The Highly Selective Production of 2-Arachidonoyl Lysophosphatidylcholine Catalyzed by Purified Calcium-independent Phospholipase A2γ (original) (raw)
Related papers
2004
Phospholipase A2 regulation of arachidonic acid levels Availability of free arachidonic acid (AA) is widely recognized as a rate-limiting step in the formation of prostaglandins. This fatty acid is an intermediate of a reacylationldeacylation cycle of membrane phospholipids, the so-called Lands pathway, in which the fatty acid is cleaved from phospholipid by phospholipase Azs (PLA z) and reincorporated by acyltransferases. Whereas in resting cells reacylation dominates, in stimulated cells the dominant reaction is the PLArmediated deacylation. Nevertheless, increased AA reacylation during cellular activation is still very significant, as manifested by the fact that only a minor portion of the free AA released by PLA z is converted into eicosanoids, the remainder being effectively incorporated back into phospholipids. Phagocytic cells generally contain multiple PLA 2 s [1, 2]. Thus the challenge in recent years has been both to identify these PLAzs and to clarify their roles in AA metabolism. A general mechanism for PLAz-regulated AA metabolism in resting and activated cells has emerged from the studies by several laboratories [3, 4], and involves participation of all three major classes of PLA z , namely cPLA z (cytosolic PLA z), iPLA z (Ca 2 + independent PLA z) and sPLA z (secreted PLA z) (Fig. 1). In resting conditions, iPLA 2 accounts for most of the PLA z activity of cells. iPLA z is therefore the dominant PLA z involved in the liberation of fatty acids, including AA, during the continuous recycling of membrane phospholipids that takes place under these conditions. Since, as indicated above, the rate of AA release by iPLA z is lesser than the rate of its reacylation back into phospholipids, no net accumulation of free fatty acid occurs. Stimulation of the cells by receptor agonists results in the activation of cPLA z , which then becomes the dominant PLA 2 involved in AA release. Under these conditions, the rate of AA release clearly exceeds that of reincorporation into phospholipids; hence net accumulation of AA occurs that is followed by its conversion into different oxygenated compounds, collectively called the eicosanoids.
Role of Calcium-Independent Phospholipases (iPLA 2) in Phosphatidylcholine Metabolism
Biochemical and Biophysical Research Communications, 2001
The proposed role of calcium-independent phospholipase A2 (iPLA2) in membrane phospholipid homeostasis was tested by examining the perturbation of phosphatidylcholine metabolism by enzyme overexpression. There are alternatively spliced forms of murine iPLA2 that were widely expressed in mouse tissues: a long form containing exon-9 that is membrane-associated and a short form lacking exon-9 that is distributed between the membrane and cytosolic fractions. Enforced expression of either iPLA2 isoform led to a significant increase in intracellular free fatty acid, lysophosphatidylcholine, and GPC without a concomitant increase in the incorporation of either exogenous arachidonic acid or choline. The accumulation of lysophosphatidylcholine in iPLA2-expressing cells illustrates the limited capacity of cells for reacylation and degradation of lysophospholipids. Since iPLA2 overexpression did not accelerate either phospholipid remodeling or phosphatidylcholine synthesis, this enzyme does play a determinant (rate-controlling?) role in either of these cellular processes.
