Christina C. Leslie

Properties and Regulation of Cytosolic Phospholipase A2

Membrane phospholipid is an important reservoir for the generation of bioactive mediators. Cellular phospholipases hydrolyze membrane phospholipid in a structurally specific manner releasing numerous lipid products that are responsible for transmitting diverse signals necessary for the induction of functional responses. Since this is a highly regulated process, the phospholipases are subject to complex mechanisms of activation and presumably deactivation. The phospholipase A2 (PLA2) 1 enzymes hydrolyze fatty acid from the sn-2 position of phospholipid with the concomitant production of lysophospholipid. Mammalian cells contain structurally diverse forms of PLA2 including secretory PLA2 (sPLA2), calcium-independent PLA2, and the 85-kDa cytosolic PLA2 (cPLA2) (1–4). PLA2 enzymes function in the digestion of dietary lipid, microbial degradation, and regulation of phospholipid acyl turnover either in a housekeeping role for membrane repair or for the production of inflammatory lipid mediators. The presence of diverse PLA2 enzymes in mammalian cells provides multiple, differentially regulated pathways for the important process of fatty acid turnover. This review will focus on the biochemical properties and regulation of cPLA2, which plays an important role in mediating arachidonic acid release. cPLA2 shares no homology with other PLA2 enzymes and is the only well characterized PLA2 that preferentially hydrolyzes sn-2 arachidonic acid (1, 5, 6). It should be noted, however, that although sPLA2 and calcium-independent PLA2 do not exhibit acyl chain specificity they can also mediate arachidonic acid release depending on the cell type and agonist involved (3, 4). Arachidonic acid is itself an important regulator of specific cellular processes including regulation of PKC and phospholipase Cg and modulation of Ca transients (7–11). Arachidonic acid can also be converted to potent inflammatory lipid mediators, the eicosanoids. This can occur enzymatically through the lipoxygenase or cyclooxygenase (COX) pathways for the production of leukotrienes, lipoxins, thromboxanes, or prostaglandins (12, 13). Arachidonic acid is also subject to non-enzymatic, free radical oxidation to bioactive isoprostanes and isoleukotrienes (14, 15). The important role of arachidonic acid in cellular activation ensures that its levels are tightly controlled. cPLA2 plays a role in maintaining arachidonate levels and is subject to complex mechanisms of regulation at both the transcriptional and post-translational levels. The involvement of cPLA2 in lipid mediator production makes it a potentially important pharmacological target for anti-inflammatory drugs. Distribution of cPLA2 and Regulation of Its Synthesis cPLA2 is a widely distributed enzyme, and the transcript is expressed at a fairly constant level in all human tissues with somewhat elevated levels in lung and hippocampus (16, 17). At the cellular level, cPLA2 is enriched in mononuclear phagocytes but has been found in most cells examined. One notable exception is mature T and B lymphocytes that do not contain cPLA2, whereas it is present in thymocytes and immature B cells (18). This suggests that expression of cPLA2 may be incompatible with the function of mature lymphocytes. The ubiquitous expression of cPLA2 is consistent with features of the 59-flanking region of the human gene, which has certain characteristics of a housekeeping promoter in that it has no TATA box (16, 19, 20). However, unlike many housekeeping promoters, it does not contain a GC-rich region or SP1 sites. The 59-flanking region of the cPLA2 gene contains a 27-base pair region with a polypyrimidine sequence that is responsible for the basal expression of cPLA2 (16). In addition, a 48-base purine/ pyrimidine repeat (CA repeat) appears to confer an inhibitory effect on cPLA2 gene transcription (20). By genetic analysis, the human cPLA2 gene has been localized to chromosome 1q31-41 between markers F13B and D1S74 (16). However, by structural analysis it has been mapped to chromosome 1q25 (21). Of interest, the COX 2 gene has also been mapped to the 1q25 region (22). A variety of cytokines and mitogens such as interleukin-1, tumor necrosis factor (TNF), colony-stimulating factor (CSF), epidermal growth factor, c-Kit ligand, and interferon g (IFNg) have been shown to induce activation and increase synthesis of cPLA2 in diverse cell models (1). In WI-38 cells, the increase in cPLA2 synthesis induced by interleukin-1 correlates with PGE2 production, and both effects are suppressed by glucocorticoids (23). In some models, there is coordinate up-regulation of both cPLA2 and COX 2 (1). The induction of cPLA2 gene expression by IFNg in an epithelial cell line is proposed to occur at the transcriptional level (24). Consistent with this, the promoter for human cPLA2 contains a putative IFNg-activated sequence and IFNg response elements (20). In addition, DNA sequence analysis has revealed two glucocorticoid response elements suggesting that steroids may act to suppress cPLA2 synthesis at the transcriptional level. There is also evidence for post-transcriptional regulation of cPLA2 synthesis. In rat mesangial cells, phorbol myristate acetate (PMA), plateletderived growth factor, and serum and epidermal growth factor increase the half-life of cPLA2 mRNA (25). An adenosine-uridinerich sequence in the 39-untranslated region of cPLA2 is thought to be responsible for the instability of the cPLA2 transcript. Stimulation of cPLA2 synthesis occurs over hours and results in the prolonged release of arachidonic acid and eicosanoid production.

