Phospholipase A2 isozymes in pregnancy and parturition

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Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 87–100

Phospholipase A2 isozymes in pregnancy and parturition M. Lappas*, G.E. Rice Mercy Perinatal Research Center, Mercy Hospital for Women, 126 Clarendon Street, East Melbourne 3002, Australia Received 1 April 2003; accepted 1 April 2003

Abstract Mammalian cells contain several structurally different phospholipase (PLA2) enzymes that exhibit distinct localisation, function and mechanisms of regulation. PLA2 isozymes have been postulated to play significant roles in the parturition process. Both secretory and cytosolic PLA2 isozymes have been identified in human gestational tissues, and there is differential expression of these PLA2 isozymes in human fetal membranes and placenta obtained at preterm and term. The aims of this commentary are: (1) to review recent data concerning the expression, role and regulation of PLA2 isozymes in human gestational tissues; and (2) to present novel data demonstrating the regulation of PLA2 isozymes in human gestational tissues by nuclear factor-kappa B (NF-kB) and peroxisome proliferator-activated receptor (PPAR)-g. r 2003 Elsevier Ltd. All rights reserved. Keywords: Secretory PLA2; Cytosolic PLA2; Pregnancy; Preterm labour; NF-kB; PPAR

1. Introduction During pregnancy, multiple pathways are established for transfer of information between mother and foetus that are essential for foetal growth and development. These pathways involve the formation and release of endocrine, paracrine and autocrine factors. One such pathway generating signals that may be interpreted by extracellular and intracellular sensors involves the metabolism of cell membrane phospholipids. The formation of phospholipid-derived mediators (PDMs) proceeds by a multiple enzyme pathway that is initiated by lipolytic enzymes that includes phospholipases A1, A2, C and D. These PDMs may act either as primary mediators (e.g., lysophosphatides, themselves manifesting biological activity), or may act as precursors for the formation of secondary mediators (such as the eicosanoids prostaglandins and leukotrienes). Prostaglandins of the 2-series are of major importance in parturition and have been implicated in the regulation of many aspects of cell function and in the processes of mammalian pregnancy and parturition. They are recognised as factors that promote myometrial contractions, cervical dilatation and membrane rupture [1]. *Corresponding author. Tel.: +61-3-9270-2557; fax: +61-3-94175406. E-mail address: [email protected] (M. Lappas). 0952-3278/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2003.04.001

Prostaglandins are formed via the action of multiple enzyme pathways, involving phospholipase (PLA2) and cyclooxygenase (COX) isozymes. Through the activity of one or more PLA2 enzymes, non-esterified arachidonic acid is released from membrane phospholipids such as phosphatidylinositol and phosphatidylethanolamine. COX are bifunctional enzymes that catalyses the first two steps in the biosynthesis of prostaglandins from the substrate arachidonic acid (reviewed in [2]). Three PLA2 isozymes have been identified: the intracellular isozyme—cytosolic PLA2 (cPLA2), and the secretory PLA2 (sPLA2) enzymes sPLA2-IIA and sPLA2-V. Their expression in human gestational tissues have been partially characterised and will be discussed in ensuing sections.

2. Phospholipase A2 isozymes PLA2 represents a ubiquitous family of esterases that hydrolyse the sn-2 acyl ester bond of 1,2 diacyl-sn-3 glycerophospholipids, thereby liberating equimolar amounts of 1-acyl lysophosphatide and free fatty acid [3]. The lysophospholipids may serve as precursors in the generation of platelet activating factor (PAF) or may themselves be pro-inflammatory mediators. As arachidonic acid is predominantly found esterified in the sn-2

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position of membrane phospholipids, PLA2 contributes significantly to its liberation. Regulation of arachidonic acid is one of the ratelimiting steps for the synthesis of eicosanoids. In the resting state, arachidonic acid is esterified in membrane glycerophospholipids at the sn-2 position [4]. The concentration of free arachidonic acid within cells is controlled by two mechanisms. Firstly, acyl-transferases are responsible for rapidly reincorporating arachidonic acid into phospholipids, which limits arachidonic acid availability to COX [5]. Secondly, phospholipases are responsible for arachidonic acid release from phospholipid stores in the cell membrane [4]. Mammalian cells contain several structurally different PLA2 enzymes that exhibit distinct localisation, function and mechanisms of regulation. To date, at least 19 enzymes that possess PLA2 activity have been identified in mammals, with four functionally distinct groups. The sPLA2 family, of which 10 isozymes have been identified, consists of low molecular weight, Ca2+ requiring, secretory enzymes that have been implicated in a number of biological functions, including eicosanoid generation, inflammation and apoptosis. The cPLA2 family is represented by 3 enzymes, of which cPLA2a plays an essential role in arachidonic acid metabolism. The sPLA2 and cPLA2 families display different biophysical characteristics and biochemical requirements for optimal enzyme activity that are consistent with their sites of action, that is, extracellular and intracellular, respectively. The calcium independent PLA2s (iPLA2) family consists of 2 enzymes, and is postulated to play a role in membrane phospholipid remodelling. An additional family of PLA2 enzymes are the PAF acetylhydrolase (PAF-AH) family that exhibit unusual substrate specificity towards PAF and/or oxidised phospholipids. This review will present an overview of the current understanding of the properties and functions of the sPLA2, cPLA2 and iPLA2 families, which have been implicated in human parturition.

3. Secretory PLA2 Secretory PLA2s are a large family of low molecular weight (14–19 kDa) extracellularly active enzymes (reviewed in [6]). These enzymes are characterised by six or seven intramolecular disulphide bonds, that give them a rigid tertiary structure that is resistant to heat and acid inactivation but sensitive to inactivation by disulphide reducing agents. sPLA2s display optimal enzymatic activity in the presence of millimolar concentrations of calcium and neutral to alkaline pH. The sPLA2 family of enzymes do not demonstrate any selectivity for the fatty acid present in the sn-2 position of the phospholipid substrate.

