Cytosolic phospholipase A₂: physiological function and role in disease.

thematic review series Thematic Review Series: Phospholipases: Central Role in Lipid Signaling and Disease

Cytosolic phospholipase A2: physiological function and role in disease Christina C. Leslie1 Department of Pediatrics, National Jewish Health, Denver, CO 80206; and Departments of Pathology and Pharmacology, University of Colorado Denver, Aurora, CO 80045

Supplementary key words arachidonic acid • calcium • Golgi apparatus • inflammation • cyclooxygenase • leukotrienes • lipoxygenase • prostaglandins • protein kinases/MAP kinase

OVERVIEW OF THE MAMMALIAN PHOSPHOLIPASE A2 FAMILY The mammalian phospholipase A2 (PLA2) family is comprised of six types of diverse enzymes: GIV PLA2 [cytosolic PLA2 (cPLA2)], GVI PLA2 [calcium-independent

This work was supported by National Institutes of Health Grant HL34303. Manuscript received 11 January 2015 and in revised form 26 March 2015. Published, JLR Papers in Press, April 2, 2015 DOI 10.1194/jlr.R057588

PLA2 (iPLA2)], GVII and GVIII PLA2 [platelet-activating factor-acetylhydrolases (PAF-AHs)], GXV PLA2 (lysosomal PLA2), GXVI PLA2 (adipose PLA2), and several groups of secreted PLA2 (sPLA2). Several reviews have described in detail the structure, activities, and known functions of the different groups of PLA2 enzymes, many of which contain several subtypes (1–8). Most of these enzymes have no structural similarity, and have different catalytic mechanisms and regulation. cPLA2s are most similar to iPLA2s (also classified as patatin-like enzymes), because they utilize an active site Ser-Asp dyad for catalysis, unlike the catalytic mechanism of other PLA2s that involves a His residue (2–4). It has been shown that the active site of the plant lipid acylhydrolase patatin contains a Ser-Asp dyad with structural similarity to cPLA2 and has homology with the iPLA2 family (4, 9). Although they have only one active site, cPLA2s and iPLA2s exhibit multiple enzymatic activities, including PLA2, PLA1, lysophospholipase, and transacylase activities, and some iPLA2s are lipases (2, 10). Many types of mammalian PLA2 enzymes can release arachidonic acid for eicosanoid production (2). An intriguing observation is that different types of PLA2s can cooperate in cells (sPLA2s with either cPLA2 or iPLA2) in promoting arachidonic acid release, although the mechanism is not understood (1). Recent studies have shown specialized roles of several sPLA2 enzymes in mediating production of eicosanoids that regulate mast cell maturation, asthma, host defense, and metabolic disorders, as detailed in a recent review (11). However, cPLA2 is the only PLA2 that

Abbreviations: COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; DSS, dextran sodium sulfate; EAE, experimental autoimmune encephalomyelitis; ER, endoplasmic reticulum; FLAP, 5-lipoxygenaseactivating protein; GI, gastrointestinal; iPLA2, calcium-independent PLA2; 5-LO, 5-lipoxygenase; LPA, lysophosphatidic acid; LTB4, leukotriene B4; mPGES-1, microsomal prostaglandin E synthase-1; NOD, nonobese diabetic; OVA, ovalbumin; PAF-AH, platelet-activating factoracetylhydrolase; PG, prostaglandin; PLA2, phospholipase A2; SCI, spinal cord injury; sPLA2, secreted PLA2; TX, thromboxane. 1 To whom correspondence should be addressed. e-mail: [email protected] Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

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Journal of Lipid Research Volume 56, 2015

This article is available online at http://www.jlr.org

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Abstract The group IV phospholipase A2 (PLA2) family is comprised of six intracellular enzymes (GIVA, -B, -C, -D, -E, and -F) commonly referred to as cytosolic PLA2 (cPLA2)␣, -␤, -␥, -␦, -␧, and -␨. They contain a Ser-Asp catalytic dyad and all except cPLA2␥ have a C2 domain, but differences in their catalytic activities and subcellular localization suggest unique regulation and function. With the exception of cPLA2␣, the focus of this review, little is known about the in vivo function of group IV enzymes. cPLA2␣ catalyzes the hydrolysis of phospholipids to arachidonic acid and lysophospholipids that are precursors of numerous bioactive lipids. The regulation of cPLA2␣ is complex, involving transcriptional and posttranslational processes, particularly increases in calcium and phosphorylation. cPLA2␣ is a highly conserved widely expressed enzyme that promotes lipid mediator production in human and rodent cells from a variety of tissues. The diverse bioactive lipids produced as a result of cPLA2␣ activation regulate normal physiological processes and disease pathogenesis in many organ systems, as shown using cPLA2␣ KO mice. However, humans recently identified with cPLA2␣ deficiency exhibit more pronounced effects on health than observed in mice lacking cPLA2␣, indicating that much remains to be learned about this interesting enzyme.—Leslie, C. C. Cytosolic phospholipase A2: physiological function and role in disease. J. Lipid Res. 2015. 56: 1386–1402.

exhibits preference for hydrolysis of arachidonic acid from phospholipid substrates that occurs in cells stimulated with diverse agonists (4). Most cells contain several types of PLA2 enzymes and studies have suggested that they have distinct functions. For example, a comparison of the function of cPLA2 and iPLA2 in mast cells and macrophages found that cPLA2, but not iPLA2, selectively hydrolyzes arachidonic acid-containing phospholipids in response to cell stimulation (12, 13).

transport (14, 19, 20). This is supported by a recent study showing that cPLA2 regulates formation of tubules involved in clathrin-independent endocytic trafficking (21). Therefore in studies designed to understand the role of cPLA2 in regulating cellular processes, it is important to consider that it has a functional role independent of eicosanoid production.

GROUP IVA cPLA2 (cPLA2) GROUP IV PLA2 FAMILY

REGULATION OF cPLA2 cPLA2 is widely expressed in cells throughout all tissues in mice and humans. It is a highly conserved enzyme with mouse and human sharing 95% amino acid identity, suggesting similarities in regulation and function (31). In addition to PLA2 activity, cPLA2 catalyzes other enzymatic reactions through the Ser-Asp catalytic dyad, including PLA1, lysophospholipase, and transacylase activity, although the physiological relevance of these activities in vivo is cPLA2: physiological function and role in disease

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The six members of the cPLA2 family (cPLA2, -, -, -, -, and -) share only about 30% homology, and have differences in enzymatic properties, tissue expression, and subcellular localization, suggesting that they are not redundant (4, 14). Analysis of the interfacial kinetic and binding properties of the cPLA2 family showed that they exhibit very different relative lysophospholipase, PLA2, and PLA1 activities, inhibitor sensitivities, calcium dependence, and activation by anionic phospholipids, highlighting potential differences in regulation and function (10). With the exception of the established role of cPLA2 in initiating production of lipid mediators, little is known about the in vivo function of other cPLA2 isoforms. However, there is evidence emerging from studies using cultured cells that members of the cPLA2 family, including cPLA2, play a role in regulating membrane trafficking that does not involve production of oxygenated metabolites of arachidonic acid. PLA2 (and PLA1) enzymes are implicated in regulating intracellular membrane trafficking by participating in the formation of transport carriers from donor membrane (15). Membrane budding requires alterations in membrane curvature that can occur by formation of lysophospholipids. Four cytoplasmic PLAs, including cPLA2, PAF-AH Ib, iPLA2, and iPLA1, are implicated in the formation of tubules from the endoplasmic reticulum (ER)-Golgi intermediate compartment and the Golgi apparatus, maintenance of Golgi structure, and transport through the Golgi. The details of the role of these enzymes have recently been reviewed (15, 16). cPLA2 regulates intra-Golgi transport by mediating the formation of tubules that connect the Golgi stacks (17). The localization of cPLA2 at the Golgi in endothelial cells has also been shown to regulate the transport of junction proteins (vascular endothelial-cadherin, occludin, claudin-5) from the Golgi to cell-cell junctions (18). In these studies it was shown that the enzymatic activity of cPLA2 is required for regulating transport, but oxygenated metabolites of arachidonic acid are not involved. Considering that these are fundamental processes important for cell function, it is surprising that the cPLA2 KO mouse does not exhibit any major phenotype. Golgi transport was found to be normal in cells deficient in cPLA2, but results of a siRNA screen suggested that GVIII PLA2 (PAF-AH) compensates for the loss of cPLA2 in regulating Golgi transport (17). Other group IV PLA2s have been shown to localize to endocytic vesicles, suggesting a possible role in regulating