Regulation of arachidonic acid release and cytosolic Phospholipase A2 activation

The 85‐kDa cytosolic PLA2 (CPLA2) mediates agonist‐induced arachidonic acid release in many cell models, including mouse peritoneal macrophages. cPLA2 is regulated by an increase in intracellular calcium, which binds to an amino‐ terminal C2 domain and induces its translocation to the nuclear envelope and endoplasmic reticulum. Phosphorylation of cPLA2 on S505 by mitogen‐activated protein kinases (MAPK) also contributes to activation. In macrophages, zymosan induces a transient increase in intracellular calcium and activation of MAPK, which together fully activate cPLA2 and synergistically promote arachidonic acid release. There are alternative pathways for regulating cPLA2 in macrophages because PMA and okadaic acid induce arachidonic acid release without increasing calcium. The baculovirus expression system is a useful model to study cPLA2 activation. Sf9 cells expressing cPLA2 release arachidonic acid to either A23187 or okadaic acid. cPLA2 is phosphorylated on multiple sites in Sf9 cells, and phosphorylation of S727 is preferentially induced by okadaic acid. However, the phosphorylation sites are non‐essential and only S505 phosphorylation partially contributes to cPLA2 activation in this model. Although okadaic acid does not increase intracellular calcium in Sf9 cells, calcium binding by the C2 domain is necessary for arachidonic acid release. A23187 and okadaic acid activate cPLA2 by different mechanisms, yet both induce translocation to the nuclear envelope in Sf9 cells. The results demonstrate that alternative regulatory pathways can lead to cPLA2 activation and arachidonic acid release. J. Leukoc. Biol. 65: 330–336; 1999.

Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7.

The lipid mediators, platelet activating factor (PAF) and the eicosanoids, can be coordinately produced from the common phospholipid precursor, 1-O-alkyl-2-arachidonoylglycerophosphocholine (1-O-alkyl-2-arachidonoyl-GPC), through the initial action of a phospholipase A2 that cleaves arachidonic acid from the sn-2 position. The mouse macrophage cell line RAW 264.7, which was used as a model macrophage system to study the arachidonoyl-hydrolyzing phospholipase A2 enzyme(s), could be induced to release arachidonic acid in response to inflammatory stimuli. A phospholipase A2 that hydrolyzed 1-O-hexadecyl-2-[3H]arachidonoyl-GPC was identified in the cytosolic fraction of these macrophages. This phospholipase activity was optimal at pH 8 and dependent on calcium. Enzyme activity could be stimulated 3-fold by heparin, suggesting the presence of phospholipase inhibitory proteins in the macrophage cytosol. Compared to 1-alkyl-2-arachidonoyl-GPC, the enzyme hydrolyzed 1-acyl-2-arachidonoylglycerophosphoethanolamine (1-acyl-2-arachidonoyl-GPE) with similar activity but showed slightly greater activity against 1-acyl-2-arachidonoyl-GPC, suggesting no specificity for the sn-1 linkage or the phospholipid base group. Although comparable activity against 1-acyl-2-arachidonoylglycerophosphoinositol (1-acyl-2-arachidonoyl-GPI) could be achieved, the enzyme exhibited much lower affinity for the inositol-containing substrate. The enzyme did, however, show apparent specificity for arachidonic acid at the sn-2 position, since much lower activity was observed against choline-containing substrates with either linoleic or oleic acids at the sn-2 position. The cytosolic phospholipase A2 was purified by first precipitating the enzyme with ammonium sulfate followed by chromatography over Sephadex G150, where the phospholipase A2 eluted between molecular weight markers of 67,000 and 150,000. The active peak was then chromatographed over DEAE-cellulose, phenyl-Sepharose, Q-Sepharose, Sephadex G150 and finally hydroxylapatite. The purification scheme has resulted in over a 1000-fold increase in specific activity (2 mumol/min per mg protein). Under non-reducing conditions, a major band on SDS-polyacrylamide gels at 70 kDa was observed, which shifted to a lower molecular weight, 60,000, under reducing conditions. The properties of the purified enzyme including the specificity for sn-2-arachidonoyl-containing phospholipids was similar to that observed for the crude enzyme. The results demonstrate the presence of a phospholipase A2 in the macrophage cell line. RAW 264.7, that preferentially hydrolyzes arachidonoyl-containing phospholipid substrates.