At present, at least 10 different types of human sPLA2 isozymes have been identified and classified according to their structural characteristics (for review see [7]), with additional sPLA2s isolated from mouse tissue and animal venoms. All sPLA2 types contain highly conserved amino acid residues and sequences which include: (1) an a-helical N-terminal segment containing the lipophilic residues Leu, Phe and Ile; (2) a Ca2+ binding loop with glycine-rich sequences; (3) an active site and (4) 12 of the 14 cysteine residues are in the same position. The genes for sPLA2-IIA, -IIC, -IID, -IIE, -IIF and V are clustered on the same chromosome locus, and are thus often referred to as the group II subfamily sPLA2s. sPLA2-III and sPLA2-XII share homology with the I/II/ V/X collection of sPLA2s only in the Ca2+-binding loop and catalytic site, thereby representing distinct group III and XII collections, respectively. 3.1. sPLA2-IB Secretory PLA2-IB isozymes were initially identified in pancreatic secretions and snake venom, and associated with digestive functions [8]. Recent studies have identified the expression of these isozymes in lung, gastric mucosa, kidney and spleen [9]. Recent data also suggest a more complex role for this protein as an intracellular signalling molecule (reviewed in [10]). 3.2. sPLA2-IIA The sPLA2-IIA isozyme shares limited overall amino acid sequence (30–40%) and three-dimensional structural homology with sPLA2-IB. The sPLA2-IIA gene is highly conserved between species. Human, rat and porcine platelet enzymes are 79% homologous in overall structure with virtually 100% homology of the catalytic and disulphide pattern [11]. The enzyme is also strongly cationic, with a pI>10. This enzyme also has a unique disulphide bond at residue 50 and an extension of several amino acid residues at the carboxyl terminus. It shows no specificity for the sn-2 fatty acid, but is selective for amino glycerophospholipids (negatively charged sn-3 functional groups). The gene for sPLA2-IIA has been cloned [11,12] and it exists as a single gene located on chromosome 1. The gene spans 4.6 kb and contains 5 exons. Exon 1 contains TATA-like and CAAT putative transcriptional regulation elements, exon 4 contains the stop codon and putative polyadenylation signal AATAAA and exon 2 contains the mature protein N-terminal and preceding 20 amino acid signal sequence [13]. The sPLA2-IIA gene encodes for a 144 amino acid protein that contains a 20 amino acid putative signal sequence [11], which is processed to a mature enzyme during translocation from the cytosolic to the luminal side of the endoplasmic

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reticulum (reviewed in [14]). The mature protein is encoded by a 1.2 kb mRNA transcript, with a calculated molecular weight of 13,939 Da and a deduced amino acid sequence consisting of 124 residues [12]. The sPLA2-IIA isozymes have been isolated from a wide variety of mammalian cells, tissues and inflamed sites (reviewed in [15]). It is stored in the granules [16] of mast cells, platelets, and eosinophils, and also expressed in spleen, thymus, tonsils, liver, heart, muscle, lung and human gestational tissues. Consistent with the presence of a signal sequence, this enzyme is released by a number of cell types. For example, sPLA2-IIA is present in lacrimal gland secretions where it functions as a bactericidal agent [17]. Soluble sPLA2-IIA is also found in high concentrations in extracellular fluids such as rheumatoid arthritic synovial fluid [11], ascitic fluid [18] and serum [19]. Secretory PLA2-IIA is regulated at the translational and transcriptional level (reviewed in [20]). The levels of sPLA2-IIA are altered in response to a number of stimuli. LPS [21], pro-inflammatory cytokines interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-a [22–25], and cyclic adenosine monophosphate (cAMP) elevating agents such as forskolin [26] increase mRNA expression, synthesis and secretion form a variety of cells [21,27] with an accompanying increase in prostaglandin production. The induction of sPLA2-IIA expression can be suppressed by glucocorticoids and anti-inflammatory cytokines (reviewed in [28]). Dexamethasone suppresses cytokine and cAMP mediated expression of sPLA2-IIA protein and mRNA expression in a variety of cells. Anti-inflammatory cytokines greatly decrease the induced expression and secretion of sPLA2IIA with a concordant decrease in prostaglandin production. Consistent with this inducibility, the promoter region of the sPLA2-IIA gene contains TATA and CAAT boxes, as well as several oligonucleotide elements homologous with consensus sequences for binding of transcription factors, including activator protein (AP)-1, nuclear factor-kappa B (NF-kB), signal transducer and activator of transcription (STAT), glucocorticoid response elements (GRE), cAMP response element (CRE), and peroxisome proliferatoractivated receptor (PPAR). Secretory PLA2 is a pluripotent enzyme that plays a role in normal cellular function such as lipid remodelling for cell membrane homeostasis, antimicrobial activity, anticoagulation and cell adhesion (reviewed in [14]). The pathological actions of sPLA2-IIA have been reviewed in detail elsewhere [14,29–31]). Type II PLA2 has been implicated in rheumatoid arthritis, inflammation, septic shock, adult respiratory distress syndrome, inflammatory bowel disease and pancreatitis. Many of these actions have been demonstrated by the presence of increased sPLA2-IIA activity in exudate fluids or serum, or by increased levels of lipid mediators present in tissues.

