Phospholipase A₂

Abstract

Phospholipase A₂s (PLA₂s) are a group of enzymes that hydrolyze the sn-2 position of phospholipids to generate fatty acids and lysophospholipids, which serve as lipid mediators or their precursors. Mammalian genomes encode genes for more than 30 PLA₂s or related enzymes, which are subdivided into several groups on the basis of their structures, enzymatic properties, and evolutional relationships. Among them, the Ca²⁺-dependent cytosolic PLA₂ (cPLA₂), Ca²⁺-independent PLA₂ (iPLA₂), and secreted PLA₂ (sPLA₂) families are regarded as the “big three.” From a general point of view, cPLA₂α (the prototypic cPLA₂) plays a major role in the initiation of arachidonic acid (AA) metabolism, the iPLA₂ family contributes to membrane homeostasis or energy metabolism, and the sPLA₂ family affects various biological events by modulating extracellular phospholipid milieus in response to given microenvironmental cues. In this chapter, we overview current understanding of the biological functions of PLA₂s as revealed by gene-manipulated mice and human diseases.

1 Introduction

Phospholipase A₂ (PLA₂) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate free fatty acids and lysophospholipids. To date, more than 30 enzymes that possess PLA₂ or related activities have been identified in mammals, and these have been subdivided into several groups based on their structures, catalytic mechanisms, localizations. and evolutionary relationships. The cPLA₂ family contains 6 enzymes (cPLA₂α−ζ), which (except for cPLA₂γ) contain an N-terminal C2 domain for Ca²⁺-dependent association with the membrane. The iPLA₂ or patatin-like phospholipase domain-containing lipase (PNPLA) family includes 9 enzymes, some of which act principally on phospholipids and others on neutral lipids such as triglyceride (TG). The sPLA₂ family, in which 10 catalytically active enzymes have been identified, are low molecular weight, extracellular enzymes that require Ca²⁺ of the mM order for optimal enzymatic activity. Because of this diversity, PLA₂ enzymes have been implicated in various biological processes such as lipid mediator production, membrane remodeling, and energy metabolism.

During the past few decades, studies of various PLA₂ transgenic and/or knockout mice as well as human diseases with PLA₂ gene mutations have provided new insights into the emerging biological roles of individual PLA₂s. The functions of individual PLA₂s may not simply reflect changes in lipid mediator signaling, or more particularly eicosanoid signaling, but may also be attributable to hydrolysis of one or a combination of various target membrane lipids. Herein, we focus on the pathophysiology of various PLA₂s as revealed by information from transgenic or knockout mice, as well as human diseases.

2 The cPLA₂ Family

2.1 General Aspects of cPLA₂s

Enzymes belonging to the cPLA₂ family are characterized by the presence of a C2 domain in their N-terminal region, with the exception of cPLA₂γ, which lacks this domain. Evolutionarily, the cPLA₂ family emerged from the ancestral iPLA₂ family at the branching point of vertebrates, correlating with the development of eicosanoid signaling cascades. cPLA₂α is no doubt the best-known PLA₂, with a major role in releasing arachidonic acid (AA), a precursor of eicosanoids (prostaglandins, PGs, and leukotrienes, LTs), from cellular membrane phospholipids.

2.2 cPLA₂α

cPLA₂α, also known as group IVA PLA₂, is localized in the cytosol of resting cells, and in response to an increase in cytosolic Ca²⁺ levels after cell activation, it translocates to the perinuclear or, more specifically, the Golgi membranes to encounter its preferred substrate, AA-containing phosphatidylcholine (PC). Ceramide-1-phosphate or phosphoinositide-4,5-bisphosphate (PIP₂) enhances the membrane interaction of cPLA₂α. Mitogen-activated protein kinases phosphorylate Ser⁵⁰⁵ on cPLA₂α, leading to its activation. The AA released by cPLA₂α is then converted to PGs and LTs by cyclooxygenases and 5-lipoxygenase, respectively. As an example, the cPLA₂α activation mechanism in mast cells (MCs) is shown in Fig. 2.1.

