Jesús Balsinde

Phospholipase A2 regulation of arachidonic acid mobilization

Phospholipase A2 (PLA2) constitutes a growing superfamily of lipolytic enzymes, and to date, at least 19 distinct enzymes have been found in mammals. This class of enzymes has attracted considerable interest as a pharmacological target in view of its role in lipid signaling and its involvement in a variety of inflammatory conditions. PLA2s hydrolyze the sn‐2 ester bond of cellular phospholipids, producing a free fatty acid and a lysophospholipid, both of which are lipid signaling molecules. The free fatty acid produced is frequently arachidonic acid (AA, 5,8,11,14‐eicosatetraenoic acid), the precursor of the eicosanoid family of potent inflammatory mediators that includes prostaglandins, thromboxanes, leukotrienes and lipoxins. Multiple PLA2 enzymes are active within and surrounding the cell and these enzymes have distinct, but interconnected roles in AA release.

Function and inhibition of intracellular calcium-independent phospholipase A2.

Our previous Minireview (1) considered the three main kinds of phospholipase A2 (PLA2) : the well characterized Groups I, II, and III small Ca-dependent secretory phospholipase A2s (sPLA2), the 85-kDa Group IV Ca -dependent cytosolic phospholipase A2 (cPLA2), and the 80-kDa Ca -independent cytosolic phospholipase A2 (iPLA2). In the ensuing years, it has become clear that PLA2 represents a growing superfamily of enzymes with many additional sPLA2s (Groups IIC, V, and IX), further definition of the 80-kDa iPLA2 (Group VI), and two Ca-independent PLA2s specific for platelet-activating factor (PAF) (Groups VII and VIII) (2). All of the well studied sPLA2s appear to use a His-Asp catalytic mechanism and require Ca to be bound tightly in the active site of the enzyme. The well characterized iPLA2 appears to require a central Ser for catalysis and of course, no Ca. Interestingly, the Group IV cPLA2 does not use Ca 21 for catalysis, but rather the Ca dependence seems to relate to a calcium lipid-binding domain (CaLB or C-2 domain) at the N-terminal end responsible for association of the enzyme with the membrane. Thus, the catalytic mechanism and active site Ser do not involve Ca (3–5); therefore a mechanistic distinction between the Group IV cPLA2 and the iPLA2s may not be warranted at this time. This is relevant because most of the inhibitors that work on the Group IV cPLA2 also act on the Group VI iPLA2 (6, 7). Inhibitor specificity will be discussed in the next section. We (8) recently surveyed all of the reported Ca-independent PLA2 activities. While there exists a group of lysosomal iPLA2s and a group of characterized ectoenzymes with broad specificity, which may actually be general lipases (8), sequenced and well characterized intracellular iPLA2s are limited to the 80-kDa Group VI iPLA2 and the 29-kDa Group VIII enzyme, which is a PAF acetyl hydrolase (9). The latter hydrolyzes the acetyl chain present at the sn-2 position of PAF and perhaps acts on oxidized phospholipids as well but not on normal phospholipids carrying unoxidized long chain fatty acids at the sn-2 position (9). This enzyme and a secreted Group VII PAF acetyl hydrolase, both of which are really iPLA2s with a particular substrate specificity, have been considered elsewhere (2). The Group VI 80-kDa iPLA2 was first identified in P388D1 macrophages (10), purified (11), further characterized (12), and then cloned and sequenced by Jones and co-workers (13) from CHO cells. The CHO iPLA2 has been shown to represent a species variant of that present in P388D1 macrophage-like cells, where the iPLA2 has also been cloned and sequenced (14). The sequence of the Group VI iPLA2 reveals the presence of eight ankyrin-like domains and the G-X-S-X-G motif commonly found in other lipases. Interestingly, no known consensus sequences for posttranslational modification, such as phosphorylation sites, are apparent in the Group VI iPLA2 (13, 14). This is compatible with the possibility that the Group VI iPLA2 acts to remodel membrane phospholipids as a sort of housekeeping enzyme as will be discussed later.

Novel Group V Phospholipase A2 Involved in Arachidonic Acid Mobilization in Murine P388D1 Macrophages

Four related genes encode four different secretory phospholipase A2 (sPLA2) enzymes in mammals, namely the well described Group I and IIA enzymes and the more recently described Groups IIC and V. A large body of research has putatively demonstrated that the Group IIA sPLA2 is involved in diverse pathologic processes, such as rheumatoid arthritis, septic shock, intestinal neoplasia, and epidermal hyperplasia, as well as in cellular signaling by regulating the formation of arachidonate-derived lipid messengers. However, we demonstrate herein the involvement of another sPLA2, i.e. the Group V sPLA2, in arachidonic acid release and prostaglandin production in the mouse macrophage-like cell line P388D1. Abundant message for Group V sPLA2 was detected in both resting and activated cells. In contrast, Group IIA sPLA2 message was undetectable as analyzed by Northern blot and reverse transcriptase-polymerase chain reaction. Moreover, blockage of Group V sPLA2 gene expression by antisense RNA oligonucleotides resulted in inhibition of prostaglandin E2 production as well as reduction of the amount of sPLA2 protein at the cellular surface. Collectively, these results uncover Group V sPLA2 as a novel effector involved in arachidonic acid-mediated signal transduction.