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3.3. New isoforms of low molecular weight sPLA2 3.3.1. sPLA2-V This enzyme shares 40% homology with the sPLA2IIA enzyme, having similar calcium binding and catalytic sites, however it lacks the prepeptide and Cterminal extension characteristic of sPLA2-IIA. Although human type IIA and type V PLA2 enzymes have distinct expression patterns, these enzymes were hypothesised to be functionally redundant [32]. However, recent data points to distinct biological roles for both enzymes (reviewed in [10]). The type V enzyme was shown to have a significantly greater affinity for phosphatidylcholine containing substrates than sPLA2IIA, making it more likely to hydrolyse extracellular mammalian membranes. 3.3.2. sPLA2-IIC The rat sPLA2-IIC gene encodes for a calciumdependent enzyme that contains a prepeptide. High expression of sPLA2-IIC is seen in the adult rat testes [12], and it is ubiquitously expressed throughout the brain [33]. The human sPLA2-IIC encodes for a non-functional peptide and is believed to be a pseudogene [12]. 3.3.3. sPLA2-X sPLA2-X has 35% homology with sPLA2-IIA, and shares structural similarities with both type IB and type IIA isoforms. Secretory PLA2-X mRNA is expressed in a number of immune cells and tissues, and may therefore be a mediator of pro-inflammatory arachidonic acid release [34]. 3.3.4. sPLA2-IID, sPLA2-IIE and sPLA2-IIF A number of other low molecular weight PLA2 isoforms have been identified and cloned. They are structurally similar to sPLA2-IIA, and are thus classified as members of the type II subgroup. They possess a carboxyl-terminal extension and have 14 cysteine residues, although the type IIF isoform has a unique C-terminus containing an additional cysteine residue [35,36]. To date, very little information is available on the functional roles of these isozymes, however sPLA2IID has been implicated in the pro-inflammatory response [35]. 3.4. Mechanism of action of sPLA2 isozymes The catalytic mechanism of sPLA2 involves the consumption of water in a general base catalysis of the ester bond in the sn-2 position of membrane phospholipids. The lysophospholipid generated is quickly reacylated. The heparin-binding group II enzymes, sPLA2-IIA, -IID, -IIE and -V, are localised intracellularly into the caveolin-rich vesicular domains via binding to the HSPG

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glypican, and internalised into the lumen side of the perinuclear membrane. The arachidonic acid released by these sPLA2 may be efficiently liberated to the adjacent enzymes COX and 5-LOX. The PC-hydrolysing enzymes, sPLA2-X and -V, act on the PC-rich outer plasma membrane to release arachidonic acid. sPLA2-IIF binds to the perturbed plasma membrane microdomain via its unique C-terminal region and releases arachidonic acid. The arachidonic acid released from the plasma membrane may diffuse or be transported across the cytosol to the perinuclear COX and 5-LOX. Evidence suggests that exogenous sPLA2 can induce cellular responses (e.g., fibroblast proliferation or smooth muscle contraction) by binding to high-affinity cell surface receptors, with these effects being independent of phospholipase activity (reviewed in [36]), although soluble receptors that bind sPLA2 have also been identified [37]. The cell surface receptors are plasma membrane glycoproteins with a molecular weight of 180 kD, that bind both types I and II PLA2 [38]. The sPLA2 receptor mRNA is expressed in several tissues including the pancreas, liver, lung, kidney [39], and placenta [40]. When sPLA2 enzymes bind this receptor through residues near their calcium binding domains, the receptor is internalised via clathrin-coated pits. The membrane bound receptor may function to traffic secreted sPLA2 back into the cell for action at intracellular locations, while the soluble form may serve as an inhibitor of sPLA2 function.

4. Cytosolic PLA2 The cPLA2 (also referred to as type IV PLA2) family are a high molecular weight (85 kDa) PLA2s that differs from other cellular phospholipases by their specificity for esterified arachidonic acid in the sn-2 position. Currently, three isozymes have been identified: cPLA2a, cPLA2b, and cPLA2g [41,37]. Although the three cPLA2 isozymes share high homology, they differ markedly in their affinity for arachidonyl substrates. cPLA2a is the best characterised and the most relevant for intracellular signalling, is highly selective for phospholipidic substrates with arachidonic acid at the sn-2 position. Enzymatic activity is optimal in the presence of low concentrations of calcium (nM-mM) and neutral-toalkaline pH. Unlike sPLA2, cPLA2 is sensitive to heatand acid-inactivation, but is resistant to disulphide reducing agents. These three enzymes have conserved catalytic sites containing a catalytic serine. 4.1. Structure of cPLA2 isozymes Human cPLA2a has been cloned [42] and encodes a 3.4 kb mRNA transcript in most cell types and is the product of a single gene. The cPLA2a gene has

been mapped to chromosome 1 [43]. The mature protein encodes a protein of 749 amino acids and a calculated molecular weight of 85 kD, although it migrates as 85–110 kD on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis [42]. The cloned cDNAs of cPLA2b and cPLA2g encode proteins of 1012 and 541 amino acids, respectively. The three cPLA2 isoforms contain two homologous catalytic domains A and B, interspaced with isoformunique sequences. The lipase consensus sequence (GXSGS) is located in the N-terminal region of the A catalytic domain. The amino terminus of cPLA2a and cPLA2b also contain a calcium-dependent ligand binding (CaLB) domain that mediates translocation of cPLA2 from the cytosol to the membrane compartment [44]. In contrast, cPLA2g lacks the CaLB domain and does not require calcium for activation. A serine at position 228 represents the catalytic centre of cPLA2a. 4.2. Expression of cPLA2 isozymes cPLA2a is constitutively expressed by most adult human tissues (reviewed in [41]). Expression is most prominent in brain, lung, kidney, heart, spleen, pancreas, placenta and smooth muscle. The enzyme is also abundant in many different cell types, including monocytes [45] and neutrophils [46], and is expressed during lymphopoiesis but absent from mature B and T cells. cPLA2b is abundantly expressed in the pancreas, brain, heart and liver, and cPLA2g in the skeletal muscle. 4.3. Regulation of cPLA2 isozymes The cPLA2a plays an important role in controlling the production of arachidonic acid, and is subject to complex regulation at the transcriptional, translational, and post-transcriptional level (reviewed in [47]). The 50 region of the cPLA2a gene is TATA-less, suggesting a housekeeping function [48]. The 50 flanking region contains a 27 base pair polypyrimidine sequence responsible for low level constitutive expression. Furthermore, ATTTA motifs in the 30 -untranslated region may indicate further regulation through transcript de-stabilisation. The promoter region for cPLA2a contains several potential binding sites for transcription factors including AP-1, AP-2, NF-kB, GRE and PPAR [48]. Cytosolic PLA2a mRNA and protein levels are altered in response to various stimuli, including IL-1 [49,50], TNF-a and LPS [109] which increase its synthesis, resulting in prolonged arachidonic acid release and eicosanoid production. In contrast to sPLA2-IIA, anti-inflammatory cytokines increase the