Fig. 2.1

Activation of cytosolic PLA₂ (cPLA₂)α in mast cells (MCs). In response to Ca²⁺ influx following FcεRI activation with IgE and cognate antigen, cPLA₂α translocates from the cytosol to the perinuclear membrane and is phosphorylated by mitogen-activated protein kinase (MAPK) for optimal activation. The arachidonic acid (AA) released from membrane phospholipids by cPLA₂α is then converted to PGD₂ by the sequential action of cyclooxygenase (COX)-1 (or COX-2 when the cells are primed by particular stimuli) and hematopoietic PGD₂ synthase (H-PGDS) to PGD₂ or by the sequential action of 5-lipoxygenase (5-LOX) incorporation with 5-LOX-activating protein (FLAP) and LTC₄ synthase (LTC4S) to LTC₄

Mice lacking cPLA₂α (Pla2g4a ^−/−) exhibit a number of striking phenotypes that can be explained by defects in pathways involving PGs, LTs, or platelet-activating factor (PAF). For instance, Pla2g4a ^−/− mice are protected from asthma, acute respiratory distress syndrome, and pulmonary fibrosis, which can be explained by marked reductions of detrimental lipid mediators such as LTs and PAF [1–3]. Pla2g4a ^−/− mice or wild-type mice treated with a cPLA₂α inhibitor are less susceptible to experimental autoimmune encephalomyelitis or collagen-induced arthritis [4, 5], are protected from brain injuries caused by ischemia or Aβ amyloid [6, 7], and have reduced incidences of intestinal and lung cancer [8, 9], all of which can be attributed to reduced PGE₂ signaling. Consistent with the protective role of PGE₂ in the gastrointestinal mucosa, the intestinal epithelium of Pla2g4a ^−/− mice has numerous small ulcerative lesions [9]. The impairment of female fertility observed in Pla2g4a ^−/− mice suggests that cPLA₂α has an important role in parturition and implantation by providing PGF_2α and PGE₂ [10, 7]. Because of reduced production of thromboxane A₂ (TXA₂) by platelets, Pla2g4a ^−/− mice are protected from thromboembolism and have prolonged bleeding times [11]. Furthermore, ablation or knockdown of cPLA₂α ameliorates metabolic disorders including atherosclerosis, hepatic fibrosis, insulin resistance, and adipose tissue inflammation [12–14]. In all cases, the levels of lipid mediators responsible for the corresponding pathophysiological events are markedly reduced in Pla2g4a ^−/− mice relative to wild-type mice. In humans, an inherited PLA2G4A mutation is linked to impaired eicosanoid biosynthesis, ulceration of the small intestine, and platelet dysfunction [15].

2.3 Other cPLA₂s

cPLA₂β, δ, ε, and ζ (group IVB, IVD, IVE, and IVF PLA₂s) map to the same chromosomal locus and are therefore evolutionally more related [16]. cPLA₂β is a dual PLA₁/PLA₂ enzyme, although cPLA₂δ has a robust PLA₁ activity in preference to PLA₂ activity. cPLA₂γ (group IVC PLA₂) is unique in that it lacks the C2 domain and displays lysophospholipase and transacylase activities in addition to PLA₂ activity [17]. The in vivo functions of these cPLA₂ isoforms are entirely unknown because knockout studies have yet to be performed. A recent study has shown that cPLA₂ε may drive recycling through clathrin-independent endocytosis [18].

3 The iPLA₂/PNPLA Family

3.1 General Aspects of iPLA₂s

The human genome encodes nine iPLA₂/PNPLA enzymes, which share a protein motif known as the “patatin domain” with an unusual folding topology that differs from classical lipases (Fig. 2.2). The cPLA₂ and iPLA₂ families seem to have evolved from a common ancestral gene, as their catalytic domains are commonly characterized by a three-layer α/β/α architecture employing a conserved Ser/Asp catalytic dyad instead of the classical catalytic triad [19]. iPLA₂/PNPLA enzymes are found in virtually all eukaryotes including yeast, plants, invertebrates, and vertebrates, suggesting that they possess fundamental roles in cellular lipid metabolism conserved in the eukaryote kingdom. The designation “PNPLA” appears to be more appropriate than “iPLA₂,” as some of the isoforms have enzymatic activities apparently distinct from bona fide PLA₂ activity. For instance, iPLA₂ζ/PNPLA2 functions as a major TG lipase in adipose and many other tissues, whereas iPLA₂ε/PNPLA3 may act mainly as an acyltransferase or transacylase for accumulation of TG, particularly in the liver [20]. Here, we focus on two particular iPLA₂s, iPLA₂β/PNPLA9 and iPLA₂γ/PNPLA8, which have robust PLA₂ activity.

