Cytosolic PLA2α Research Articles

The IRE1α Inhibitor KIRA6 Blocks Leukotriene Biosynthesis in Human Phagocytes

The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) has been linked to inflammation and lipid mediator production. Here we report that the potent IRE1α inhibitor, KIRA6, blocks leukotriene biosynthesis in human phagocytes activated with lipopolysaccharide (LPS) plus N-formyl-methionyl-leucyl-phenylalanine (fMLP) or thapsigargin (Tg). The inhibition affects both leukotriene B4 (LTB4) and cysteinyl leukotriene (cys-LTs) production at submicromolar concentration. Macrophages made deficient of IRE1α were still sensitive to KIRA6 thus demonstrating that the compound’s effect on leukotriene production is IRE1α-independent. KIRA6 did not exhibit any direct inhibitory effect on key enzymes in the leukotriene pathway, as assessed by phospholipase A2 (PLA2), 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S) enzyme activity measurements in cell lysates. However, we find that KIRA6 dose-dependently blocks phosphorylation of p38 and ERK, mitogen-activated protein kinases (MAPKs) that have established roles in activating cytosolic PLA2α (cPLA2α) and 5-LOX. The reduction of p38 and ERK phosphorylation is associated with a decrease in cPLA2α phosphorylation and attenuated leukotriene production. Furthermore, KIRA6 inhibits p38 activity, and molecular modelling indicates that it can directly interact with the ATP-binding pocket of p38. This potent and unexpected, non-canonical effect of KIRA6 on p38 and ERK MAPKs and leukotriene biosynthesis may account for some of the immune-modulating properties of this widely used IRE1α inhibitor.

Macrophage polarization is linked to Ca2+-independent phospholipase A2β-derived lipids and cross-cell signaling in mice

Phospholipases A2 (PLA2s) catalyze hydrolysis of the sn-2 substituent from glycerophospholipids to yield a free fatty acid (i.e., arachidonic acid), which can be metabolized to pro- or anti-inflammatory eicosanoids. Macrophages modulate inflammatory responses and are affected by Ca2+-independent phospholipase A2 (PLA2)β (iPLA2β). Here, we assessed the link between iPLA2β-derived lipids (iDLs) and macrophage polarization. Macrophages from WT and KO (iPLA2β-/-) mice were classically M1 pro-inflammatory phenotype activated or alternatively M2 anti-inflammatory phenotype activated, and eicosanoid production was determined by ultra-performance LC ESI-MS/MS. As a genotypic control, we performed similar analyses on macrophages from RIP.iPLA2β.Tg mice with selective iPLA2β overexpression in β-cells. Compared with WT, generation of select pro-inflammatory prostaglandins (PGs) was lower in iPLA2β-/- , and that of a specialized pro-resolving lipid mediator (SPM), resolvin D2, was higher; both changes are consistent with the M2 phenotype. Conversely, macrophages from RIP.iPLA2β.Tg mice exhibited an opposite landscape, one associated with the M1 phenotype: namely, increased production of pro-inflammatory eicosanoids (6-keto PGF1α, PGE2, leukotriene B4) and decreased ability to generate resolvin D2. These changes were not linked with secretory PLA2 or cytosolic PLA2α or with leakage of the transgene. Thus, we report previously unidentified links between select iPLA2β-derived eicosanoids, an SPM, and macrophage polarization. Importantly, our findings reveal for the first time that β-cell iPLA2β-derived signaling can predispose macrophage responses. These findings suggest that iDLs play critical roles in macrophage polarization, and we posit that they could be targeted therapeutically to counter inflammation-based disorders.

Respective contribution of cytosolic phospholipase A2α and secreted phospholipase A2 IIA to inflammation and eicosanoid production in arthritis

Phospholipase A2s (PLA2) play a key role in generation of eicosanoids. Cytosolic PLA2α (cPLA2α) is constitutively expressed in most cells, whereas IIA secreted PLA2 (sPLA2-IIA) is induced during inflammation and is present at high levels in the synovial fluid of rheumatoid arthritis patients. In mice, both cPLA2α and sPLA2-IIA have been implicated in autoimmune arthritis; however, the respective contribution of these two enzymes to the pathogenesis and production of eicosanoids is unknown. We evaluated the respective role of cPLA2α and sPLA2-IIA with regard to arthritis and eicosanoid profile in an in vivo model of arthritis. While arthritis was most severe in mice expressing both enzymes, it was abolished when both cPLA2α and sPLA2-IIA were lacking. cPLA2α played a dominant role in the severity of arthritis, although sPLA2-IIA sufficed to significantly contribute to the disease. Several eicosanoids were modulated during the course of arthritis and numerous species involved sPLA2-IIA expression. This study confirms the critical role of PLA2s in arthritis and unveils the distinct contribution of cPLA2α and sPLA2-IIA to the eicosanoid profile in arthritis.