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expression of cPLA2a, confirming that these enzymes are regulated independently. Consistent with the presence of a putative glucocorticoid response element in the promoter region of the cPLA2a gene (reviewed in [28]), glucocorticoids are strong suppressors of induced cPLA2a expression [49]. Matrix metalloproteinase (MMP) constituents have also been shown to act as suppressors of cPLA2a [51]. Post-transcriptional regulation of cPLA2a has been reviewed in detail elsewhere [52,41]. Briefly, cPLA2a is regulated by: (i) calcium binding via the CaLB domain which is responsible for the translocation of cPLA2a in the presence of submicromolar Ca2+ concentrations; (ii) phosphorylation of serine at position 505, a process mediated by MAP kinases, and (iii) guanine nucleotidebinding proteins (G proteins), which transduce receptor mediated signals for numerous effector systems (reviewed in [110]). 4.4. Mechanism of action Cytosolic PLA2a activity occurs at two distinct levels, affinity for substrate and catalytic activity (reviewed in [14,41]). Cytosolic PLA2a is translocated from the cytosol to the perinuclear membrane by changes in intracellular calcium, where it associates with membrane phospholipids through the CaLB domain. A single phospholipid molecule binds to the active site, and following enzyme substrate complex formation, the catalytic centre (Ser 228 or Ser 727) of cPLA2a attacks the sn-2 ester bond to form the arachidonyl enzyme intermediate. Thereafter, it is hydrolysed by water to yield free arachidonic acid. Finally, either dissociation of cPLA2a occurs from the membrane or the catalytic cycle is repeated. 4.5. Biological functions of cPLA2 isozymes Cytosolic PLA2a is implicated in a wide variety of cellular responses, most of which are mediated indirectly by the eicosanoids and PAF generated by the action of cPLA2a (reviewed in detail in [14]). Although cPLA2a exhibits lysophospholipase and transacylase activities, their physiological significance is unknown, although lysophospholipase activity may help to maintain cellular homeostasis (reviewed in [53]). The cellular function of both cPLA2b and cPLA2g are poorly understood. Cytosolic PLA2a knockout mice have been a valuable tool in elucidating the role of cPLA2a in normal and pathological physiology (reviewed in [54]). Collectively, these studies suggest that cPLA2a has an important role in stroke, Parkinson’s disease, asthma, normal fertility and generation of eicosanoids from inflammatory cells. Importantly, many other forms of PLA2 could not replace many of the functions of cPLA2a.

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5. Phospholipase A2 isozymes and human labour and delivery 5.1. Secretory PLA2 in human labour and delivery In human gestational tissues, most studies have focused on the presence of sPLA2-IIA, although other PLA2 enzymes have also been identified (reviewed in [55,56]). Much of the data that has previously been published on the role of sPLA2 in human parturition requires considered interpretation. Previous studies have quantified net PLA2 enzymatic activity ex situ where the contribution made by the individual PLA2 isozymes is difficult to establish and dependent on the specificity of inhibitors or inhibitory conditions (e.g. calcium free or reducing environment) utilised. In addition, sPLA2 isozymes have been reported to be strongly cationic proteins (BpI 10) that associate with negatively charged components of the cell membrane and extracellular matrix [57]. High ionic strength or chaotropic media are required to dissociate these PLA2 isozymes from tissue components. Many studies have used low ionic strength homogenisation media for the extraction of PLA2 enzymatic activity. Farrugia et al. [58] demonstrated that only 5–15% of tissue immunoreactive sPLA2-IIA was recovered using low ionic strength media. It is likely that the data obtained using such extraction techniques therefore have significantly underestimated total tissue PLA2 enzymatic activity. The presence of sPLA2-IIA mRNA [59–61] and immunoreactive protein and enzymatic activity [58,62] in placenta, amnion and choriodecidua has been established. No significant labour associated changes in protein content, enzymatic activity or gene expression have been detected in placenta, amnion and choriodecidua [63,64], although a 2- to 3-fold increase was detected in amnion [64]. Type IIA and type V sPLA2 isozymes are more abundantly expressed in placenta than foetal membranes [65]. In placenta, it has been demonstrated that sPLA2-IIA accounts for up to 80% of the total PLA2 enzymatic activity [56]. Consistent with its extracellular site of action, sPLA2-IIA is secreted by human gestational tissues in vitro [66]. Furthermore, sPLA2-IIA activity has also been detected in human myometrium, although no changes were found to occur with advanced gestation or labour [67]. In human gestational tissues, sPLA2-IIA has been immunohistochemically localised to the vascular smooth muscle, endothelial cells, mesenchymal connective cells and trophoblast [68,111]. Immunoreactive sPLA2-IIA has also been identified in maternal plasma during pregnancy at the time of labour, both term and preterm. Rice et al. [69] quantified immunoreactive sPLA2-IIA concentrations in plasma obtained from women at 9–41 weeks of pregnancy and from women in labour. Although no significant changes