Fig. 2.2

Evolutional relationship between the cPLA₂ and iPLA₂ families. The iPLA₂ family is present in all eukaryotes, whereas the cPLA₂ family emerged from the iPLA₂ family at the stage of divergence of vertebrates

3.2 iPLA₂β

iPLA₂β (PNPLA9 or group VIA PLA₂), the best characterized iPLA₂, has long been thought to be involved in homeostatic phospholipid remodeling through deacylation of phospholipids in the Lands’ cycle. Indeed, the composition of phospholipids, particularly those containing docosahexaenoic acid (DHA), is noticeably altered in the brain (but not other tissues) of mice lacking iPLA₂β (Pla2g6 ^−/−) [21]. Notably, human PLA2G6 mutations are associated with neurodegenerative diseases such as infantile neuroaxonal dystrophy (INAD), neurodegeneration with brain iron accumulation (NBIA), and Schindler’s disease, which share the distinctive pathological feature of axonal degeneration with spheroid bodies in the nervous system [22]. Similar neurodegenerative phenotypes are also evident in Pla2g6 ^−/− mice or Pla2g6 mutant mice (Pla2g6-inad, in which the Pla2g6 gene harbors a point mutation), which show motor dysfunction caused by widespread degeneration of axons and synapses, accompanied by the appearance of spheroids and vacuoles [23, 24]. iPLA₂β has also been proposed to have more diverse signaling roles. These Pla2g6 ^−/− phenotypes include male infertility [25], defective opening of store-operated Ca²⁺ entry, probably caused by reduced production of lysophosphatidylcholine (LPC) [26], impaired insulin secretion by pancreatic β-cells [27], reduced apoptosis [28], decreased eicosanoid generation in vascular cells [29], and protection from ovarian cancer, possibly through reduction of lysophosphatidic acid (LPA) [30]. In most cases, however, the iPLA₂β-driven lipid metabolic processes underlying these events are poorly characterized.

3.3 iPLA₂γ

iPLA₂γ, also known as PNPLA8 or group VIB PLA₂, is localized to mitochondria or peroxisomes and displays PLA₂ or PLA₁ activity depending on the substrates involved [31]. Mice null for iPLA₂γ (Pnpla8 ^−/−) exhibit bioenergetic dysfunctional phenotypes, including growth retardation, cold intolerance, reduced exercise endurance, increased mortality from cardiac stress, and abnormal mitochondrial function with an altered cardiolipin composition [32]. Furthermore, Pnpla8 ^−/− mice are resistant to diet-induced obesity, fatty liver, and hyperlipidemia [33, 34]. These mice also display lipodystrophy, impaired glucose-stimulated insulin secretion, and decreased mitochondrial β-oxidation. Myocardium-specific Pnpla8-transgenic mice show a dramatic reduction of myocardial phospholipid mass, marked accumulation of TG, impaired mitochondrial function, and hemodynamic dysfunction [35]. Thus, iPLA₂γ appears to be crucial for maintaining efficient bioenergetic mitochondrial function by tailoring mitochondrial lipid metabolism. However, considering that defective β-oxidation usually leads to increased fat accumulation in peripheral tissues, the protective effect of iPLA₂γ ablation against diet-induced metabolic disorders might involve an as yet unknown mechanism. Pnpla8 ^−/− mice also display a profound alteration in hippocampal mitochondrial homeostasis, leading to cognitive dysfunction [36]. The Pnpla8 ^−/− hippocampus has an increased level of cardiolipin and a decrease of plasmalogen, implying a function of iPLA₂γ in remodeling of these phospholipids. Overall, the neurological abnormalities in Pnpla8 ^−/− mice are reminiscent of features in patients with Barth syndrome, a disease caused by disturbed cardiolipin metabolism [37].