It has been over 25 years since a soluble calcium-regulated high-molecular-weight PLA2 that exhibited arachidonic acid selectivity was identified in cells and purified (22–29). Cloning of PLA2 revealed the C2 domain for calcium regulation (30, 31). The identification of the active site residues, characterization of stimulus-dependent phosphorylation by MAPKs, and structural elucidation then followed (32–36). Over the years, we have learned a great deal about the regulation of cPLA2 and its role in initiating the production of bioactive lipid mediators. cPLA2 activation results in the production of diverse lipid mediators derived from its products, arachidonic acid and lysophospholipids. Leukotrienes are potent pro-inflammatory mediators produced by cells involved in the inflammatory response, particularly neutrophils, macrophages, and mast cells (37). Prostaglandins (PGs) are made by many cell types and have very diverse functions. In addition to their role in regulating homeostatic processes, they can promote all the cardinal signs of acute inflammation and participate in the maintenance of chronic inflammation (38, 39). However they facilitate the resolution of inflammation by altering the balance of pro-inflammatory and antiinflammatory cytokines, and by enhancing clearance of apoptotic neutrophils (38, 40–43). cPLA2 is also implicated in the production of the pro-resolving lipid mediators (lipoxins) and bioactive lysophospholipids (or derived from lysophospholipids) such as platelet-activating factor and lysophosphatidylinositol (44–46). The generation of mice deficient in cPLA2 has revealed its role in regulating normal physiological processes and disease pathogenesis in rodents (47, 48). More recently, the identification of humans with cPLA2 deficiency has emphasized its important role in human health (49–51). This review will provide a brief overview of the regulation of cPLA2 and primarily focus on reviewing its function in specific organs of mice and humans.

analysis of the human cPLA2 gene (73). This IL-1responsive distal regulatory element contains an AP-1 site, which regulates both basal and IL-1-induced expression of cPLA2. Binding of c-Jun to this site acts as a repressor in the basal state and an activator in response to IL-1, and an important role for C/EBP as a transcriptional activator of cPLA2 was demonstrated. cPLA2 is rapidly activated in cells by posttranslational processes, including increases in intracellular calcium and phosphorylation by MAPK (Fig. 1). These signaling pathways are activated in cells through engagement of many types of receptors, indicating that cPLA2 activation and arachidonic acid release occur commonly in response to cell stimulation. There have been a number of reviews that detail the regulation of cPLA2 by posttranslational processes (2–4, 74). In brief, increases in intracellular calcium promote the translocation of cPLA2 from the cytosol to intracellular membrane (75–78). Calcium binds to the Nterminal C2 domain of cPLA2 that increases the hydrophobicity of the calcium binding loops, which penetrate the membrane bilayer (79–83) (Fig. 2). Differences in phospholipid binding specificity of the C2 domains of protein kinase C (anionic phospholipids) and cPLA2 (phosphatidylcholine) play an important role in determining

Fig. 1. Regulation of cPLA2. cPLA2-mediated arachidonic acid (AA) release occurs following cell stimu2+ lation that results in increases in intracellular Ca and kinase activation. In the cytosol, cPLA2 is bound to p11/annexin A2 and is released from this complex when phosphorylated on S727. Calcium binds to the calcium binding loops (CBL, green) at the membrane binding face of the C2 domain. This increases the hydrophobicity of the C2 domain and promotes preferential binding of cPLA2 to the Golgi and, at higher levels of intracellular calcium, to the ER and nuclear envelope. Basic residues (orange patch) in the C2 domain are implicated in binding to ceramide-1-P in the Golgi. Association of the catalytic domain with membrane is mediated in part by a tryptophan residue in the catalytic domain (W464, red patch) that stabilizes cPLA2 on the membrane. Phosphorylation of S505 by MAPKs in the catalytic domain enhances cPLA2 activity. Interaction of basic residues in the catalytic domain (blue patch) with anionic components in the membrane, such as polyphosphoinositides, optimizes catalytic activity. AA released by cPLA2 is metabolized by COX-1 and -2, which are primarily localized to the ER, but also occur on the Golgi. Leukotriene production occurs at the nuclear envelope, the site of 5-LO translocation in response to increases in calcium. 5-LO binds to the scaffold protein FLAP, an AA binding protein, which also binds LTC4 synthase (syn) for production of LTC4.

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unknown (2, 4, 32, 35). The intense focus on cPLA2 stems from its preferential hydrolysis of sn-2 arachidonic acid, and its well-established role in initiating the release of arachidonic acid for the production of lipid mediators (52). cPLA2 KO mice have provided a source for cells, including (but not limited to) mast cells, neutrophils, macrophages, platelets, endothelial cells, and lung fibroblasts, to establish a role for cPLA2 in mediating arachidonic acid release and production of lipid mediators (47, 53–59). cPLA2 is regulated at the transcriptional level and by posttranslational mechanisms. It is basally expressed in many cells, due in part to transcription factor II D binding to the TATA-less promoter (60–62). cPLA2 expression is also induced transcriptionally through a number of signaling pathways involving Ras and MAPKs, and transcriptional activators NF-B, Krüppel-like factor, hypoxiainducible factor, Sp1, and c-Jun have been described (63–70). Pro-inflammatory cytokines induce expression of human cPLA2 mRNA that is blocked by glucocorticoids (71). However, glucocorticoids are not universally suppressive because they paradoxically enhance cPLA2 expression in human amnion fibroblasts (see Reproduction section below) (72). Recently the first cytokine-dependent enhancer element has been identified by DNase I hypersensitive site

their distinct subcellular targeting to the plasma membrane and intracellular membranes, respectively (84–86). The catalytic domain is then positioned on the membrane by calcium-independent mechanisms, in part through a tryptophan residue (W464) that stabilizes membrane binding (87–90). cPLA2 preferentially translocates to the Golgi apparatus and, at higher intracellular calcium concentrations, to the ER and nuclear envelope (76–78, 91, 92) (Fig. 1). Binding of cPLA2 to the Golgi may, in part, involve interaction of the C2 domain with sphingolipids, including ceramide-1-P and lactosylceramide (93–96). Translocation of cPLA2 to membrane is necessary but not sufficient for cPLA2 to release arachidonic acid (92). The catalytic activity of cPLA2 is enhanced by phosphorylation of S505 by the MAPKs, extracellular regulated protein kinases, and p38 (33, 34, 97, 98). More recent studies suggest that cPLA2 is also activated by c-jun N-terminal kinases (99, 100). Activation of cPLA2 in vascular smooth muscle cells by calcium/calmodulin-dependent kinase II that phosphorylates S515 has been described (101). Phosphorylation of cPLA2 on S727 by MAPK-interacting kinase does not enhance catalytic activity per se, but blocks its binding to an inhibitory complex in the cytosol composed of p11(S100A10)/annexin A2 (74, 102–105) (Fig. 1). In addition to phosphorylation, the activity of cPLA2 is regulated by basic residues in the catalytic domain that are the site for activation by polyphosphoinositides (89, 106– 108). The basic residues are required for the ability of cPLA2 to release arachidonic acid in cells but are not required for translocation of cPLA2 to the Golgi (92). Collectively, the