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in concentration were detected during gestation, elevated concentrations were associated with labour, both at term and preterm. 5.1.1. Type II PLA2 and preterm labour While the role of specific PLA2 isozymes in the aetiology and progression of various pathologies is well established, only limited information is available concerning their involvement in preterm labour. The concentration of immunoreactive sPLA2-IIA in maternal plasma [69] and amniotic fluid [70] is elevated in women delivering preterm. Bacterial endotoxin has been demonstrated to induce sPLA2-IIA expression [71] and release [66] in human gestational tissues in vitro with a concomitant increase in prostaglandin production. These findings are consistent with the hypothesis that sPLA2-IIA is induced and released in association with bacterial infection-associated preterm labour and that this results in the formation of uterotonic prostaglandins and thus the activation of premature labour. Recent investigations in our laboratory have identified a higher sPLA2-IIA tissue content in preterm not-inlabour group compared to preterm in-labour group in the amnion and choriodecidua [72]. Consistent with the localisation of sPLA2-IIA in secretory granules [16,73] the reduced sPLA2-IIA content in the in-labour group may reflect the exocytotic release of cellular stores of this enzyme during labour. Evidence to support this comes from the localisation of sPLA2-IIA within vascular smooth muscle cells [68], and consistent with its extracellular site of action, it is secreted by placental explants in vitro [66]. This release may contribute to an increased release of arachidonic acid and its subsequent conversion to the well characterised labour-associated increase in uterotonic prostaglandins that facilitate labour and delivery. This is consistent with elevated concentrations of sPLA2-IIA observed in association with preterm labour in peripheral plasma [69,74], and elevations of sPLA2-IIA activity observed in serum and amniotic fluid associated with infection-induced premature labour [70]. 5.1.2. Functions of sPLA2 in human parturition Although sPLA2 is released from human gestational placenta and foetal membranes during pregnancy, its function remains enigmatic. It has been implicated, however, in the release of arachidonic acid for eicosanoid synthesis, removal of cell surface procoagulant phospholipids, and regulation of cell adhesion, migration and apoptosis. 5.1.3. Eicosanoid generation Exogenously added PLA2 [75] or arachidonic acid [76] are known to stimulate prostaglandin synthesis. Thus, a labour-associated increase in sPLA2-IIA secretion from gestational tissues may act in an autocrine,

paracrine or juxtacrine fashion to metabolise cell membrane phospholipids to liberate fatty acid substrates. These substrates are then available for further processing, and may account for the observed labour associated increase in prostaglandins. Therefore, the rate of sPLA2-IIA release and its subsequent extracellular processing represents a significant determinant of phospholipid metabolism and PDM formation in human gestational tissues and the development of inhibitors may be efficacious in the clinical management of human labour. Inhibition of sPLA2 activity in human intrauterine tissues dramatically reduces prostaglandin formation. Recently, it was reported that sPLA2-IIA is released from term placenta and represents the major PLA2 activity recovered in incubation medium [66]. Most significantly, preventing the release of sPLA2-IIA from placenta or inhibiting the activity of this isozyme released, suppressed prostaglandin release by up to 80% in vitro [64,77,78]. These data further support the hypothesis that increased secretion of sPLA2 promotes phospholipid metabolism and eicosanoid production at the time of labour. These data are also consistent with the observed increase in maternal plasma concentrations of immunoreactive sPLA2-IIA at the time of labour [69]. The contribution of sPLA2 to prostaglandin formation in other human gestational tissues (amnion, choriodecidua and myometrium) remains to be determined. While it has been established that sPLA2-IIA may affect cell function via the catabolism of cell membrane phospholipids and the subsequent formation of prostaglandins, sPLA2 isozymes may also influence cell function by at least two other processes, that is, a receptor mediated pathway or through controlling the exposure of aminophospholipids on the cell surface (reviewed in [56]). The presence of a sPLA2 receptor transcript represents another mechanism by which sPLA2 isozymes may mediate their physiological effects in human gestational tissues, i.e., via a phospholipid independent pathway. Using degenerate oligonucleotide primers based on conserved regions of the rabbit and bovine sPLA2-IIA receptors, mRNA transcripts have been amplified from human placental and foetal membrane mRNA [40]. Although there is a paucity of information concerning the function of these receptors, in non-gestational tissues the available data are consistent with the hypothesis that these receptors may be involved in PLA2 induced contraction of smooth muscle, cell proliferation, eicosanoid formation and chemokine cell migration [79–82]. The expression and tissue localisation of these receptors, and their effects on human intrauterine tissues have not been investigated. Future studies are required to elucidate the functional significance of these receptors and their specificity and affinity for sPLA2 isozymes in human gestational tissues.