4 The sPLA₂ Family

4.1 General Aspects of sPLA₂s

More than one third of the PLA₂ enzymes belong to the sPLA₂ family, which contains ten catalytically active isoforms (IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA). Individual sPLA₂s exhibit unique tissue and cellular localizations and enzymatic properties, suggesting their distinct pathophysiological roles. Classical group I/II/V/X sPLA₂s are closely related, 14- to 19-kDa secreted enzymes with a highly conserved Ca²⁺-binding loop and a His/Asp catalytic dyad as well as conserved disulfide bonds, whereas group III and XII sPLA₂s are atypical and classified into distinct classes. As sPLA₂s are secreted, their target membranes should reside in the extracellular spaces. Individual sPLA₂s contribute to various biological events through production of lipid mediators, promotion of membrane remodeling, modification of extracellular noncellular lipid components such as surfactant, microparticles, and lipoproteins, or degradation of foreign phospholipids such as those originating from microbes and dietary components. Here we overview the pathophysiological functions of several classical sPLA₂s (IB, IIA, IID, IIE, V, X) and an atypical sPLA₂ (group III), as revealed by their transgenic overexpression or gene targeting in mice. Several examples of these sPLA₂-mediated lipid networks are illustrated in Fig. 2.3.

Fig. 2.3

Several examples of sPLA₂-driven lipid networks. (a) sPLA₂-V in M2 macrophages facilitates the Th2 response and that in airway epithelial cells degrades lung surfactant. sPLA₂-X from the airway epithelium acts on eosinophils to augment LTC₄ generation. Accordingly, the two sPLA₂s independently promote asthma. (b) sPLA₂-III released from immature MCs acts on fibroblasts to promote L-PGDS-dependent generation of PGD₂, which in turn acts on the PGD₂ receptor DP1 on MCs to promote MC maturation. Accordingly, PLA2G3 facilitates MC-dependent anaphylaxis. Activation of cPLA₂α in mature MCs is highlighted in Fig. 2.1. (c) In lymph nodes, sPLA₂-IID in DCs hydrolyzes PE to release DHA, which is then converted to the pro-resolving lipid mediator resolving D1 (RvD1) to sequester Th1 immunity. Accordingly, sPLA₂-IID ameliorates Th1-dependent contact dermatitis

4.2 sPLA₂-IB

sPLA₂-IB, often called “pancreatic sPLA₂,” is abundantly expressed in the pancreas. After secretion into the duodenal lumen, an N-terminal heptapeptide of the inactive zymogen is cleaved by trypsin to yield an active enzyme. The main role of this enzyme is digestion of dietary and biliary phospholipids. Gene disruption of sPLA₂-IB (Pla2g1b ^−/−) results in decreased phospholipid digestion and absorption in the gut [38]. The reduced intestinal production of LPC, which is a causal factor for hepatic insulin resistance, results in protection from diet-induced obesity, glucose intolerance, hyperlipidemia, and atherosclerosis in Pla2g1b ^−/− mice [39–41].

4.3 sPLA₂-IIA

sPLA₂-IIA is the only isozyme detectable in the circulation, particularly under pathological conditions. It is often referred to as “inflammatory sPLA₂” as its levels in sera or inflammatory exudates correlate with the severity of inflammatory diseases, and it is robustly induced by pro-inflammatory stimuli in various cells [42]. However, the precise role of sPLA₂-IIA in inflammation remains debatable, as a natural mutation in its gene (Pla2g2a) in C57BL/6 and 129Sv mice [43] prevents adequate evaluation of its functions by gene targeting. So far, therefore, most of the in vivo functions of sPLA₂-IIA have currently been addressed mainly using Pla2g2a-transgenic mice.