2004
Group VIA calcium-independent phospholipase A 2 (iPLA 2 ) has been shown to play a major role in regulating basal phospholipid deacylation reactions in certain cell types. More recently, roles for this enzyme have also been suggested in the destruction of membrane phospholipid during apoptosis and after oxidant injury. Proposed iPLA 2 roles have rested heavily on the use of bromoenol lactone as an iPLA 2 -specific inhibitor, but this compound actually inhibits other enzymes and lipid pathways unrelated to PLA 2 , which makes it difficult to define the contribution of iPLA 2 to specific functions. In previous work, we pioneered the use of antisense technology to decrease cellular iPLA 2 activity as an alternative approach to study iPLA 2 functions. In the present study, we followed the opposite strategy and prepared U937 cells that exhibited enhanced iPLA 2 activity by stably expressing a plasmid containing iPLA 2 cDNA. Compared with control cells, the iPLA 2 -overexpressing U937 cells showed elevated responses to hydrogen peroxide with regard to both arachidonic acid mobilization and incorporation of the fatty acid into phospholipids, thus providing additional evidence for the key role that iPLA 2 plays in these events. Long-term exposure of the cells to hydrogen peroxide resulted in cell death by apoptosis, and this process was accelerated in the iPLA 2overexpressing cells. Increased phospholipid hydrolysis and fatty acid release also occurred in these cells. Unexpectedly, however, abrogation of U937 cell iPLA 2 activity by either methyl arachidonyl fluorophosphonate or an antisense oligonucleotide did not delay or decrease the extent of apoptosis induced by hydrogen peroxide. These results indicate that, although iPLA 2
Journal of Biological Chemistry, 1998
We examined the relative contributions of five distinct mammalian phospholipase A 2 (PLA 2) enzymes (cytosolic PLA 2 (cPLA 2 ; type IV), secretory PLA 2 s (sPLA 2 s; types IIA, V, and IIC), and Ca 2؉-independent PLA 2 (iPLA 2 ; type VI)) to arachidonic acid (AA) metabolism by overexpressing them in human embryonic kidney 293 fibroblasts and Chinese hamster ovary cells. Analyses using these transfectants revealed that cPLA 2 was a prerequisite for both the calcium ionophore-stimulated immediate and the interleukin (IL)-1-and serum-induced delayed phases of AA release. Type IIA sPLA 2 (sPLA 2-IIA) mediated delayed AA release and, when expressed in larger amounts, also participated in immediate AA release. sPLA 2-V, but not sPLA 2-IIC, behaved in a manner similar to sPLA 2-IIA. Both sPLA 2 s-IIA and-V, but not sPLA 2-IIC, were heparin-binding PLA 2 s that exhibited significant affinity for cell-surface proteoglycans, and site-directed mutations in residues responsible for their membrane association or catalytic activity markedly reduced their ability to release AA from activated cells. Pharmacological studies using selective inhibitors as well as co-expression experiments supported the proposal that cPLA 2 is crucial for these sPLA 2 s to act properly. The AA-releasing effects of these sPLA 2 s were independent of the expression of the M-type sPLA 2 receptor. Both cPLA 2 , sPLA 2 s-IIA, and-V were able to supply AA to downstream cyclooxygenase-2 for IL-1-induced prostaglandin E 2 biosynthesis. iPLA 2 increased the spontaneous release of fatty acids, and this was further augmented by serum but not by IL-1. Finally, iPLA 2derived AA was not metabolized to prostaglandin E 2. These observations provide evidence for the functional cross-talk or segregation of distinct PLA 2 s in mammalian cells in regulating AA metabolism and phospholipid turnover.
Archives of Biochemistry and Biophysics, 1997
and involves a peroxide-induced stimulation of cPLA 2 phosphorylation. ᭧ 1997 Academic Press Cytosolic phospholipase A 2 (cPLA 2 ) is a signal-re-Key Words: cytosolic phospholipase A 2 ; protein kisponsive enzyme that is highly selective to the nature nase C; calcium; oxidative stress; enzyme phosphorylaof phospholipid substrates. A mechanism for cPLA 2 tion; phospholipid hydroperoxide. activity regulation through a signal transduction pathway has been proposed and this signaling appears to be influenced by oxidants. Oxidant-mediated signaling of PLA 2 may serve as an alternative mecha-Phospholipase A 2 (PLA 2 ) 2 plays an important role in nism for enzyme regulation; however, the manner of the production of proinflammatory lipid mediators as regulation has yet to be delineated. In this report we well as participating in cell signaling events (1). Mamdemonstrate that there is a direct effect of membrane malian PLA 2 is classified into extra-and intracellular oxidation on cPLA 2 phosphorylation and activity. A forms that include a variety of secreted PLA 2 (sPLA 2 ) simple in vitro system consisting of purified cPLA 2 and intracellular enzymes of which cytosolic PLA 2 and phospholipid vesicles was used to facilitate pro-(cPLA 2 ) has received considerable recent attention (2).