A cytosolic phospholipase in human neutrophils that hydrolyzes arachidonoyl-containing phosphatidylcholine

In stimulated neutrophils the production of eicosinoids and the lipid mediator, platelet-activating factor, is thought to be initiated by the activation of a phospholipase A2 which cleaves arachidonic acid from choline-containing glycerophospholipids. Accordingly, studies were undertaken in human neutrophils to characterize phospholipase enzymes that can hydrolyze 1-acyl- and 1-alkyl-linked arachidonoyl-containing phosphatidylcholine (PC). Cellular homogenates were incubated with sonicated dispersions of the arachidonoyl-labeled phospholipid substrates and the hydrolysis of radiolabeled arachidonate was measured. The phospholipase activity was cytosolic, optimal at pH 8.0, and calcium dependent. The homogenization conditions used were important in determining the amount of recoverable enzymatic activity. Vigorous sonication and the presence of calcium during homogenization were strongly inhibitory, whereas the presence of EGTA, heparin and proteinase inhibitors during homogenization increased the activity. Competitive experiments with unlabeled substrates suggested that the phospholipase hydrolyzed arachidonic acid equally well from either 1-acyl- or 1-alkyl-linked PC. However, the phospholipase did show specificity for arachidonic acid, compared to oleic or linoleic acids, at the sn-2 position of 1-acyl-linked PC. When neutrophils were first stimulated with the ionophore A23187, the phospholipase activity against 1-O-hexadecyl-2-[3H]arachidonoylglycerophosphocholine (GPC) increased in a time-dependent fashion up to 3.5-fold over the unstimulated level. The activity against 1-palmitoyl-2-[3H]arachidonoyl-GPC also increased after ionophore stimulation but to a lesser extent. The results demonstrate the presence of a cytosolic, activatable phospholipase that may be involved in PC turnover, arachidonic acid release, and platelet-activating factor production in human neutrophils.

Regulation of Cytosolic Phospholipase A2 Activation and Cyclooxygenase 2 Expression in Macrophages by the β-Glucan Receptor

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A2 is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the β-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-β-d-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C4 production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2–/– macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-β-d-glucan. The results suggest that cytosolic phospholipase A2 activation triggered by the β-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.

Group V Secretory Phospholipase A2 Translocates to the Phagosome after Zymosan Stimulation of Mouse Peritoneal Macrophages and Regulates Phagocytosis

We have previously reported that group V secretory phospholipase A2 (sPLA2) amplifies the action of cytosolic phospholipase A2(cPLA2) α in regulating eicosanoid biosynthesis by mouse peritoneal macrophages stimulated with zymosan (Satake, Y., Diaz, B. L., Balestrieri, B., Lam, B. K., Kanaoka, Y., Grusby, M. J., and Arm, J. P. (2004) J. Biol. Chem. 279, 16488-16494). To further understand the role of group V sPLA2, we studied its localization in resting mouse peritoneal macrophages before and after stimulation with zymosan and the effect of deletion of the gene encoding group V sPLA2 on phagocytosis of zymosan. We report that group V sPLA2 is present in the Golgi apparatus and recycling endosome in the juxtanuclear region of resting peritoneal macrophages. Upon ingestion of zymosan by mouse peritoneal macrophages, group V sPLA2 is recruited to the phagosome. There it co-localizes with cPLA 2 α, 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C4 synthase. Using immunostaining for the cysteinyl leukotrienes in carbodiimide-fixed cells, we show, for the first time, that the phagosome is a site of cysteinyl leukotriene formation. Furthermore, peritoneal macrophages from group V sPLA2-null mice demonstrated a >50% attenuation in phagocytosis of zymosan particles, which was restored by adenoviral expression of group V sPLA2 but IIA not group sPLA2. These data demonstrate that group V sPLA2 contributes to the innate immune response both through regulation of eicosanoid generation in response to a phagocytic stimulus and also as a component of the phagocytic machinery.