Pla2g2a-transgenic mice have skin abnormalities manifested by hair loss and epidermal hyperplasia [44] and by increased carcinogen-induced skin cancer [45]. In line with clinical evidence that the serum level of sPLA₂-IIA correlates with cardiovascular diseases [46], Pla2g2a-transgenic mice develop advanced atherosclerotic lesions [47]. Given that atherosclerosis represents chronic inflammation in the aorta, sPLA₂-IIA can be regarded as a pro-inflammatory enzyme in atherosclerosis. The most probable physiological role of sPLA₂-IIA is degradation of bacterial membranes, thereby providing a first line of antimicrobial defense [48]. sPLA₂-IIA is capable of hydrolyzing phosphatidylethanolamine (PE) and phosphatidylglycerol in marked preference to PC, which can account for the preferential action of this enzyme on bacteria rather than on mammalian cells. Accordingly, Pla2g2a-transgenic mice or wild-type mice treated with sPLA₂-IIA are resistant to pneumonia and sepsis following bacterial infection [49]. For this reason, sPLA₂-IIA is often referred to as a “bactericidal sPLA₂.”

Mouse strains with natural disruption of the Pla2g2a gene (see foregoing) are more sensitive to intestinal tumorigenesis [50]. Transgenic transfer of the Pla2g2a gene into these strains reduces the incidence of intestinal polyposis [51]. Thus, sPLA₂-IIA appears to have an antitumorigenic role in the gastrointestinal tract. Presumably, bactericidal sPLA₂-IIA may affect the gastrointestinal microflora, thereby influencing tumor development. On the other hand, sPLA₂-IIA expression is positively correlated with the malignancy of prostate cancer [52], revealing distinct impacts of sPLA₂-IIA on different types of cancer. Recently, the mutated Pla2g2a allele in the C57BL/6 mouse strain was delivered into the BALB/c mouse strain to produce Pla2g2a ^−/− BALB/c mice. Autoantibody-induced arthritis is attenuated in these Pla2g2a ^−/− mice relative to Pla2g2a-sufficent mice, whereas it is conversely aggravated in Pla2g2a-transgenic mice [53]. This study has provided the first compelling evidence for the pro-inflammatory role of sPLA₂-IIA. Recently, it has been shown that sPLA₂-IIA targets phospholipids in extracellular mitochondria, and thereby amplifies inflammation by producing eicosanoids as well as mitochondrial DNA, a kind of danger-associated molecular pattern (DAMP) [54].

4.4 sPLA₂-IID

sPLA₂-IID is structurally most related to sPLA₂-IIA and is expressed preferentially in dendritic cells (DCs) in secondary lymphoid organs [55], suggesting its immunoregulatory role. In a model of Th1-dependent contact dermatitis, resolution of inflammation is compromised in skin and lymph nodes of mice lacking sPLA₂-IID (Pla2g2d ^−/−) [55]. sPLA₂-IID in regional lymph nodes mobilizes a pool of polyunsaturated fatty acids that can be metabolized to pro-resolving lipid mediators such as DHA-derived resolvin D1, which reduces Th1 cytokine production and DC activation. sPLA₂-IID preferentially hydrolyzes DHA-containing PE in lymph node membranes. In accordance with its antiinflammatory role, sPLA₂-IID expression in DCs is downregulated after cell activation. Furthermore, administration of sPLA₂-IID-Fc protein attenuates autoimmune diseases in mice [56]. Together, the existing data suggest that sPLA₂-IID is a “resolving sPLA₂” that ameliorates inflammation by mobilizing DHA-derived pro-resolving lipid mediators.

4.5 sPLA₂-IIE

Similar to sPLA₂-IID, sPLA₂-IIE is structurally most homologous to sPLA₂-IIA. Expression of sPLA₂-IIE is markedly induced in adipocytes during adipogenesis in vitro and after high-fat feeding in vivo. Mice deficient in sPLA₂-IIE (Pla2g2e ^−/−) are modestly protected from diet-induced obesity, fatty liver, and hyperlipidemia [57]. sPLA₂-IIE preferentially hydrolyzes minor lipoprotein phospholipids, phosphatidylserine (PS), and PE, with no apparent fatty acid selectivity. As such, sPLA₂-IIE alters lipid composition in lipoproteins, thereby affecting fat deposition in adipose tissue and liver. Thus, sPLA₂-IIE is a “metabolic sPLA₂” that controls systemic metabolic states by modulating lipoprotein phospholipids. These findings shed light on the importance of the minor lipoprotein phospholipids (PS and PE) in metabolic regulation.

