino]butanoate) > AX010 (methyl 4-[(2-oxohexadecanoyl)amino]butanoate) and for inhibiting group VIA iPLA2 was AX048, AX057 > AX006, and AX010. No agent altered recombinant cyclooxygenase activity. In vivo, i.t. (30 μg) and systemic (0.2-3 mg/kg i.p.) AX048 blocked carrageenan hyperalgesia and after systemic delivery in a model of spinally mediated hyperalgesia induced by i.t. substance P (SP). The other agents were without activity. In rats prepared with lumbar i.t. loop dialysis catheters, SP evoked spinal prostaglandin E2 (PGE2) release. AX048 alone inhibited PGE2 release. Intrathecal SR141617, a cannabinoid CB1 inhibitor at doses that blocked the effects of i.t. anandamide had no effect upon i.t. AX048. These results suggest that AX048 is the first systemically bioavailable compound with a significant affinity for group IVA cPLA2, which produces a potent antihyperalgesia. The other agents, although demonstrating enzymatic activity in cell-free assays, appear unable to gain access to the intracellular PLA2 toward which their action is targeted.

Differing roles for members of the phospholipase A2 superfamily in experimental autoimmune encephalomyelitis

The phospholipase A(2) (PLA(2)) superfamily hydrolyzes phospholipids to release free fatty acids and lysophospholipids, some of which can mediate inflammation and demyelination, hallmarks of the CNS autoimmune disease multiple sclerosis. The expression of two of the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) and two of the secreted PLA(2)s (sPLA(2) GIIA and sPLA(2) GV) are increased in different stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We show using small molecule inhibitors, that cPLA(2) GIVA plays a role in the onset, and iPLA(2) GVIA in the onset and progression of EAE. We also show a potential role for sPLA(2) in the later remission phase. These studies demonstrate that selective inhibition of iPLA(2) can ameliorate disease progression when treatment is started before or after the onset of symptoms. The effects of these inhibitors on lesion burden, chemokine and cytokine expression as well as on the lipid profile provide insights into their potential modes of action. iPLA(2) is also expressed by macrophages and other immune cells in multiple sclerosis lesions. Our results therefore suggest that iPLA(2) might be an excellent target to block for the treatment of CNS autoimmune diseases, such as multiple sclerosis.

Intracellular phospholipase A2 group IVA and group VIA play important roles in Wallerian degeneration and axon regeneration after peripheral nerve injury

We provide evidence that two members of the intracellular phospholipase A(2) family, namely calcium-dependent group IVA (cPLA(2) GIVA) and calcium-independent group VIA (iPLA(2) GVIA) may play important roles in Wallerian degeneration in the mouse sciatic nerve. We assessed the roles of these PLA(2)s in cPLA(2) GIVA(-/-) mice, and mice treated with a selective inhibitor of iPLA(2) GVIA (FKGK11). Additionally, the effects of both these PLA(2)s were assessed by treating cPLA(2) GIVA(-/-) mice with the iPLA(2) inhibitor. Our data suggest that iPLA(2) GVIA may play more of a role in the early stages of myelin breakdown, while cPLA(2) GIVA may play a greater role in myelin clearance by macrophages. Our results also show that the delayed myelin clearance and Wallerian degeneration after sciatic nerve crush injury in mice lacking cPLA(2) and iPLA(2) activities is accompanied by a delay in axon regeneration, target re-innervation and functional recovery. These results indicate that the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) contribute significantly to various aspects of Wallerian degeneration in injured peripheral nerves, which is then essential for successful axon regeneration. This work has implications for injury responses and recovery after peripheral nerve injuries in humans, as well as for understanding the slow clearance of myelin after CNS injury and its potential consequences for axon regeneration.

An Integrated Model of Eicosanoid Metabolism and Signaling Based on Lipidomics Flux Analysis

There is increasing evidence for a major and critical involvement of lipids in signal transduction and cellular trafficking, and this has motivated large-scale studies on lipid pathways. The Lipid Metabolites and Pathways Strategy consortium is actively investigating lipid metabolism in mammalian cells and has made available time-course data on various lipids in response to treatment with KDO(2)-lipid A (a lipopolysaccharide analog) of macrophage RAW 264.7 cells. The lipids known as eicosanoids play an important role in inflammation. We have reconstructed an integrated network of eicosanoid metabolism and signaling based on the KEGG pathway database and the literature and have developed a kinetic model. A matrix-based approach was used to estimate the rate constants from experimental data and these were further refined using generalized constrained nonlinear optimization. The resulting model fits the experimental data well for all species, and simulated enzyme activities were similar to their literature values. The quantitative model for eicosanoid metabolism that we have developed can be used to design experimental studies utilizing genetic and pharmacological perturbations to probe fluxes in lipid pathways.

Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.