ANIONIC PHOSPHOLIPIDS STIMULATE AN ARACHIDONOYL-HYDROLYZING PHOSPHOLIPASE A2 FROM MACROPHAGES AND REDUCE THE CALCIUM REQUIREMENT FOR ACTIVITY

Mechanisms involved in regulating the activity of intracellular phospholipase A2 enzymes that function in eicosanoid and platelet-activating factor production are poorly understood. The properties of the substrate in the membrane may play a role in modulating phospholipase A2 activity. In this study, the effect of anionic phospholipids, diacylglycerol (DAG) and phosphatidylethanolamine (PE) on the activity of a partially purified, intracellular, arachidonoyl-hydrolyzing phospholipase A2 from the macrophage cell line, RAW 264.7 was studied. For these experiments phospholipase A2 activity was assayed in the presence of 1 microM calcium by measuring the hydrolysis of [3H]arachidonic acid from sonicated dispersions of the ether-linked substrate, 1-O-hexadecyl-2[3H]arachidonoylglycerophosphocholine. All the anionic phospholipids tested, including phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2), stimulated phospholipase A2 activity. At the lowest concentration of anionic phospholipids tested. PIP2 was the most stimulatory, resulting in a 7-fold increase in phospholipase A2 activity at 1 mol%. Co-dispersion of either DAG or PE with the substrate also induced a dose-dependent increase in phospholipase A2 activity, whereas sphingomyelin was inhibitory suggesting that the phospholipase A2 more readily hydrolyzed the ether-linked substrate when there was a decrease in the packing density of the bilayer. PIP2, together with either DAG or PE, synergistically stimulated phospholipase A2 activity by about 20-fold, and dramatically decreased the calcium concentration (from mM to nM) required for full activity of the enzyme. The results of this study demonstrate that the presence of anionic phospholipids and the packing characteristics of the bilayer can have pronounced effects on the activity and calcium requirement of an intracellular, arachidonoyl-hydrolyzing phospholipase A2 from macrophages.

A pyrrolidine-based specific inhibitor of cytosolic phospholipase A2α blocks arachidonic acid release in a variety of mammalian cells

We analyzed a recently reported (K. Seno, T. Okuno, K. Nishi, Y. Murakami, F. Watanabe, T. Matsuur, M. Wada, Y. Fujii, M. Yamada, T. Ogawa, T. Okada, H. Hashizume, M. Kii, S.-H. Hara, S. Hagishita, S. Nakamoto, J. Med. Chem. 43 (2000)) pyrrolidine-based inhibitor, pyrrolidine-1, against the human group IV cytosolic phospholipase A(2) alpha-isoform (cPLA(2)alpha). Pyrrolidine-1 inhibits cPLA(2)alpha by 50% when present at approx. 0.002 mole fraction in the interface in a number of in vitro assays. It is much less potent on the cPLA(2)gamma isoform, calcium-independent group VI PLA(2) and groups IIA, X, and V secreted PLA(2)s. Pyrrolidine-1 blocked all of the arachidonic acid released in Ca(2+) ionophore-stimulated CHO cells stably transfected with cPLA(2)alpha, in zymosan- and okadaic acid-stimulated mouse peritoneal macrophages, and in ATP- and Ca(2+) ionophore-stimulated MDCK cells.

Enzymatic Properties of Human Cytosolic Phospholipase A2γ

The enzymatic properties of cytosolic phospholipase A2γ (cPLA2γ), an isoform of 85-kDa group IV cPLA2α (cPLA2α) were studied in vitro and when the enzyme was expressed in cells. cPLA2γ expressed in Sf9 cells is associated with membrane. Membranes isolated from [3H]arachidonic acid-labeled Sf9 cells expressing cPLA2γ, constitutively release [3H]arachidonic acid. The membrane-associated activity is inhibited by the group IV PLA2 inhibitor methylarachidonyl fluorophosphonate, but not effectively by the group VI PLA2 inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cPLA2γ has higher lysophospholipase activity than PLA2 activity. Purified His-cPLA2γ does not exhibit phospholipase A1 activity, but sequentially hydrolyzes fatty acid from the sn-2 and sn-1 positions of phosphatidylcholine. cPLA2γ overexpressed in HEK293 cells is constitutively active in isolated membranes, releasing large amounts of oleic, arachidonic, palmitic, and stearic acids; however, basal fatty acid release from intact cells is not increased. cPLA2γ overexpressed in lung fibroblasts from cPLA2α-deficient mice is activated by mouse serum resulting in release of arachidonic, oleic, and palmitic acids, whereas overexpression of cPLA2α results primarily in arachidonic acid release.

Group IV Phospholipase A2α Controls the Formation of Inter-Cisternal Continuities Involved in Intra-Golgi Transport

The enzyme phospholipase A2 (cPLA2α) is involved in the formation of intercisternal tubules that mediate transport of proteins within the Golgi complex.