4.6 sPLA₂-V

Because sPLA₂-V is able to hydrolyze PC more efficiently than sPLA₂-IIA, most investigators in this research field have been interested in the potential roles of this enzyme in inflammation in the context of AA metabolism. Indeed, zymosan-induced peritonitis or lipopolysaccharide (LPS)-induced air pouch inflammation is partially attenuated in mice lacking sPLA₂-V (Pla2g5 ^−/−) [58, 59]. sPLA₂-V is highly expressed in the myocardium, and Pla2g5 ^−/− mice exhibit a markedly decreased infarct size in a myocardial ischemia and reperfusion model [60]. sPLA₂-V is expressed in bronchial epithelial cells and alveolar macrophages, and Pla2g5 ^−/− mice are protected from airway disorders such as antigen-induced asthma and LPS-induced respiratory distress syndrome [61, 62]. Moreover, in keeping with the view that hydrolysis of phospholipids in low density lipoprotein (LDL) by sPLA₂-V can promote foam cell formation by macrophages in vitro [63], Ldlr ^−/− mice transplanted with Pla2g5 ^−/− bone marrow cells are partially protected from atherosclerosis [64]. Although most of these studies support the offensive roles of sPLA₂-V, the underlying mechanisms by which sPLA₂-V regulates each of these pathologies have remained controversial. Of note, sPLA₂-V prefers phospholipids bearing fatty acids with a lower degree of unsaturation (e.g., oleate and linoleate) to those containing highly polyunsaturated fatty acids (e.g., AA and DHA), making it unclear whether sPLA₂-V indeed mobilizes AA-derived eicosanoids in vivo. Because increased inflammation is generally accompanied by cPLA₂α activation, the observed changes in eicosanoid levels in Pla2g5 ^−/− mice might simply reflect disease-associated changes in cPLA₂α activation, rather than hydrolytic liberation of AA by sPLA₂-V. Indeed, transgenic overexpression of sPLA₂-V leads to respiratory distress and neonatal death without alterations in pulmonary eicosanoid levels [65]. This phenotype has been ascribed to aberrant hydrolysis of surfactant phospholipids (dipalmitoyl-PC) and is apparently eicosanoid independent.

The roles of sPLA₂-V in inflammation have been proven to be more complex. Although sPLA₂-V was thought to be induced by pro-inflammatory stimuli (as in the case of sPLA₂-IIA), it has recently become obvious that its expression is induced by the Th2 cytokines IL-4 and IL-13, rather than proinflammatory stimuli including LPS, zymosan, and Th1 cytokines, which decrease sPLA₂-V expression [57, 66]. sPLA₂-V is expressed in IL-4-driven M2 macrophages and Th2 cells, which facilitate Th2-type inflammation while attenuating Th1 or Th17 immunity. Importantly, Th2 responses, as monitored by IL-4 expression and IgE production, are greatly reduced in Pla2g5 ^−/− mice, thus underscoring the reduced asthmatic phenotype from the lack of sPLA₂-V. Thus, sPLA₂-V appears to function in at least two regulatory steps in asthma: (1) in antigen-presenting cells to regulate antigen processing and thereby the Th2 response, and (2) in airway-resident cells to promote airway inflammation that may involve surfactant degradation. Pla2g5 ^−/− mice are more susceptible to Candida infection (Th1 immunity) and arthritis (Th17 immunity) [53, 67], which could also be partly explained by the ability of sPLA₂-V to promote Th2 immunity (and therefore to suppress Th1/Th17 immunity).

The function of sPLA₂-V as a “Th2-prone sPLA₂” also influences obesity, as Th2 or M2 response dampens adipose tissue inflammation. In obesity, sPLA₂-V is induced in hypertrophic adipocytes [57]. When fed a high-fat diet, Pla2g5 ^−/− mice display hyperlipidemia with higher plasma levels of lipid-rich LDL and increased obesity, fatty liver, and insulin resistance. sPLA₂-V has a protective function against metabolic disorders by hydrolyzing and thereby normalizing PC in LDL and by tipping the immune balance toward an Th2/M2 state that counteracts adipose tissue inflammation. Mechanistically, sPLA₂-V-driven oleate and linoleate from PC in LDL dampen M1 macrophage polarization by saturated fatty acids (e.g., palmitate), probably through attenuation of endoplasmic reticulum stress. Clinically also, sPLA₂-V expression in human visceral adipose tissue inversely correlates with plasma LDL levels. These studies underscore the physiological relevance of lipoprotein hydrolysis by sPLA₂s, highlight two adipocyte-driven “metabolic sPLA₂s” (sPLA₂-IIE and sPLA₂-V) as integrated regulators of immune and metabolic responses, and bring about a paradigm shift toward a better understanding of the roles of the sPLA₂ family as a metabolic coordinator.