results show that the ability of cPLA2 to release arachidonic acid in cells involves calcium-dependent membrane binding and optimization of catalytic activity by phosphorylation and interaction of basic residues in the catalytic domain with anionic components, perhaps polyphosphoinositides, in the membrane (92). The translocation of cPLA2 to intracellular membranes raises the interesting question of how cPLA2-derived arachidonic acid couples to the downstream enzymes, 5-lipoxygenase (5-LO) and cyclooxygenase (COX)-1 and -2 (Fig. 1). There are general similarities in the regulation of cPLA2 and 5-LO involving both calcium and phosphorylation (109). 5-LO, a soluble enzyme (cytoplasmic or intranuclear) in resting cells, translocates to the nuclear envelope in response to calcium, which binds to an N-terminal C2like domain (110–115). Leukotriene production requires interaction of 5-LO with 5-LO-activating protein (FLAP), an integral nuclear envelope protein that functions as an arachidonic acid binding protein (116). It has recently been shown that leukotriene synthesis involves activationdependent formation of supramolecular complexes on the nuclear envelope composed of FLAP with 5-LO and FLAP with leukotriene C4 synthase (117, 118). In addition, cPLA2-derived arachidonic acid was shown to affect the conformation of FLAP and enhance its ability to recruit 5-LO for complex formation (119). It is not known whether cPLA2 physically associates with these complexes or whether arachidonic acid released in their vicinity diffuses through the membrane to initiate leukotriene production. cPLA2: physiological function and role in disease

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Fig. 2. Ribbon diagram of cPLA2 structure. Two ions of calcium bind to the calcium-binding loops (CBL) on the membrane binding face of the C2 domain. The catalytic domain with the active site nucleophile S228 contains residues involved in cPLA2 regulation (pink) that include phosphorylation sites (S505, S727), basic residues (K488, K544, K543), and W464 important for membrane interaction. The residues mutated in humans with cPLA2 deficiencies are shown in yellow. The residue V707 is predicted to be the start of the deletion in patients with cryptogenic multifocal ulcerating stenosing enteritis.

ROLE OF cPLA2 IN NORMAL PHYSIOLOGICAL PROCESSES AND DISEASE The first characterization of mice with cPLA2 gene disruption published in 1997 was followed by a large body of work that provides a comprehensive view of cPLA2 function in normal processes and disease pathogenesis in mice (47, 48). The cPLA2 KO mouse develops normally and lives a normal life span with no overt adverse health effects. Considering the evolutionary conservation of cPLA2 and its role in eicosanoid production, similarities in the regulation and function of cPLA2 in rodents and humans is expected. However, as discussed in this review, there are deleterious effects on the health of humans with cPLA2 deficiency, suggesting differences in the role of cPLA2 and eicosanoids in mouse models and humans. This could be due to a number of factors, including differences in expression of other types of PLA2 enzymes that could compensate and differences in the tissue-specific expression of receptors for lipid mediators that could influence their function. In many cases, the mechanisms responsible for observed phenotypes in human and mouse cPLA2 deficiencies are not well understood. This is understandable considering that cPLA2 is the first regulatory enzyme that releases the lipid mediators, arachidonic acid and lysophospholipids, which are precursors for an enormous number of diverse bioactive lipid metabolites that can have opposing effects. Gastrointestinal tract The consequences of inherited cPLA2 deficiency in several human patients have now been described (49–51). 1390


In all of these patients, a common clinical finding is abnormalities in the gastrointestinal (GI) tract. However, there are differences in the clinical presentations and severity of disease. In one subject, two rare heterozygous single bp mutations in PLA2G4A were found; one mutation (S111P) occurred in the C2 domain of cPLA2 and the other allele had a mutation in the catalytic domain (R485H) (Fig. 2) (49). The expression of cPLA2 protein and PLA2 activity in platelet lysates was reduced compared with normals. The patient exhibited a global reduction in eicosanoid production with reduced urinary metabolites of thromboxane (TX)A2, PGI2, PGD2, and PGE2, and undetectable leukotriene E4 in urine. The production of leukotriene B4 (LTB4) from leukocytes in A23187-stimulated blood was reduced by 97%. The patient had occult GI blood loss and anemia in childhood with more acute GI bleeding in his fourth decade with recurrent ulcerations in the ileum and jejunum of the small intestine. The beneficial effect of misoprostol suggests that cPLA2-mediated PGE2 production is important for function of the small intestine. Unlike the effect of nonsteroid anti-inflammatory drug toxicity, the patient did not exhibit gastroduodenal ulcerations, and the colon was normal. The results support a role for cPLA2 in providing arachidonic acid for a large proportion of eicosanoid production in humans. Other PLA2s may contribute to the residual eicosanoids produced, or the patient may retain a low level of cPLA2 activity. To investigate the effect of the mutations on cPLA2 function in more detail, we compared the catalytic activity of WT and mutant forms of purified cPLA2 in vitro, and compared their cellular function by expression in cells lacking endogenous cPLA2 (124). cPLA2 with the S111P mutation showed slightly reduced catalytic activity and interfacial binding in vitro at saturating calcium. When expressed in cells, there was less translocation of cPLA2 with the S111P mutation to the Golgi in response to serum stimulation and reduced arachidonic acid release. In contrast, cPLA2 with the R485H mutation translocated to the Golgi in serum-stimulated cells, but failed to release arachidonic acid and was inactive in vitro. Consequently, the mutations in the C2 and catalytic domains of cPLA2 compromise function, but do not result in a complete loss of protein or activity. Another patient, and his twin sister, with inherited cPLA2 deficiency also exhibited GI abnormalities from early childhood characterized by duodenal ulcers that were corrected by misoprostol treatment (51). The incidence of small intestinal ulcers was not evaluated. Homozygous bp mutations (D575H) in the cPLA2 catalytic domain were identified (Fig. 2). cPLA2 protein was not detected in lysates from patients with the homozygous mutations. Unlike the patients described above, the mutations resulted in global diathesis (see Hemostasis section below). The most severe clinical manifestation of inherited cPLA2 deficiency occurred in siblings diagnosed with cryptogenic multifocal ulcerating stenosing enteritis due to homozygous deletion mutations in PLA2G4A (50). The affected male had severe peptic ulcers and bleeding beginning at an early age (4 years) then over the next several years


COX-1 and -2 are also found in internal membranes, but unlike cPLA2 and 5-LO, they are integral membrane proteins primarily localized in the ER (120). However, it was recently shown that COX-2 localizes to the Golgi in cancer cell lines detected by using a COX-2-specific fluorescent probe (121). Another study investigating the posttranslational processing and degradation of COX-2 found catalytically active COX-2 (but not COX-1) localized to the Golgi as a result of anterograde trafficking from the ER, a step required for posttranslational modification prior to COX-2 degradation through the ER-associated degradation pathway (122). It is interesting that microsomal PGE synthase-1 (mPGES-1) was also found at the Golgi, suggesting that this site has a system for PGE2 production involving cPLA2/COX-2/mPGES-1 (122). Another report showed that COX-1, but not COX-2, is constitutively found at the Golgi in airway epithelial cells, where it colocalizes with cPLA2 (123). The localization of cPLA2, COX, mPGES-1, and leukotriene synthetic enzymes at intracellular membranes, including the ER, nuclear envelope, and Golgi, may efficiently coordinate the production of certain eicosanoids. However, several downstream enzymes involved in PG and leukotriene production are cytosolic, and not apparently subject to stimulus-dependent translocation, although the sub-cellular organization of enzymes involved in eicosanoid production is only beginning to be elucidated.