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5.1.4. Removal of coagulant phospholipids In human gestational tissues, sPLA2 isozymes may play other important roles such as maintaining blood flow to the foetus and regulating trophoblast differentiation required for placental establishment and differentiation through controlling aminophospholipid exposure on the cell surface (for review see [56]). Under normal conditions, the outer surface of the cell is composed of neutral, choline containing phospholipids. This presents a relatively inactive surface to its external environment, such that it is protected against nonspecific association with exogenous proteins, fusion with other cell membranes and recognition by phagocytes. Upon cellular activation and increased calcium concentration, however, there is an increased expression of negatively charged phospholipids (aminophospholipids) on the cell surface. This exposure dramatically alters cellular characteristics as these aminophospholipids are able to act as cell surface binding sites, for example with proteins involved in clot formation. Efficient and localised activation of blood coagulation requires the expression of negatively charged phospholipids on cell surfaces. As sPLA2 isozymes are strongly cationic and contain a cluster of positively charged amino acid residues, they may compete with coagulation factors for the negatively charged cell surface phospholipids (reviewed in [14]). In human gestational tissues, sPLA2IIA is primarily localised to the perivascular and trophoblast regions ([68], Inoue et al., 1997) and may thus play a role in preventing clot formation within the placental vascular bed and, in particular, in the intervillous space. The expression of phospholipids on the cell surface of trophoblast cells, and their role in coagulopathy during pregnancy has not been established. 5.1.5. Regulation of apoptosis Apoptosis, also known as programmed cell death, involves cell shrinkage, nuclear condensation, and endonucleolytic cleavage of DNA into oligonucleosomal fragments, as well as plasma membrane blebbing. The fact that apoptosis is accompanied by plasma membrane blebbing suggests that there are also significant changes in the phospholipids that form a major component of the plasma membrane. Atsumi et al. [83] demonstrated that cells undergoing apoptosis are highly sensitive to exogenous sPLA2-IIA. Specifically, neuron-like cells deprived of nerve growth factor, mast cells deprived of hematopoietic cytokines, and anti-Fas antibody-treated U937 monocytic leukaemia cells, all of which display the classical changes of apoptosis, become sensitive to sPLA2-IIA-mediated membrane glycerophospholipid hydrolysis and liberate arachidonic acid. The fact that three different systems have yielded similar results strongly suggests that apoptotic cells are potential pathophysiological targets

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for extracellular sPLA2-IIA. When these apoptotic cells were cultured in the presence of sPLA2-IIA, release of arachidonic acid increased markedly in a time- and dose-dependent manner. These results suggest that although sPLA2-IIA has little effect on the plasma membranes of intact cells, it recognises and degrades the ‘perturbed’ structure of apoptotic cell membranes. Since incorporation of arachidonic acid into cells decreased by half during apoptosis, increase in free arachidonic acid level may be a reflection of both increased release and decreased incorporation into cells. Other studies have demonstrated that sPLA2 induces apoptosis. In primary cultures, sPLA2-IIA caused marked neuronal cell death [84]. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA2-IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA2-IIA liberated arachidonic acid and generated PGD2 from neurons. Indoxam significantly suppressed not only arachidonic acid release, but also PGD2 generation. Indoxam prevented neurons from sPLA2IIA-induced neuronal cell death, with the neuroprotective effect of indoxam observed even when it was administered after sPLA2-IIA treatment. Activation of sPLA2 has also been observed in apoptosis triggered by agonists such as TNF-a and oxidative stress. For example, Zhao et al. [85] demonstrated a causal association between sPLA2 activation and delayed oxidant-induced cell death, and suggested that Bcl-2 may suppress apoptosis by preventing sPLA2 activation. Short-term exposure of cells to a low steadystate concentration of H2O2 causes no immediate cell death but apoptosis occurs several hours later. The PLA2 inhibitor 4-bromophenacyl bromide diminished both delayed lysosomal rupture and apoptosis while direct micro-injection of sPLA2 induced lysosomal rupture and apoptosis. Furthermore, B-cell leukaemia/ lymphoma 2 (Bcl-2) over-expression prevents oxidantinduced activation of sPLA2, delayed lysosomal destabilisation and apoptosis. This raises the possibility that delayed cell death following oxidant challenge might similarly involve activated PLA2 which, in turn, could progressively destabilise the membranes of organelles, such as lysosomes and mitochondria, either through simple depletion of phospholipid from organellar membranes, or through the accumulation of chaotropic products, such as free fatty acids or lysophosphatides. The resulting rupture of these organelles would then constitute a sort of auto-digestive death spiral culminating in delayed apoptosis. As human gestational tissues undergo apoptosis, it is therefore possible that the arachidonic acid released from apoptotic cells is delivered to neighbouring cells and is then converted into prostanoids. This provides a new insight into the target cells used by sPLA2-IIA to

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provide arachidonic acid at sites of inflammation where both sPLA2-IIA and apoptotic cells accumulate in high concentrations. 5.1.6. PLA2 and cell migration Cellular migration and extracellular matrix (ECM) invasion are critical steps in embryonic implantation, birth, inflammation, wound healing, and cancer metastasis. PLA2s have been reported to stimulate dramatic invasion of an artificial ECM (Matrigel) by NIH 3T3, mouse fibrosarcoma, and sarcoma cells in vitro [86]. There effects may be mediated by ligation and activation of cell-surface sPLA2 receptors. This hypothesis is supported by the observations that catalytically inactivated type I PLA2 was as effective as the active enzyme in stimulating invasion; down regulation of sPLA2 receptor expression inhibits ECM invasion; and treatment with type I PLA2 receptor-antibody produces identical effects. These data are consistent with a receptor-mediated function of PLA2-I and raise the possibility that sPLA2s play important physiological as well as pathological roles via this receptor. With the recent identification of sPLA2 receptors in human gestational tissues [40], the involvement of sPLA2 in cell migration events that attend labour and delivery warrant further investigation (e.g. leukocytic infiltration, activation of ECM remodelling enzymes). 5.2. Cytosolic PLA2 in human labour and delivery In human gestational tissues, cPLA2 has been detected in placenta, amnion, chorion and myometrium [65,87,88]. It is expressed in greater abundance in the foetal membranes than in the placenta [65], accounting for 50–70% of total PLA2 enzymatic activity [56]. In the amnion, cPLA2 activity has been shown to increase with gestational age, being highest at term prior to labour, but the activity actually decreases in this tissue during labour at term and preterm [89]. This decrease in cPLA2 activity during delivery has been attributed to the depletion of this isozyme during labour. As was the case with sPLA2, no changes in cPLA2 activity were found to occur in human myometrium with advanced gestation or labour [67]. Data obtained from cPLA2 knockout mice further support a critical role for PLA2 isozymes in the process of labour and delivery. Uozumi et al. [90] reported that cPLA2 (/) female mice failed to labour at term (18.5– 20.5 days of pregnancy), delivering a few live offspring at 21.5–22.5 days of gestation. Viable neonates, however, could be delivered by Caesarean section. The role of cPLA2 in intrauterine prostaglandin production has not been studied as extensively as sPLA2. In amnion-derived WISH cells, IL-1b induces cPLA2 synthesis and activity [50], thus facilitating the large increase in PGE2 production by these cells. By

contrast, the cPLA2 specific inhibitor, arachidonyl trifluoromethyl ketone, inhibits 70–90% of cytokine stimulated PGE2 production in these cells [50,91]. Thus, cPLA2 is a regulatory point for cytokine-stimulated arachidonate release and prostaglandin production in cells resident in the foetal membranes.