Irreversible inhibition of Ca2+-independent phospholipase A2 by methyl arachidonyl fluorophosphonate

Methyl arachidonyl fluorophosphonate (MAFP) has been recently reported to be a selective, active-site directed, irreversible inhibitor of the Group IV 85 kDa cytosolic phospholipase A2 (cPLA2). We have now shown that this compound also potently inhibits the Ca(2+)-independent cytosolic phospholipase A2 (iPLA2). MAFP inhibited iPLA2 in a concentration-dependent manner with half-maximal inhibition observed at 0.5 microM after a 5 min preincubation at 40 degrees C. This inhibition was not reversed upon extensive dilution of the enzyme into the assay mixture. Preincubation of iPLA2 with MAFP resulted in a linear, time-dependent inactivation of enzyme activity, and the enzyme was protected from inactivation by the reversible inhibitor PACOCF3. The ability of MAFP to inhibit the iPLA2 suggests that this enzyme proceeds through an acyl-enzyme intermediate as has been proposed for the cPLA2. Further testing indicated that MAFP did not inhibit the arachidonoyl-CoA synthetase, CoA-dependent acyltransferase, or CoA-independent transacylase activities from P388D1 cells. Thus, MAFP is not a general inhibitor for enzymes which act on arachidonoyl substrates. Instead, the inhibitor appears to show some selectivity for PLA2, although it does not discriminate between cPLA2 and iPLA2. Particular caution must be exercised to distinguish these activities if this inhibitor is used in intact cells.

Calcium-independent phospholipase A2 : structure and function

The classical Ca(2+)-independent phospholipase A(2) enzyme, now known as Group VIA PLA(2), was initially purified and characterized from the P388D(1) macrophage-like cell line. The corresponding cDNA was subsequently cloned from a variety of sources, and it is now known that multiple splice variants of the enzyme are expressed, some of which may act as negative regulators of the active enzyme. Group VIA PLA(2) has a consensus lipase motif (GTSTG) containing the catalytic serine, is 85-88 kDa, and exists in an aggregated form. The enzyme contains multiple ankyrin repeats, which may play a role in oligomerization. The Group VIA enzyme exhibits lysophospholipase activity as well as phospholipase A(2) activity, and it is capable of hydrolyzing a wide variety of phospholipid substrates. A major function of Group VIA PLA(2) is to mediate phospholipid remodeling, but the enzyme may play other roles as well. Other Ca(2+)-independent PLA(2) enzymes have more recently been identified, and it may be possible to discriminate between the various Ca(2+)-independent PLA(2) enzymes based on sequence or inhibitor-sensitivity. However, the physiological functions of the newly identified enzymes have yet to be elucidated.

Antisense Inhibition of Group VI Ca2+-independent Phospholipase A2 Blocks Phospholipid Fatty Acid Remodeling in Murine P388D1 Macrophages

A major issue in lipid signaling relates to the role of particular phospholipase A2 isoforms in mediating receptor-triggered responses. This has been difficult to study because of the lack of isoform-specific inhibitors. Based on the use of the Group VI Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL), we previously suggested a role for the iPLA2 in mediating phospholipid fatty acid turnover (Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92: 8527–8531). We have now further evaluated the role of the iPLA2 in phospholipid remodeling by using antisense RNA technology. We show herein that inhibition of iPLA2 expression by a specific antisense oligonucleotide decreases both the steady-state levels of lysophosphatidylcholine and the capacity of the cell to incorporate arachidonic acid into membrane phospholipids. These effects correlate with a decrease in both iPLA2 activity and protein in the antisense-treated cells. Collectively these data provide further evidence that the iPLA2 plays a major role in regulating phospholipid fatty acyl turnover in P388D1 macrophages. In stark contrast, experiments with activated cells confirmed that the iPLA2 does not play a significant role in receptor-coupled arachidonate mobilization in these cells, as manifested by the lack of an effect of the iPLA2antisense oligonucleotide on PAF-stimulated arachidonate release.