against colon cancer in mice induced by the colon carcinogen, azoxymethane, which induces increased tumor multiplicity in heterozygous and KO mice compared with WT mice (131). The increased colon tumorigenesis in cPLA2 mutant mice occurs despite a decrease in PGE2 production. In contrast, genetic ablation of cPLA2 protects against cancer development in mouse models of adenomatous polyposis (Apc 716 KO mice and ApcMin mice) (125, 132). Apc 716 KO mice develop small intestinal polyps that express high levels of COX-2 and cPLA2 mRNA compared with normal small intestine (125, 133). Genetic deletion of cPLA2 in Apc 716 KO mice leads to a reduction in size of the polyps but has no effect on the number of polyps unlike COX-2 KO mice (125, 133). In contrast, knocking out cPLA2 in ApcMin mice results in a reduction in size and a dramatic decrease in the number of small intestinal polyps, although there is no effect on tumor number in the large intestine (132). The results highlight differences in the role of cPLA2 in promoting tumorigenesis in the small and large intestine. Several lines of evidence suggest that tumorigenesis in the small intestine involves the cPLA2/COX-2/PGE2 pathway rather than a role for arachidonic acid itself in promoting apoptosis (132, 134). Understanding how cPLA2 regulates tumorigenesis is complex because it is upstream of many lipid mediators including arachidonic acid, diverse PGs, leukotrienes, and lysophospholipids. In addition, the large repertoire of receptors for lipid mediators is differentially expressed in tissues and promotes signaling pathways with distinct effects on cell function. This adds another level of complexity in understanding the role of cPLA2-derived bioactive lipids in organ-specific cancer development. Liver Studies have shown that PGs promote increased triglyceride storage in adipocytes and hepatocytes, prompting investigations on the role of cPLA2 in regulating lipid storage and liver damage (135–140). It is of interest that even under normal dietary conditions, cPLA2 regulates lipid storage in adipose tissue and liver (138). cPLA2 KO mice fed a normal diet have reduced adipose tissue mass compared with WT mice, and lower triglyceride deposition in the liver, that correlates with lower serum levels of PGE2. An extension of this work showed that cPLA2 KO mice are protected from fatty liver damage induced by a high-fat diet (139). Mice with cPLA2 deficiency have lower serum PGE2 levels, which may afford protection because the administration of PGE2 enhances hepatic triacylglycerol content, whereas inhibition of COX-2 prevents fat deposition in rodents (137, 141). Obesity is associated with development of nonalcoholic fatty liver disease with pathological manifestations including nonalcoholic steatohepatitis, fibrosis, or cirrhosis. Recently, it was shown that cPLA2 KO mice fed a high-fat diet are protected from hepatic liver deposition and development of hepatic fibrosis (140). Similar results are observed in mice subjected to repeated administration of carbon tetrachloride that induces fibrosis without lipid deposition. The mechanism is cPLA2: physiological function and role in disease

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developed more severe disease involving the ileum, stomach, esophagus, duodenum, biliary system, and liver, although the colon remained normal. He developed type 2 diabetes, peripheral neuropathy, and osteoporosis. His sister exhibited similar severe GI disease beginning at age 2 years that worsened over time. She experienced infections with Campylobacter enteritis and Salmonella enteriditis, and developed Candida septicemia, staphylococcus lung infection, and acute respiratory distress syndrome. Other extra-intestinal disease included acute renal failure, endometriosis, left ventricular concentric hypertrophy, fibrotic bladder with stones, and infertility. The siblings contained homozygous 4 bp deletions in the PLA2G4A gene predicted to result in a frameshift and premature stop codon with the loss of 43 amino acids at the C terminus of cPLA2 (from V707) (Fig. 2). cPLA2 was undetectable by immunohistochemical analysis of small bowel biopsy and could not be detected in peripheral blood mononuclear cells by Western blot. Many factors could contribute to the individual phenotypic differences in disease severity associated with inherited cPLA2 deficiency, but the studies show a critical role for cPLA2 and eicosanoids in maintaining the integrity of the gut, particularly the small intestine. The small intestine of the cPLA2 KO mouse has numerous small ulcers, but there appears to be little effect of the lesions on the health of mutant mice (125). However, there is a role for cPLA2 in protecting the GI tract of mice from chemical toxicity induced by COX inhibitors and dextran sodium sulfate (DSS) (126, 127). Injury induced by COX inhibitors in cPLA2 KO mice did not occur in the colon, but was extensive in the small intestine, the site of greatest drug absorption (126). Differences in toxicity of cPLA2 WT and KO mice did not correlate with PGE2 levels, but may have been due to greater mitochondrial toxicity in KO mice. In the DSS model, there was extensive damage in the colons of cPLA2 KO mice that correlated with lower levels of PGs in colon tissue compared with DSS-treated WT mice (127). Mice deficient in mPGES-1 also exhibited greater DSS toxicity in the colon, suggesting an important protective role for PGE2. In contrast to cPLA2 KO mice, which recovered from DSS toxicity to a similar extent as WT mice, the recovery of mPGES-1 KO mice was compromised. The studies in mice and humans show an important role for cPLA2 in protecting the GI tract. However, levels of eicosanoids in the GI tract of cPLA2 KO mice are only partially reduced, suggesting a role for other PLA2s. This may contribute to the less severe health effects of cPLA2 deficiency in mice compared with humans. There has not been a systematic comparison of the relative levels of the various types of PLA2 enzymes expressed in different tissues of mice and humans to speculate on the PLA2s that may account for eicosanoid production in the GI tract of mice. Although results support a role for PGs in protecting the integrity of the intestine, COX-2 expression and PGE2mediated inflammation are associated with development of human colorectal cancer, consistent with epidemiological evidence that nonsteroid anti-inflammatory drug use is protective (128–130). However, cPLA2 protects

thought to involve a role for cPLA2 in production of monocyte chemoattractant protein-1 and infiltration of macrophages in these models. Previous results have shown that leukotrienes and PGs regulate induction of monocyte chemoattractant protein-1, a chemotactic factor implicated in development of hepatic fibrosis (142–145). Eicosanoids secreted by cells influence gene expression through paracrine or autocrine interaction with their specific G proteincoupled receptors (146, 147). For example in an infectious disease model using macrophages from WT and cPLA2 KO mice, we found that cPLA2 activation promotes an autocrine loop involving production of PGs that engage prostanoid receptors, increase cAMP, and globally affect expression of genes involved in host defense and inflammation (43).