4.7 sPLA₂-X

As in the case of sPLA₂-IB, sPLA₂-X is synthesized as a zymogen, and removal of an N-terminal pro-peptide produces an active mature enzyme [68]. Among the sPLA₂s, sPLA₂-X has the highest binding affinity for PC and thus exhibits the most potent ability to hydrolyze plasma membrane phospholipids in intact cells [69]. Because of this property, many investigators have speculated that sPLA₂-X has a major role in inflammation. In line with this scenario, mice lacking sPLA₂-X (Pla2g10 ^−/−) are refractory to antigen-induced asthma, with markedly reduced infiltration of eosinophils and lymphocytes, attenuated goblet cell hyperplasia and smooth muscle layer thickening, and decreased levels of Th2 cytokines and pro-asthmatic eicosanoids [70]. The attenuated asthmatic responses in Pla2g10 ^−/− mice are fully restored by knock-in of human sPLA₂-X, and treatment of the knock-in mice with an inhibitor specific for human sPLA₂-X suppresses airway inflammation [71]. Mechanistically, sPLA₂-X secreted from the airway epithelium may act on infiltrating eosinophils to augment LT production in a process involving LPC-dependent activation of cPLA₂α [72]. Pla2g10 ^−/− mice are also protected from the early phase of influenza infection [73], further highlighting the role of this enzyme in the airway. Moreover, sPLA₂-X is one of the major sPLA₂ isoforms detected in the airway of patients with asthma [74], thus directing attention to sPLA₂-X as a novel therapeutic target for asthma. In contrast to sPLA₂-V, however, sPLA₂-X does not influence the Th2 response itself, as antigen-sensitized Pla2g10 ^−/− mice have normal IgE and IL-4 levels.

Several phenotypes have been reported for Pla2g10 ^−/− mice, but the data are controversial. These phenotypes include protection from myocardial infarction or aneurysm [75, 76], exacerbation or attenuation of atherosclerosis [77, 78], increased or decreased adiposity [79, 80], altered macrophage responses [81], and lower response to peripheral pain [79]. In some of these studies, experiments were performed under the assumption that sPLA₂-X is expressed in immune cells such as neutrophils and macrophages. However, the expression of sPLA₂-X in such immune cells is very low or almost undetectable [75, 79], raising a question as to the physiological relevance of studies involving adoptive transfer of Pla2g10 ^−/− bone marrow-derived cells. Rather, the possibility that paracrine sPLA₂-X may alter the properties of inflammatory cells should be taken into account. Because sPLA₂-X is abundantly expressed in the gut epithelium, it is likely that the decreased digestion and absorption of dietary and biliary phospholipids are eventually linked to reduced fat accumulation in adipose tissue of Pla2g10 ^−/− mice [79], a situation similar to Pla2g1b ^−/− mice (see foregoing).

sPLA₂-X is most abundantly expressed in the testis, where it is stored in acrosomes (secretory granules) in the head of sperm cells [82]. Pla2g10 ^−/− spermatozoa display an impaired acrosome reaction and low fertility despite showing a normal number and motility [83, 82]. Thus, sPLA₂-X plays a specific role in sperm activation, boosting the acrosome reaction by producing LPC from sperm membranes in a paracrine or autocrine manner. Last, a striking skin phenotype characterized by alopecia in Pla2g10-transgenic mice points to a unique role of sPLA₂-X in hair homeostasis [84]. Although grossly the coat hairs of Pla2g10 ^−/− mice appear normal, they have ultrastructural abnormalities including a hypoplasic outer root sheath and reduced melanin granules in their hair follicles.