Regulation of Delayed Prostaglandin Production in Activated P388D1 Macrophages by Group IV Cytosolic and Group V Secretory Phospholipase A2s

Group V secretory phospholipase A2 (sPLA2) rather than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin E2 (PGE2) production in the macrophage-like cell line P388D1. When a new clone of these cells, P388D1/MAB, selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6–24 h) in response to lipopolysaccharide alone occurred in parallel with the induction of Group V sPLA2 and cyclooxygenase-2 (COX-2). No changes in the level of cytosolic phospholipase A2(cPLA2) or COX-1 were observed, and Group IIA sPLA2 was not detectable. Use of a potent and selective sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2revealed that delayed PGE2 was largely dependent on the induction of Group V sPLA2. Also, COX-2, not COX-1, was found to mediate delayed PGE2 production because the response was completely blocked by the specific COX-2 inhibitor NS-398. Delayed PGE2 production and Group V sPLA2expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca2+-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response as well. Collectively, the results suggest a model whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2 production via COX-2.

Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells.

A novel Ca2+-independent phospholipase A2 (iPLA2) has recently been purified and characterized from P388D1 macrophages (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme appears to play a key role in regulating basal phospholipid remodeling reactions. Also an iPLA2 from Chinese hamster ovary (CHO) cells has been purified, molecularly cloned, and expressed (Tang, J., Kriz, R., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. S. (1997) J. Biol. Chem. 272, 8567-8575). We report herein that the cloned CHO iPLA2 is equivalent to the mouse enzyme purified from P388D1 cells. Polymerase chain reaction amplification of cDNA fragments from P388D1 cells using primers based on the CHO iPLA2 sequence, revealed a high degree of homology between the mouse and hamster enzymes at both the nucleotide and amino acid levels (92 and 95%, respectively). Identity between the two proteins was further demonstrated by using immunochemical, pharmacological, and biochemical approaches. Thus, an antiserum generated against the CHO enzyme recognized the P388D1 cell enzyme and gave similar molecular masses (about 83 kDa) for the two enzymes under the same experimental conditions. Further, the CHO enzyme has exactly the same sensitivity to inhibition by a variety of compounds previously shown to inhibit the P388D1 enzyme, including bromoenol lactone, palmitoyl trifluoromethyl ketone, and methyl arachidonyl fluorophosphonate. Additionally, covalent modification of the CHO enzyme by [3H]bromoenol lactone is dependent on active enzyme as is the P388D1 iPLA2. Finally, both enzymes have the same specific activities under identical experimental conditions.

Control of free arachidonic acid levels by phospholipases A2 and lysophospholipid acyltransferases

Arachidonic acid (AA) and its oxygenated derivatives, collectively known as the eicosanoids, are key mediators of a wide variety of physiological and pathophysiological states. AA, obtained from the diet or synthesized from linoleic acid, is rapidly incorporated into cellular phospholipids by the concerted action of arachidonoyl-CoA synthetase and lysophospholipid acyltransferases. Under the appropriate conditions, AA is liberated from its phospholipid storage sites by the action of one or various phospholipase A(2) enzymes. Thus, cellular availability of AA, and hence the amount of eicosanoids produced, depends on an exquisite balance between phospholipid reacylation and hydrolysis reactions. This review focuses on the enzyme families that are involved in these reactions in resting and stimulated cells.

Involvement of calcium-independent phospholipase A2 in hydrogen peroxide-induced accumulation of free fatty acids in human U937 cells.

Previous studies have demonstrated that U937 cells are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in response to receptor-directed and soluble stimuli by a mechanism that involves the activation of Group IV cytosolic phospholipase A2α. In this paper we show that these cells also mobilize AA in response to an oxidative stress induced by H2O2 through a mechanism that appears not to be mediated by cytosolic phospholipase A2α but by the calcium-independent Group VI phospholipase A2(iPLA2). This is supported by the following lines of evidence: (i) the response is essentially calcium-independent, (ii) it is inhibited by bromoenol lactone, and (iii) it is inhibited by an iPLA2 antisense oligonucleotide. Enzyme assays conducted under a variety of conditions reveal that the specific activity of the iPLA2 does not change as a result of H2O2 exposure, which argues against the activation of a specific signaling cascade ending in the iPLA2. Rather, the oxidant acts to perturb membrane homeostasis in a way that the enzyme susceptibility/accessibility to its substrate increases, and this results in altered fatty acid release. In support of this view, not only AA, but also other fatty acids, were found to be liberated in an iPLA2-dependent manner in the H2O2-treated cells. Collectively, these studies underscore the importance of the iPLA2 in modulating homeostatic fatty acid deacylation reactions and document a potentially important route under pathophysiological conditions for increasing free fatty acid levels during oxidative stress.