Reproduction PGs produced through the COX-1 and COX-2 pathways play an important role in regulating female reproduction, therefore it is not surprising that cPLA2 KO female mice 1392


Kidney cPLA2 KO female mice exhibit defects in renal function, possibly due to a deficiency in PGE2 production, which regulates various aspects of kidney physiology (161). Mice lacking cPLA2 have a urine-concentrating defect that develops with age. Based on abnormal aequorin staining in proximal tubules, the defect in cPLA2 KO female mice may be due to diminished water reabsorption. There is also evidence that cPLA2 regulates Na+-HCO3- transport by angiotensin II in kidney proximal tubules (162). The role of cPLA2 in kidney function of humans is poorly understood. The female sibling with the cPLA2 deletion developed acute renal failure requiring hemodialysis, although whether this is a direct effect of cPLA2 deletion is not clear (50). It has recently been


Hemostasis Considering the importance of eicosanoids in regulating hemostasis, the role of cPLA2 in TXA2 production, platelet activation, and hemostasis has been investigated. Platelets from cPLA2 KO mice stimulated with collagen produce much less TXA2, and bleeding time is increased compared with WT mice (59). However, the low level of TXA2 produced by other phospholipases in collagen-stimulated cPLA2 KO platelets is sufficient for platelet aggregation. In vivo, cPLA2 KO mice have increased bleeding time and are protected from thromboembolism that correlates with reduced TXA2 levels and vasoconstriction. cPLA2 appears to play a discrete role in the complex regulation of platelet activation in mice. Studies of humans with inherited cPLA2 deficiency have shown an important role of cPLA2 in regulating platelet function and hemostasis. In the patient with two heterologous bp mutations in the coding regions of PLA2G4A, the PLA2 activity in platelet lysates was reduced and levels of TXB2 and 12-hydroxyeicosatetraenoic acid in serum during blood clotting (platelet-derived) were much lower than normal levels (49). Platelet aggregation and ATP release were compromised indicating impaired function. In patients with homozygous deletion mutants of cPLA2 associated with cryptogenic multifocal ulcerating stenosing enteritis (50), similar abnormalities of platelet function were observed, including defects in collageninduced aggregation and reduced levels of TXB2 production. The results indicate that cPLA2 plays a dominant role in providing arachidonic acid for the COX-1 and 12-lipoxygenase products in platelets, although the patients described above did not exhibit generalized clotting deficiency. In contrast, the siblings with inherited cPLA2 deficiency due to homozygous bp mutations had lifelong bleeding diathesis characterized by spontaneous mucocutaneous bleeding in addition to GI bleeding. The bleeding defect correlated with defective platelet function and extremely low levels of serum TXB2 (51).

exhibit decreased fertility, small litter size, and delayed onset of labor (47, 48, 148–150). In cPLA2 KO mice, blastocyst attachment is delayed due to a loss of maternal cPLA2, but decidualization is unaffected (151). Implantation beyond the normal “window” results in defective embryo development, abnormal uterine spacing of the embryos and resorption contributing to the small litter size, and defects in parturition (151). A later study emphasized the importance of cPLA2 function early in pregnancy because the defect in parturition in cPLA2 KO mice could be attributed to the deferred embryo implantation (152). cPLA2 KO mice showed less pronounced defects in ovulation and fertilization compared with COX-2 KO mice (149, 151, 152). A more detailed study showed that cPLA2 modestly regulates ovulation and fertilization by promoting the induction of COX-2 for PG production (153). A role for cPLA2 in regulating male sexual maturation has also been described (154). Male cPLA2 KO mice are fertile but exhibit abnormal histology of the testes showing a reduced number of interstitial cells. Compared with WT mice, cPLA2 KO males have delayed spermatogenesis and reduced testosterone production, although these defects normalized with age. It is difficult to extrapolate from studies in mouse models in understanding the specific role of cPLA2 in human reproduction, which has many unique regulatory features not shared by other mammalian species (155). The female patient with homozygous deletion mutations in cPLA2 associated with cryptogenic multifocal ulcerating stenosing enteritis is infertile, although the basis for this is unknown, and may be due to the severe consequences of the disease (50). PGs regulate labor and birth in humans, and an important source of PGs is amnion fibroblasts in fetal membranes (156, 157). There is an increase in cortisol secretion and PG production during parturition, a paradoxical event because cortisol suppresses PG production in most tissues (158). Glucocorticoids have been shown to induce mRNA and protein expression of cPLA2 and COX-2 in human amnion fibroblasts (72, 159). The induction of cPLA2 by glucocorticoids involves activation of Gs leading to cyclic AMP response element-binding protein-1 phosphorylation, which interacts with the glucocorticoid receptor on the glucocorticoid response element in the cPLA2 promoter (160).

shown that cPLA2 plays a role in mediating the onset of nonobstructive neonatal hydronephrosis, induced in mice by treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which increases expression of cPLA2 in the kidneys (163). Results showed that cPLA2 KO neonates have reduced incidence and severity of hydronephrosis that correlates with reduced expression of COX-2, mPGES-1, and lower urinary PGE2 compared with 2,3,7,8-tetrachlorodibenzo-p-dioxintreated WT pups. The COX-2/PGE2 pathway is also implicated in the pathogenesis of dioxin-induced hydronephrosis (164). The toxic effects of dioxins are mediated by the arylhydrocarbon receptor, which binds to the second intron of Pla2g4a and mediates its induction (165, 166). The arylhydrocarbon receptor pathway also promotes signaling cascades for posttranslational activation of cPLA2 by increasing calcium and phosphorylation (167, 168).

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Nervous system Many studies have implicated cPLA2 in mediating several disease processes in the nervous system including postischemic brain injury, spinal cord injury (SCI), and neurodegenerative diseases, as will be discussed below. However, there is a role for cPLA2 in regulating neuronal homeostasis, particularly long-term depression and longterm potentiation involved in synaptic plasticity. Longterm depression is blocked in brain slices from cPLA2 KO mice by treatment with cPLA2 (pyrrolidine-1) and COX-2 inhibitors (169). The defect in long-term depression by cPLA2 deficiency or COX-2 inhibition attenuates optokinetic eye movement adaptation, suggesting that cPLA2regulated long-term depression is important for motor learning. Long-term potentiation is also important for motor learning and a recent report found that long-term potentiation in cerebellar parallel fiber-Purkinje cell synapses is abolished in cPLA2 KO mice (170, 171). The earliest work investigating the role of cPLA2 in brain injury found that cPLA2 KO mice are protected from postischemic brain injury induced by middle cerebral artery occlusion (47). Infarct volumes are reduced in KO mice after reperfusion; this is particularly evident in posterior brain slices. The KO mice also exhibit fewer neurological deficits compared with WT mice. No differences in the vascular anatomy of the brain in KO and WT mice were noted. An extension of this work found that cPLA2 contributes to the early events leading to injury during cerebral ischemia (172). cPLA2 WT mice exhibit greater COX-2 expression, PGE2 production, and reactive oxygen species after ischemia, and more disruption of neuron morphology than KO mice. Other studies have reported a role for cPLA2 in regulating the expression of COX-2 in the brain (173). After systemic LPS, there is less COX-2 expression and PGE2 production in cPLA2 KO compared with WT mice (174). COX-2 is induced in neuronal and nonneuronal cells of the brain during inflammation and promotes injury (175). A role for cPLA2 in regulating SCI has also been investigated. One report found that cPLA2 expression is increased in SCI, particularly in neurons and oligodendrocytes (176). It was shown that inhibiting cPLA2 (AACOCF3) or