4.8 sPLA₂-III

sPLA₂-III, an atypical sPLA₂, more closely resembles bee venom sPLA₂ rather than other mammalian sPLA₂s [85]. Transgenic overexpression of sPLA₂-III in mice with an ApoE ^−/− background results in increased atherosclerosis from accelerated LDL hydrolysis and increased TXA₂ synthesis [86]. These mice also develop systemic inflammation as they age because of elevated eicosanoid formation [87]. Thus, beyond the overexpression strategy, sPLA₂-III has a pro-inflammatory potential.

sPLA₂-III is highly expressed in the epididymal epithelium. Studies using mice lacking sPLA₂-III (Pla2g3 ^−/−) have revealed that epididymal sPLA₂-III acts on immature sperm cells passing through the duct in a paracrine manner to regulate phospholipid remodeling. During epididymal transit of spermatozoa, PC in the sperm membrane undergoes a dramatic shift in its acyl groups from oleate, linoleate, and AA to docosapentaenoic acid (DPA) and DHA, and the increased proportion of DPA/DHA consequently contributes to increased sperm membrane fluidity and thereby sperm motility. In Pla2g3 ^−/− mice, this sperm membrane remodeling is severely compromised. Accordingly, spermatozoa from Pla2g3 ^−/− mice have a low DPA/DHA content, aberrant acrosomes and flagella with an abnormal axoneme configuration, and display hypomotility and reduced fertility [88]. Thus, the two “reproductive sPLA₂s” (sPLA₂-III and sPLA₂-X), which are expressed in different locations within the male genital organs, exert nonredundant but interrelated functions in two major steps of male fertility, the former during sperm maturation in the epididymis and the latter during capacitation and the acrosome reaction, likely after ejaculation in the uterus and oviduct.

Microenvironmental alterations in MC phenotypes affect susceptibility to allergy, yet the mechanisms underlying the proper maturation of MCs toward an allergy-sensitive phenotype were poorly understood. sPLA₂-III is stored in and released from MC granules, and MC-associated passive and active anaphylactic responses are markedly attenuated in Pla2g3 ^−/− mice, whereas they are augmented in Pla2g3-transgenic mice [89]. Tissue MCs in Pla2g3 ^−/− mice are immature and are therefore resistant to IgE-dependent and -independent activation. Similar MC abnormalities are also seen in mice lacking lipocalin-type prostaglandin D₂ (PGD₂) synthase (L-PGDS) or those lacking the PGD₂ receptor DP1, suggesting their functional relationship. Indeed, genetic or pharmacological ablation of DP1 in MCs or L-PGDS in fibroblasts phenocopies that of sPLA₂-III in MCs in terms of defective MC maturation and anaphylaxis. Taken together, the data suggest that sPLA₂-III secreted from immature MCs is coupled with fibroblastic L-PGDS to provide microenvironmental PGD₂, which in turn promotes MC maturation via DP1. The sPLA₂-III/L-PGDS/DP1 paracrine loop is a novel lipid-orchestrated mechanism, providing a missing microenvironmental cue that underlies the proper maturation of MCs.

5 Concluding Remarks

With the growing list of knockout and transgenic mouse strains for PLA₂s, much progress has been made in delineating the physiological functions of each PLA₂. It is now becoming obvious that cPLA₂α is a central regulator of AA metabolism, supported by the view that the molecular evolution of cPLA₂α coincided with that of eicosanoid receptors when vertebrates evolved, that the iPLA₂ family is a fundamental regulator of membrane homeostasis and energy metabolism, and that individual sPLA₂s exert unique and tissue-specific biological functions by acting on extracellular phospholipids, which include adjacent cell membranes, noncellular lipid components, and foreign phospholipids such as those in microbes and the diet. The diversity of target phospholipids and products may explain why each PLA₂ family contains many isoforms. Further advances in this research field and their integration for therapeutic applications are likely to benefit from improved, time- and space-resolved lipidomics technology that will allow monitoring of individual PLA₂s and their associated forms of lipid metabolism within specific tissue niches. Hopefully, the next decade will yield a comprehensive map of the PLA₂-driven lipid networks, which will allow the therapeutic application of inhibitors for some PLA₂s central to human diseases.