Prostaglandins & other lipid mediators, 1999
Ca 2ϩ-independent phospholipase A 2 (iPLA 2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA 2 in AA remodeling in different cells was evaluated by studying the Ca 2ϩ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA 2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca 2ϩ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca 2ϩ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca 2ϩ. Both events were fully dependent on extra and intracellular Ca 2ϩ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca 2ϩ at short times (Ͻ30 min). The involvement of iPLA 2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced Յ50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA 2 plays a major role in regulating AA turnover in different cell types.
The Journal of Lipid Research, 2012
Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LD are formed by two different routes, namely (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60-70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A 2 α (cPLA 2 α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA 2 α activation by phosphorylation. Collectively, these results suggest that concomitant activation of both p38 and JNK by AA cooperate to activate cPLA 2 α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis. ABBREVIATIONS-LD, lipid droplets; AA, arachidonic acid; CE, cholesteryl esters; cPLA 2 α, group IVA cytosolic phospholipase A 2 α; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PLA 2 , phospholipase A 2 ; TAG, triacylglycerol. 2 by guest, on September 4, 2012 www.jlr.org Downloaded from Reagents-Cell culture medium and BODIPY® 493/503 were obtained from Molecular Probes-Invitrogen (Carlsbad, CA). Chloroform and methanol (HPLC grade) were from Fisher Scientific (Hampton, NH). [5,6,8,9,11,12,14,15-3 H]AA (sp. act. 211 Ci/mmol) was purchased from GE Healthcare (Buckinghamshire, UK). [1,2-14 C]acetic acid (sp. act. 54.3 mCi/mmol) was from Perkin Elmer (Waltham, MA). Silicagel thin layer chromatography plates were from Macherey-Nagel (Düren, Germany). The p38 MAP kinase inhibitor SB 203580 was from Calbiochem/Merck KGaA (Darmstadt, Germany). Triacsin C was purchased from Enzo Life Sciences (Farmingdale, NY). Paraformaldehyde was from Electron Microscopy Sciences (Hartfield, PA). Antibodies against p-cPLA 2 (Ser505), p-p38(Thr180/Tyr182) and p-JNK(Thr183/Tyr185) were purchased from Cell Signaling (Danvers, MA). The cPLA 2 α inhibitor
Journal of Biological Chemistry, 2003
Here we report cellular arachidonate (AA) release and prostaglandin (PG) production by novel classes of secretory phospholipase A 2 s (sPLA 2 s), groups III and XII. Human group III sPLA 2 promoted spontaneous AA release, which was augmented by interleukin-1, in HEK293 transfectants. The central sPLA 2 domain alone was sufficient for its in vitro enzymatic activity and for cellular AA release at the plasma membrane, whereas either the unique Nor C-terminal domain was required for heparanoid-dependent action on cells to augment AA release, cyclooxygenase-2 induction, and PG production. Group III sPLA 2 was constitutively expressed in two human cell lines, in which other sPLA 2 s exhibited different stimulus inducibility. Human group XII sPLA 2 had a weak enzymatic activity in vitro and minimally affects cellular AA release and PG production. Cells transfected with group XII sPLA 2 exhibited abnormal morphology, suggesting a unique functional aspect of this enzyme. Based on the present results as well as our current analyses on the group I/II/V/X sPLA 2 s, general properties of cellular actions of a full set of mammalian sPLA 2 s in regulating AA metabolism are discussed.