genetic ablation (cPLA2 KO C57Bl/6 mice) results in improved motor deficiencies and less tissue damage from SCI. Another study reported a complex role for different PLA2 enzymes (cPLA2, sPLA2 GIIA, and iPLA2 GVIA) in protecting or exacerbating SCI (177). All of the PLA2s were upregulated after SCI. In contrast to Liu et al. (176), cPLA2 was found to protect from SCI, which was determined using specific inhibitors and cPLA2 KO mice (Balb/c), but sPLA2 GIIA and iPLA2 GVIA were detrimental. Results suggested that the beneficial effect of cPLA2 in SCI involves PGE2 signaling through the EP1 receptor. A confounding factor in evaluating the role of cPLA2 in these mouse models is the influence of genetic differences in mouse strains. For example, the commonly used mouse strains, BALB/c and C57BL/6, generally exhibit distinct polarization of helper T cell responses, with BALB/c predisposed to Th2 and C57BL/6 to Th1 responses (178). In addition, C57BL/6 mice lack sPLA2 GIIA due to a natural deletion in the gene that does not occur in BALB/c mice (179). A role for cPLA2 in mouse models of neurodegenerative disease such as Alzheimer’s disease has been investigated (180, 181). The A peptides that are implicated in Alzheimer’s neurodegeneration exert toxic effects in neurons, which are in part mediated by cPLA2 (182, 183). Genetic ablation of cPLA2 improves cognitive function in a mouse model of familial Alzheimer’s disease, and protects from the toxic effects of intra-cerebroventricular injection of A oligomers (184, 185). A recent study found that A peptides activate cPLA2 in rat primary cortical neurons, resulting in increased expression of -amyloid precursor protein by an autocrine pathway involving PGE2 production and increases in cAMP (186). Additional studies also suggest that cPLA2 activation is involved in promoting neurodegeneration that occurs in prion diseases. In primary cortical neurons, the prion-derived peptide (PrP82-146) induces cPLA2 activation and arachidonic acid release that correlates with synapse damage and cell death (187 ). Treatment of neurons with PLA2 inhibitors (AACOCF3, MAFP) and a PGE2 receptor antagonist (AH13205), protects from synapse degeneration (188). In a model of Parkinson’s disease, cPLA2 contributes to neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (189). The reduction in dopamine induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is significantly less in KO than in WT mice. It is not known how cPLA2 contributes to neurodegeneration in these models, but mechanisms involving free radical production, effects on intracellular membrane trafficking, and arachidonic acid metabolites have been proposed. It is important to consider underlying differences that have been found in the architecture and lipid composition of the brain in cPLA2 WT and KO mice. A recent study showed by microscopy that cortical neurons in cPLA2 KO mice exhibit abnormal architecture including larger nuclei, increased number of nucleoli, and aggregated intra-nuclear ribosomes (190). Abnormalities in the nuclear envelope, nuclear pore, and synapses were observed. The authors speculated that this could be due to the role of

Lung There is extensive information investigating the role of eicosanoids in regulating lung diseases, including fibrosis, acute lung injury, and cancer. Comparisons of cPLA2 KO and WT mice have provided interesting insight into how cPLA2 regulates these processes. When the cPLA2 KO mouse was initially developed, the role of cPLA2 in regulating type I allergic response (anaphylaxis) in the lung was investigated (48). To induce systemic anaphylaxis KO and WT mice were immunized by intraperitoneal injection of ovalbumin (OVA) followed by intravascular OVA injection to elicit anaphylaxis. In WT mice, there was narrowing of the airway and alveolar thickening that was not evident in KO mice. Lung resistance was lower and recovery was faster in KO mice than WT mice after OVA challenge. Bronchial reactivity to methacholine challenge was dramatically reduced in KO mice. Airway hyperreactivity is also reduced in 5-LO KO mice, suggesting that cPLA2mediated production of leukotrienes mediates allergic responses (192). In contrast, allergic lung responses are exacerbated in COX-1 and COX-2 KO mice compared with WT mice, suggesting PGs play a protective role (193). Over the past few years, Wyeth has developed indole compounds that selectively inhibit human cPLA2 and have been tested in a sheep model of allergic asthma (194). Because diverse lipid mediators are implicated in mediating asthmatic responses in this model, they reasoned that broad inhibition with a cPLA2 inhibitor would be beneficial. The indole cPLA2 inhibitors reduced late phase bronchoconstriction and airway hyperreactivity in allergen-challenged sheep. The indole inhibitor also blocked allergen-dependent induction of novel genes expressed in cells from asthmatics (195). One of the most potent indole 1394


inhibitors, PF-5212372, blocks eicosanoid production by stimulated human lung mast cells and lung homogenates. However, in human lung homogenates, which contain a complex cell mix, the inhibitor was less effective at blocking PGE2 production than other eicosanoids, suggesting that other PLA2s, particularly sPLA2s, may contribute to arachidonic acid release for eicosanoid production (1, 196). The inhibitor also blocked contraction of isolated human bronchial rings induced by AMP. The results provide convincing evidence for a role of cPLA2 in mediating responses in allergic asthma. Analysis of the promoter region of PLA2G4A has shown that microsatellite fragments are shorter in patients with severe asthma than healthy controls (197). A comparison of peripheral blood monocytes from severe asthmatics with short and long microsatellite sequences in the PLA2G4A promoter found a correlation between the shorter sequences with increased expression of cPLA2 mRNA and protein, and greater eicosanoid production (197, 198). Results of studies comparing cPLA2 WT and KO mice have also supported a role for cPLA2 in mediating acute lung injury (199). One model induced lung injury by intravenous administration of endotoxin and fungal cell walls, zymosan; the other model involved intratracheal administration of hydrochloric acid. These models simulate endotoxemia and acid aspiration, which are causes of acute respiratory distress syndrome. In both models of lung injury, the cPLA2 KO mice were protected, exhibiting less protein leakage and neutrophil recruitment into the lung than WT mice, that correlated with reduced levels of TXB2, LTB4, and cysteinyl leukotrienes. However, in a cecal ligation and puncture model of experimental sepsis, there was no difference in survival of cPLA2 WT and KO mice, although there was lower production of eicosanoids (with the exception of 12-hydroxy-eicosatetraenoic acid) and cytokines (IL-6 and CCL2) in the mutant mice (200). It is possible that lipid mediators do not play a dominant role in regulating this sepsis model, or that just targeting one PLA2 isoform is not sufficient (201). A study using an antisense approach to inhibit both sPLA2-IIa and cPLA2 showed improved survival of rats in a cecal ligation and puncture model (202). Another consideration is that cPLA2 mediates the production of diverse lipid mediators that may have opposing effects, some are pro-inflammatory and others anti-inflammatory (201). The opposing role of cPLA2-derived lipid mediators is illustrated in the regulation of pulmonary fibrosis, a chronic disease characterized by the accumulation of myofibroblasts and deposition of extracellular matrix (203). Chronic inflammation often leads to fibrosis due to aberrant wound healing resulting in permanent scarring and organ failure (204). A study of pulmonary fibrosis induced by bleomycin found that fibrosis, collagen synthesis, and inflammation were reduced in cPLA2 KO mice compared with WT mice (205). In WT mice, the effects of bleomycin were accompanied by increases in eicosanoids in bronchoalveolar lavage fluid that were markedly lower in bleomycin-treated cPLA2 KO mice. Although disease parameters were not completely attenuated in KO mice,


cPLA2 in regulating membrane structure and curvature. In addition, a study comparing the brain lipid content of cPLA2 WT and KO mice found altered brain phospholipid composition in mutant mice but no differences in the concentrations of nonesterified fatty acids in the plasma or brain (191). There were decreased levels of esterified arachidonic acid in phosphatidylinositol and decreases of several fatty acids including linoleic, arachidonic, and docosahexaenoic acids in phosphatidylcholine in cPLA2 KO mice compared with WT mice. However, an interesting finding was the increased incorporation and turnover of AA in phosphatidylinositol and phosphatidylcholine in cPLA2 KO mice, perhaps due to compensation from other PLA2s. Consequently the differences observed in phospholipid brain composition in cPLA2 WT and KO mice may reflect both a lack of cPLA2 and compensation from other enzymes with differences in lipid specificity as noted by the authors (191). The lipid alterations in KO mice may influence membrane properties contributing to the structural differences noted in cortical neurons, and could also influence the response of mutant mice to brain injury. This again raises the question of whether responses of cPLA2 KO mice are due to a primary effect of the loss of cPLA2 or secondary compensatory changes that occur.