6. Transcriptional regulation of phospholipases in human gestational tissues Two transcription factors, NF-kB and peroxisome proliferator-activated receptor (PPAR)-g, have been demonstrated to positively and negatively regulate the inflammatory response in non-gestational tissues, respectively (reviewed in [92,93]). Furthermore, the promoter region for the sPLA2 and cPLA2a gene contain NF-kB and PPAR-g binding sites. However, not much is known about the transcription factors involved in regulation of PLA2 isozymes in human reproductive tissues. Therefore, studies in our laboratory were undertaken to investigate if NF-kB and PPAR-g also regulate PLA2 isozymes in human placenta, amnion and choriodecidua. 6.1. Nuclear factor-kappa B (NF-kB) NF-kB is a transcription factor that upon activation (e.g., LPS and TNF-a) leads to the coordinated expression of many inflammatory gene products. NFkB subunits p50 and RelA have been demonstrated in amnion-derived WISH cells [5,26], human myometrial cells [94], human cytotrophoblasts [95,96], and human gestational tissues [97]. King et al. [98] have demonstrated the differential expression of mRNA transcripts encoding for NF-kB pathway intermediates throughout the peripartum period. The identification of the NF-kB signalling pathway in human intrauterine tissues suggests that it plays a functional role in these tissues, however there is very little evidence in support of this. The available data have however demonstrated the importance of NF-kB in prostaglandin formation in human myometrial cells and amnion WISH cells [99,94,95], and IL-8 gene expression in amnion cells obtained before and after labour [100]. Following challenge with TNF-a, placental trophoblasts showed activated NF-kB and increased COX-2 gene and protein expression with concurrent increases in the production of PGE2 and PGF2a [95]. Furthermore, we have recently demonstrated that NF-kB also regulates the release of LPS-stimulated IL-6, IL-8 and TNF-a from human placenta, amnion and choriodecidua [97]. As the promoter region of both sPLA2-IIA and cPLA2 contains putative binding sites for NF-kB, we used a human tissue explant model to investigate the involvement of the NF-kB signalling pathway on PLA2

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80 60 40 * 20

(a)

0 LPS

Type II PLA2 Content (ng/mg protein)

1

5

60 40 *

*

(b)

20 0 0

(b) Type II PLA2 Content (ng/mg protein)

0.1

Sulphasalazine Concentration (mM)

(a)

1 5 10 Sulphasalazine Concentration (mM)

30 (c) 20 * 10

*

0 0

1

5

10

Fig. 3. This figure is a representative Western blot gel demonstrating the effect of SASP on cPLA2 tissue protein content from human (a) placenta, (b) amnion and (c) choriodecidua. Explants (n ¼ 6) were incubated for 6 h in the presence of 10 mg/ml LPS. SASP concentrations greater that 5 mM reduced cPLA2 levels from placenta, amnion and choriodecidua.

Sulphasalazine Concentration (mM)

(c)

Fig. 1. This figure illustrates the mean (SEM) levels of type II PLA2 content from (a) placental, (b) amnion and (c) choriodecidual explants incubated in the presence of 0, 0.1, 1, 5 or 10 mM SASP. Explants (n ¼ 9) were incubated for 6 h in the presence of 10 mg/ml LPS. Significant differences, as compared to control, are represented by (Po0:05; ANOVA). Type II PLA content was significantly inhibited 2 by SASP.

Type II PLA2 release (ng/mg protein)

95

10 8

[97]. Following a 6 h incubation, tissues were collected and (i) nuclear protein was immediately extracted to determine NF-kB activity by gel shift assays and (ii) assayed for sPLA2-IIA and cPLA2 tissue content. The incubation media was collected and assayed for sPLA2IIA release. The data we have obtained to date demonstrate that SASP inhibited sPLA2-IIA tissue content (Fig. 1) and release (Fig. 2), and cPLA2 tissue content (Fig. 3).

6 4

*

6.2. Peroxisome proliferator-activated receptor (PPAR)-g

5

PPARs are a subset of the nuclear receptor superfamily that mediate diverse metabolic functions through ligand-dependent transcription. In mammals, there are three subtypes: PPAR-a, PPAR-b/d and PPAR-g. PPARs bind to DNA as heterodimers with retinoid X receptors (RXR). The PPAR/RXR heterodimers play a key role in the control of the expression of genes involved in lipid metabolism. They are activated by several synthetic and natural low molecular weight ligands. Although the identities of the ligands that in vivo regulate their activity remain to be definitely elucidated, 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2) and leukotriene B4 (LTB4) have been demonstrated to stimulate PPAR-g and PPAR-a, respectively.