The GIVA phospholipase A2 (PLA2) contains two domains: a calcium-binding domain (C2) and a catalytic domain. These domains are linked via a flexible tether. GIVA PLA2 activity is Ca2+-dependent in that calcium binding promotes protein docking to the phospholipid membrane. In addition, the catalytic domain has a lid that covers the active site, presumably regulating GIVA PLA2 activity. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium (H/D) exchange coupled with liquid chromatographymass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the C2 domain, the catalytic domain, and the intact GIVA PLA2. We also analyzed the changes in H/D exchange of the intact GIVA PLA2 upon Ca2+ binding. The DXMS results showed a fast H/D-exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the “cleft” region between the C2 and catalytic domains. The slow exchanging region of the C2 domain is in tight proximity to the catalytic domain. The effects of Ca2+ binding on GIVA PLA2 are localized in the C2 domain and suggest that binding of two distinct Ca2+ ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step in GIVA PLA2 activation.

Lipidomics Analysis of Essential Fatty Acids in Macrophages

The Lipid Metabolites and Pathway Strategy (LIPID MAPS) Consortium is a nationwide initiative that has taken on the task of employing lipidomics to advance our understanding of lipid metabolism at the molecular and mechanistic level in living organisms. An important step toward this goal is to craft enabling analytical procedures to comprehensively measure all lipid species, to establish the precise structural identity of the lipid molecules analyzed, and to generate accurate quantitative information. The LIPID MAPS Consortium has succeeded in the implementation of a complete infrastructure that now provides tools for analysis of the global lipidome in cultured and primary cells. Here we illustrate the advancement of a gas chromatography mass spectrometry (GC/MS) procedure for the analysis of essential fatty acids in RAW 264.7 cells. Our method allows for the specific identification and quantification of over 30 fatty acids present in cells in their free form in a single analytical GC/MS run. Free fatty acids are selectively extracted in the presence of deuterated internal standards, which permit subsequent estimation of extraction efficiencies and quantification with high accuracy. Mass spectrometer conditions were optimized for single-ion monitoring, which provides an extremely sensitive technology to measure fatty acids from biological samples in trace amounts. These methods will be presented in the context of our broader effort to analyze all fatty acids as well as their metabolites in inflammatory cells.

Phospholipase A2 superfamily members play divergent roles after spinal cord injury

Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be prevenTABLE as it occurs, secondary to the trauma. We show that the phospholipase A2 (PLA2) superfamily plays important roles in SCI. PLA2 enzymes hydrolyze membrane glycerophospholipids to yield a free fatty acid and lysophospholipid. Some free fatty acids (arachidonic acid) give rise to eicosanoids that promote inflammation, while some lysophospholipids (lysophosphatidylcholine) cause demyelination. We show in a mouse model of SCI that two cytosolic forms [calcium‐dependent PLA2 group IVA (cPLA2 GIVA) and calcium‐independent PLA2 group VIA (iPLA2 GVIA)], and a secreted form [secreted PLA2 group IIA (sPLA2 GIIA)] are up‐regulated. Using selective inhibitors and null mice, we show that these PLA2s play differing roles. cPLA2 GIVA mediates protection, whereas sPLA2 GIIA and, to a lesser extent, iPLA2 GVIA are detrimental. Furthermore, completely blocking all three PLA2s worsens outcome, while the most beneficial effects are seen by partial inhibition of all three. The partial inhibitor enhances expression of cPLA2 and mediates its beneficial effects via the prostaglandin EP1 receptor. These findings indicate that drugs that inhibit detrimental forms of PLA2 (sPLA2 and iPLA2) and upregulate the protective form (cPLA2) may be useful for the treatment of SCI.—López‐Vales, R., Ghasemlou, N., Redensek, A., Kerr, B. J., Barbayianni, E., Antonopoulou, G., Baskakis, C., Rathore, K. I., Constantinou‐Kokotou, V., Stephens, D., Shimizu, T., Dennis, E. A., Kokotos, G., David, S. Phospholipase A2 superfamily members play divergent roles after spinal cord injury. FASEB J. 25, 4240–4252 (2011). www.fasebj.org

Structure-activity relationships of natural and non-natural amino acid-based amide and 2-oxoamide inhibitors of human phospholipase A2 enzymes

A variety of 2-oxoamides and related amides based on natural and non-natural amino acids were synthesized. Their activity on two human intracellular phospholipases (GIVA cPLA(2) and GVIA iPLA(2)) and one human secretory phospholipase (GV sPLA(2)) was evaluated. We show that an amide based on (R)-gamma-norleucine is a highly selective inhibitor of GV sPLA(2).

Synthesis of 2‐oxoamides based on sulfonamide analogs of γ‐amino acids and their activity on phospholipase A2

A variety of lipophilic 2‐oxoamides containing sulfonamide analogs of γ‐amino acids as well as acyl sulfonamides of γ‐aminobutyric acid were synthesized. Their ability to inhibit intracellular GIVA cPLA2 and GVIA iPLA2 as well as secreted GV sPLA2 was evaluated. The sulfonamide group seems a bioisosteric group suitable to replace the carboxyl group in 2‐oxoamide inhibitors of GVIA cPLA2. Copyright