Autoimmunity Chronic inflammation is a characteristic of certain autoimmune diseases, such as rheumatoid arthritis. Collageninduced arthritis in mice has pathological features resembling rheumatoid arthritis and the role of eicosanoids has been studied extensively. Interestingly, there is evidence that both leukotrienes and PGs contribute to disease pathogenesis based on studies using FLAP inhibitors, LTB4 receptor antagonist, and COX-2 KO mice (225–227). Therefore, it is not surprising that cPLA2 KO mice are also protected

from collagen-induced arthritis (228). Based on the collective involvement of eicosanoids in this autoimmune disease, it has been suggested that a cPLA2 inhibitor would have therapeutic value by simultaneously blocking eicosanoids from the COX and 5-LO pathways (228). In rheumatoid arthritis, bone homeostasis is disrupted due, in part, to chronic inflammation resulting in joint destruction and bone loss. The role of cPLA2 in regulating inflammatory bone resorption induced in mice with endotoxin has been investigated (229). In response to endotoxin (ip), cPLA2 KO mice exhibit less bone loss and reduced PGE2 production. The results indicate that cPLA2 mediates osteoclastic bone resorption due to endotoxininduced PGE2 production by osteoblasts. The role of cPLA2 in regulating autoimmune disease in nonobese diabetic (NOD) mice has also been investigated (230). NOD mice spontaneously develop autoimmune diabetes that has similarities to human type 1 diabetes. In contrast to collageninduced arthritis, cPLA2 plays a protective role in the development of diabetes. NOD mice with genetic ablation of cPLA2 have a higher incidence of diabetes than cPLA2 WT NOD mice. Results suggest that cPLA2 plays a protective role during the diabetic stage, possibly due to the ability of PGE2 produced by macrophages to suppress the production of the pro-inflammatory cytokine, TNF. Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease in mice that has features of the human autoimmune disease multiple sclerosis (231). There are conflicting data on the role of specific eicosanoids in regulating EAE based on studies with 5-LO KO mice, in which EAE is exacerbated, or using an LTB4 receptor antagonist, which protects (232). The role of PGs is not clear because COX-2 KO mice are not protected but a COX-2 inhibitor is beneficial in EAE (233, 234). A study of EAE induced in cPLA2 WT and KO mice on the susceptible C57Bl/6 background found that KO mice are resistant to disease (235). Results suggested that cPLA2 contributes in the induction and effector phase by contributing to the development of Th1 responses. Because the development of the immune system may be altered in KO mice, a follow up study tested the effect of cPLA2, 5-LO, and COX inhibitors on the development of EAE in WT mice (236). The cPLA2 inhibitor, WAY-196025, used at the time of immunization to promote EAE blocked the development of EAE, and when used at the start of disease reduced the duration of EAE relapses. Inhibitors of COX1/2 and 5-LO also reduced disease severity. Results using the selective cPLA2 inhibitor are consistent with the studies using the cPLA2 KO mice. A recent study also showed that another selective cPLA2 inhibitor (2-oxoamide, AX059) protected rats from EAE that correlated with increased production of Tregs (237). However, the conflicting studies on the role of specific eicosanoids in EAE highlight potential difficulties in interpreting results from KO mice, which can exhibit compensatory effects or changes that occur during embryogenesis. However, there are also complications in the use of inhibitors involving dosing and time of administration, as well as unknown off-target effects. cPLA2: physiological function and role in disease

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cPLA2 may contribute to the pathogenesis of fibrosis due to production of pro-inflammatory lipid mediators. This is supported by studies using mice with targeted disruption of 5-LO and the cysteinyl leukotriene receptors 1 and 2, which show reduced bleomycin-induced pulmonary fibrosis (206–208). In contrast, COX-2 deficiency promotes pulmonary fibrosis, and an important role for PGE2 and PGI2 in protecting the lung has emerged (209–212). Consequently cPLA2 is upstream of pathways that produce mediators with positive and negative effects on lung disease. There has been considerable interest in the role of lysophosphatidic acid (LPA) in promoting fibrosis in many organ systems, including the lung (213, 214). LPA levels increase during fibrosis and it exerts a profibrotic effect primarily through the LPA1 receptor. Several pathways for the synthesis of LPA have been described (215, 216). Although there is correlative data that cPLA2 may play a role in the initial production of lysophospholipids that are metabolized to LPA by autotaxin, there is as yet little definite data to support a role for cPLA2 in LPA production (217, 218). Mouse models have been used to investigate the role of cPLA2 in regulating lung cancer. Using the chemical carcinogen urethane to initiate lung cancer, cPLA2 KO mice developed fewer lung tumors than WT mice, and much lower levels of tumor-associated PGs (219). Another study investigated whether the presence of cPLA2 in the tumor microenvironment influenced lung cancer progression and metastasis (220). The injection of mouse lung tumor cells into mouse lungs induced primary tumors of similar size in cPLA2 WT and KO mice. However, KO mice had fewer secondary tumor metastases, less lymph node spread and increased survival. cPLA2 is thought to contribute to tumorigenesis, in part by enhancing the viability and proliferation of endothelial cells leading to formation of vascular networks in the tumor microenvironment (221, 222). This process diminishes the effectiveness of radiotherapy. It has recently been shown that a cPLA2 inhibitor (PLA-695) blocks pro-survival signaling in endothelial cells and renders cancer cells radiosensitive in a simulated tumor microenvironment (223). Combined therapy (PLA-695 and irradiation) also reduces lung tumor growth in mice. Results suggest that cPLA2-mediated production of lysophospholipid metabolites promotes vascular endothelial cell proliferation, migration, and tumor vascularization (224). Therefore a number of lipid mediators, including arachidonic acid, eicosanoids, and lysophospholipids, may contribute to cancer development.

Therefore multiple approaches are required to evaluate the role of these pathways in disease pathogenesis. As an example, a recent study used bone marrow transplantation to specifically block PGE2 synthesis, or EP receptor expression, in peripheral immune cells and showed that PGE2/EP4 signaling promotes development of EAE in mice (238). This approach demonstrates the importance of investigating the role of specific cell types and receptor pathways for understanding how lipid mediators regulate disease processes in vivo.