2 0 0

0.1 1 Sulphasalazine Concentration (mM)

Fig. 2. This figure illustrates the mean (SEM) release of type II PLA2 from placental explants incubated in the presence of 0, 0.1, 1 or 5 mM SASP. Explants (n ¼ 9) were incubated for 6 h in the presence of 10 mg/ ml LPS. Significant differences, as compared to control, are represented by (Po0:05; ANOVA). Compared to control, SASP significantly inhibited placental type II PLA2 release.

isozymes in human gestational tissues. Human placenta, amnion and choriodecidua tissues were incubated in the absence (control) or presence of increasing concentrations of a known NF-kB inhibitor sulphasalazine

ARTICLE IN PRESS PPAR-g plays a pivotal role in regulating the expression of multiple genes involved in immune and inflammatory responses. Ligand activation of PPAR-g has an anti-inflammatory role in many cellular systems by inhibiting the production of components of the phospholipid metabolising pathway such as COX-2 [101,102] and PLA2 [103]. The recent identification of PPAR-g mRNA expression in human gestational tissues [104] suggests that it plays a functional role in these tissues. Eicosanoid products of the phospholipase/cyclooxygenase biosynthetic pathway are potent ligand activators of PPAR-g (reviewed in [105]). Of particular interest is 15d-PGJ2, a member of the cyclopentenone group of prostaglandins that is an end-product of the PGD2 pathway. 15d-PGJ2 is the most potent of the naturally occurring ligands with a high affinity for PPAR-g (low micromolar range). Cyclopentenone prostaglandins affect cell proliferation and inhibit inflammation and virus replication. Recent investigations into the function of 15d-PGJ2 suggest that it acts as a physiological negative feedback regulator of prostaglandin synthesis and thus the inflammatory response. Using a rat model of pleurisy, Gilroy et al. [106] demonstrated that in inflamed pleural cavity exudates, 15d-PGJ2 was produced during the late phase of the inflammatory response where it was involved in the resolution of inflammation. It was proposed that 15d-PGJ2 may contribute to the resolution of acute inflammation through PPAR-g activation and/or inhibition of NF-kB activation. Consistent with the finding that 15d-PGJ2 acts as a negative regulator of prostaglandin biosynthesis, it repressed the synthesis of COX-2 and inhibited the production of PGE2 by macrophages [107], and caused almost complete inhibition of COX-2 expression in TNF-a treated trophoblast cells [108]. Furthermore, Tsubouchi et al. [103] demonstrated that 15d-PGJ2, suppressed IL-1b induced PGE2 synthesis in rheumatoid synovial fibroblasts by inhibiting cPLA2 and COX-2 expression. Thus, although the data to date demonstrate that 15dPGJ2 acts as a negative regulator of prostaglandin biosynthesis, there is a paucity of information on the role of 15d-PGJ2 on PLA2 isozymes from human gestational tissues. Therefore, a human explant system was used to establish the effect of the PPAR-g ligands 15d-PGJ2 on the release of sPLA2-IIA, PGF2a and NFkB DNA binding activity from human placenta. Placental tissues were incubated in the absence (control) or presence of 30 mM 15d-PGJ2. Following a 6 h incubation, tissues were collected and nuclear protein was immediately extracted, and gel shift assays were performed to determine NF-kB DNA binding activity. Incubation media was collected and assayed for sPLA2IIA and PGF2a release.

Type II PLA2 Release (ng/mg protein)

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15 10

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5 0 LPS control

LPS + 15d-PGJ2

(a) 20 PGF2α Release (pmol/mg protein)

96

15 10

*

5 0

(b)

LPS control

LPS + 15d-PGJ2

Fig. 4. This figure illustrates the mean (SEM) levels of (a) type II PLA2 release and (b) PGF2a release from placental explants incubated in the absence (control) and presence of 30 mM 15d-PGJ2. Explants (n ¼ 9) were incubated for 6 h in the presence of 10 mg/ml LPS. Significant differences, as compared to control, are represented by (Po0:05; ANOVA). Both type II PLA and PGF release were 2 2a significantly inhibited by 15d-PGJ2.

The data to date demonstrate that 15d-PGJ2 significantly decreases sPLA2-IIA tissue release from human placenta (Fig. 4a), and this reduction was associated with a decrease in PGF2a production (Fig. 4b). Furthermore, 15d-PGJ2 suppresses NF-kB DNA binding activity in nuclear protein extracts from placenta [97]. Therefore it is possible that the generation of prostaglandins in response to an inflammatory signal may thus act as an autoregulator involved in the maintenance of human parturition.

7. Summary The aim of this review was to highlight recent advances in establishing the role of PLA2 isozymes in human pregnancy and, in particular, labour and delivery. Available evidence confirms the importance

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of cytosolic PLA2 isozymes to the labour-associated increase in eicosanoid formation. In addition to the wellestablished role of this family of lipolytic enzymes in the liberation of arachidonic acid from membrane phospholipids, there is increasing evidence that the secretory forms of PLA2 may also affect cell function via receptormediated pathways. Ligation of sPLA2 receptors has been implicated in aspects of cellular eicosanoid formation, proliferation and migration. The role of such pathways during pregnancy and labour remain to be established. PLA2 isozymes also may affect the outcome of pregnancy by modifying the reactivity of the cell surface. One of the most fundamental and phylogenetically conserved factors that determines how cells interact with their environment is the phospholipid composition of the outer monolayer of the cell membrane. Modifying the structure, composition and/or spatial distribution of phospholipid moieties (e.g. phosphatidylserine) affords a direct and simple mechanism for regulating cellmembrane-dependent events. Secretory, extracellularly active PLA2s may play a role in maintaining the outerleaflet of the cell membrane bilayer in a quiescent, nonreactive state. By this mechanism sPLA2s may regulate cell-membrane-dependent events, including, terminal cellular differentiation, apoptosis, cell-surface assembly of clotting factors, immuno-recognition and association with exogenous proteins and foreign cell membranes (independent of their well-characterised roles in second messenger generation). The significance of such an sPLA2-dependent pathway in human gestational tissue warrants further investigation.

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