CONCLUDING REMARKS

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1. Lambeau, G., and M. H. Gelb. 2008. Biochemistry and physiology of mammalian secreted phopholipases A2. Annu. Rev. Biochem. 77: 495–520. 2. Dennis, E. A., J. Cao, Y. H. Hsu, V. Magrioti, and G. Kokotos. 2011. Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chem. Rev. 111: 6130–6185. 3. Murakami, M., Y. Taketomi, Y. Miki, H. Sato, T. Hirabayashi, and K. Yamamoto. 2011. Recent progress in phospholipase A2 research: from cells to animals to humans. Prog. Lipid Res. 50: 152–192. 4. Ghosh, M., D. E. Tucker, S. A. Burchett, and C. C. Leslie. 2006. Properties of the group IV phospholipase A2 family. Prog. Lipid Res. 45: 487–510. 5. Shayman, J. A., R. Kelly, J. Kollmeyer, Y. He, and A. Abe. 2011. Group XV phospholipase A2, a lysosomal phospholipase A2. Prog. Lipid Res. 50: 1–13. 6. Stafforini, D. M. 2009. Biology of platelet-activating factor acetylhydrolase (PAF-AH, lipoprotein associated phospholipase A2). Cardiovasc. Drugs Ther. 23: 73–83. 7. Marathe, G. K., C. Pandit, C. L. Lakshmikanth, V. H. Chaithra, S. P. Jacob, and C. J. D’Souza. 2014. To hydrolyze or not to hydrolyze: the dilemma of platelet-activating factor acetylhydrolase. J. Lipid Res. 55: 1847–1854. 8. Abbott, M. J., T. Tang, and H. S. Sul. 2010. The role of phospholipase A2-derived mediators in obesity. Drug Discov. Today Dis. Mech. 7: e213–e218. 9. Rydel, T. J., J. M. Williams, E. Krieger, F. Moshiri, W. C. Stallings, S. M. Brown, J. C. Pershing, J. P. Purcell, and M. F. Alibhai. 2003. The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad. Biochemistry. 42: 6696–6708. 10. Ghomashchi, F., G. S. Naika, J. G. Bollinger, A. Aloulou, M. Lehr, C. C. Leslie, and M. H. Gelb. 2010. Interfacial kinetic and binding properties of mammalian group IVB phospholipase A2 (cPLA2B) and comparison with other cPLA2 isoforms. J. Biol. Chem. 285: 36100–36111. 11. Murakami, M., Y. Taketomi, Y. Miki, H. Sato, K. Yamamoto, and G. Lambeau. 2014. Emerging roles of secreted phospholipase A2 enzymes: the 3rd edition. Biochimie. 107: 105–113. 12. Ueno, N., Y. Taketomi, K. Yamamoto, T. Hirabayashi, D. Kamei, Y. Kita, T. Shimizu, K. Shinzawa, Y. Tsujimoto, K. Ikeda, et al. 2011. Analysis of two major intracellular phospholipases A2 (PLA2) in mast cells reveals crucial contribution of cytosolic PLA2alpha, not Ca(2+)-independent PLA2beta, to lipid mobilization in proximal mast cells and distal fibroblasts. J. Biol. Chem. 286: 37249–37263. 13. Gil-de-Gómez, L., A. M. Astudillo, C. Guijas, V. Magrioti, G. Kokotos, M. A. Balboa, and J. Balsinde. 2014. Cytosolic group IVA and calcium-independent group VIA phospholipase A2s act on distinct phospholipid pools in zymosan-stimulated mouse peritoneal macrophages. J. Immunol. 192: 752–762. 14. Ohto, T., N. Uozumi, T. Hirabayashi, and T. Shimizu. 2005. Identification of novel cytosolic phospholipase A2s, murine cPLA2delta, epsilon, and zeta, which form a gene cluster with cPLA2beta. J. Biol. Chem. 280: 24576–24583. 15. Bechler, M. E., P. de Figueiredo, and W. J. Brown. 2012. A PLA1-2 punch regulates the Golgi complex. Trends Cell Biol. 22: 116–124. 16. Ha, K. D., B. A. Clarke, and W. J. Brown. 2012. Regulation of the Golgi complex by phospholipid remodeling enzymes. Biochim. Biophys. Acta. 1821: 1078–1088. 17. San Pietro, E., M. Capestrano, E. V. Polishchuk, A. DiPentima, A. Trucco, P. Zizza, S. Mariggiò, T. Pulvirenti, M. Sallese, S. Tete, et al. 2009. Group IV phospholipase A2 controls the formation of inter-cisternal continuities involved in intra-Golgi transport. PLoS Biol. 7: e1000194. 18. Regan-Klapisz, E., V. Krouwer, M. Langelaar-Makkinje, L. Nallan, M. Gelb, H. Gerritsen, A. J. Verkleij, and J. A. Post. 2009. Golgiassociated cPLA2alpha regulates endothelial cell-cell junction integrity by controlling the trafficking of transmembrane junction proteins. Mol. Biol. Cell. 20: 4225–4234. 19. Ghosh, M., R. Loper, M. H. Gelb, and C. C. Leslie. 2006. Identification of the expressed form of human cytosolic phospholipase A2beta (cPLA2beta): cPLA2beta3 is a novel variant localized to mitochondria and early endosomes. J. Biol. Chem. 281: 16615–16624.


Due to the key role of cPLA2 in mediating lipid mediator production and its widespread tissue expression, it has been implicated in regulating homeostatic processes and disease pathogenesis throughout all organ systems. The function of cPLA2 in regulating certain physiological systems, such as female reproduction and hemostasis, is more straightforward and can be attributed to specific types of lipid mediators. However, elucidating the specific mechanism for cPLA2 action in disease processes in many cases is not known and understandably difficult to evaluate. The cPLA2 KO mouse has provided a wealth of information; however, multiple approaches are needed to determine cPLA2 function. Genetic differences in mouse strains lead to difficulties in interpretation of results, and compensatory changes in the conventional KO mouse are a complicating issue. The use of cPLA2 conditional KO mice with ablation in specific tissues would be useful to bypass changes occurring during embryogenesis and development. The availability of specific inhibitors is also important to understand cPLA2 function in animal models in vivo, and in human cells (2). The primary function of cPLA2 is due to its role in mediating production of eicosanoids, however, there is evidence that cPLA2 regulates processes such as membrane trafficking that are not linked to production of eicosanoids, adding an additional complexity to understanding its function (16, 17, 74). Many studies, primarily from comparisons of cPLA2 WT and KO mice, have suggested that cPLA2 is a useful therapeutic target for treating a plethora of diseases. There are a number of issues in considering cPLA2 as a therapeutic target, especially because most of the studies are based on disease models comparing cPLA2 WT and KO mice, which in some cases may not accurately reflect processes contributing to human disease. This is apparent from the differences observed between mice and humans as a consequence of cPLA2 deficiency. Another complication is that cPLA2 is the first regulatory enzyme in the pathway for production of numerous lipid mediators that often have opposing effects, although targeting cPLA2 may be beneficial in some diseases where both 5-LO and COX metabolites contribute to disease, as suggested in studies of asthma and CIA (194, 228). However, the essential role of cPLA2 in human health, particularly for maintenance of the small intestine and female reproduction, is also a consideration for targeting cPLA2 in disease.

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Cytosolic phospholipase A2 and eicosanoids modulate life, death and function of human osteoclasts in vitro.

Regulatory role of cytosolic phospholipase A2 alpha in the induction of CD40 in microglia.

Predominant role of cytosolic phospholipase A2α in dioxin-induced neonatal hydronephrosis in mice.

Human neutrophil cytosolic phospholipase C: partial characterization.

Role of phospholipase d in g-protein coupled receptor function.

Physiological and pathophysiological roles for phospholipase D.

Cytosolic phospholipase A2α promotes pulmonary inflammation and systemic disease during Streptococcus pneumoniae infection.

Ontogeny of cytosolic phospholipase A2 activity in rat brain.

Cytosolic phospholipase A2 modulates TLR2 signaling in synoviocytes.

Ryanodine receptors: physiological function and deregulation in Alzheimer disease.

NO signaling during microglial activation through the lipoxygenase pathway.

Study of the role of cytosolic phospholipase A2 alpha in eicosanoid generation and thymocyte maturation in the thymus.

The step further to understand the role of cytosolic phospholipase A2 alpha and group X secretory phospholipase A2 in allergic inflammation: pilot study.

Phospholipase Cβ interacts with cytosolic partners to regulate cell proliferation.

Glucocorticoids inhibit TNF alpha-induced cytosolic phospholipase A2 activity.

Physiological and molecular functions of the cytosolic peptide:N-glycanase.

The role of cytosolic phospholipase A2 α in amyloid precursor protein induction by amyloid beta1-42 : implication for neurodegeneration.

Purification and characterization of a cytosolic phosphoinositide-phospholipase C (gamma 2-type) from human platelets.

Synthesis and Evaluation of Cytosolic Phospholipase A(2) Activatable Fluorophores for Cancer Imaging.

Cytosolic pH and pancreatic beta-cell function.

Developmental changes in energy metabolism ofSchistosoma mansoni and physiological role of oxygen in maintaining parasite function.

Cytosolic phospholipase A2 protein as a novel therapeutic target for spinal cord injury.

Purification and characterization of cytosolic phospholipase A2 activities from canine myocardium and sheep platelets.

Modeling mitochondrial function and its role in disease.