Neutrophil-Derived Cytosolic PLA2α Contributes to Bacterial-Induced Neutrophil Transepithelial Migration.

Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2, and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa-induced neutrophil transepithelial migration has not been explored. Human and mouse neutrophil-epithelial cocultures were used to evaluate the role of neutrophil-derived cPLA2α in infection-induced transepithelial signaling by pharmacological and genetic approaches. Primary human airway basal stem cell-derived epithelial cultures and micro-optical coherence tomography, a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil transepithelial migration, were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils, but not epithelial cells, plays a significant role in infection-induced neutrophil transepithelial migration by mediating LTB4 synthesis during migration, which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection.

Chronic intermittent hypoxia affects the cytosolic phospholipase A2α/cyclooxygenase 2 pathway via β2-adrenoceptor-mediated ERK/p38 stimulation.

Cardiac resistance against acute ischemia/reperfusion (I/R) injury can be enhanced by adaptation to chronic intermittent hypoxia (CIH), but the changes at the molecular level associated with this adaptation are still not fully explored. Phospholipase A2 (PLA2) plays an important role in phospholipid metabolism and may contribute to membrane destruction under conditions of energy deprivation during I/R. The aim of this study was to determine the effect of CIH (7000m, 8h/day, 5weeks) on the expression of cytosolic PLA2α (cPLA2α) and its phosphorylated form (p-cPLA2α), as well as other related signaling proteins in the left ventricular myocardium of adult male Wistar rats. Adaptation to CIH increased the total content of cPLA2α by 14% in myocardial homogenate, and enhanced the association of p-cPLA2α with the nuclear membrane by 85%. The total number of β-adrenoceptors (β-ARs) did not change but the β2/β1 ratio markedly increased due to the elevation of β2-ARs and drop in β1-ARs. In parallel, the amount of adenylyl cyclase decreased by 49% and Giα proteins increased by about 50%. Besides that, cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2) increased by 36 and 84%, respectively. In parallel, we detected increased phosphorylation of protein kinase Cα, ERK1/2 and p38 (by 12, 48 and 19%, respectively). These data suggest that adaptive changes induced in the myocardium by CIH may include activation of cPLA2α and COX-2 via β2-AR/Gi-mediated stimulation of the ERK/p38 pathway.

Progesterone-induced Acrosome Exocytosis Requires Sequential Involvement of Calcium-independent Phospholipase A2β (iPLA2β) and Group X Secreted Phospholipase A2 (sPLA2)

Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA2 isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA2β and cytosolic PLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2β is critical for spontaneous AR, whereas both iPLA2β and group X secreted PLA2 are involved in P4-induced AR. Cytosolic PLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 pathways to achieve AR. At low P4 concentration (2 μm), sperm undergoing early AR (0-5 min post-P4) rely on iPLA2β, whereas sperm undergoing late AR (20-30 min post-P4) rely on group X secreted PLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, with the PLA2s being key actors at low physiological P4 concentrations (≤2 μm) but not at higher P4 concentrations (~10 μm).

Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils

Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. Arabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. Weused confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1β increased the expression of PLA2G10 by eosinophils. These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulatedby proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications forthe treatment of eosinophilic disorders such as asthma.

Emerging roles of phospholipase A2s in mast cell biology

Tissue-resident mast cells are derived from circulating committed progenitors, which are originated from pluripotent hematopoietic stem cells in bone marrow. These progenitors migrate into extravascular tissues, where they undergo differentiation and maturation into tissue-specific mast cell phenotypes. When activated by antigen or microenvironmental factors, mast cells release various biologically active products, including pre-formed mediators stored in secretory granules, de novo synthesized lipid mediators, and newly transcribed cytokines and chemokines, thereby promoting anaphylactic inflammation as well as other acute and chronic inflammatory diseases. Here, I will highlight the newest understanding of the phospholipase A2 (PLA2)-driven lipid networks in the maturation and effector functions of mast cells and attendant allergic responses. Group III secreted PLA2, the sole mammalian homolog of the potent extrinsic anaphylaxis inducer bee venom PLA2, regulates mast cell maturation through the paracrine prostaglandin D2 (PGD2) circuit. While cytosolic PLA2α is essential for the generation of PGD2 and leukotriene C4 by mast cells, it is also functionally coupled, through the arachidonic acid transfer mechanism, with PGE2 synthase in stromal fibroblasts to provide anti-anaphylactic PGE2. In addition, the roles of two particular mast cell maturation-responsible genes, NDRG1 and NLRP3, in mast cells will be discussed.

The Role of Sphingolipids in Arachidonic Acid Metabolism

The arachidonic acid (AA) cascade is regulated mainly by the actions of two rate-limiting enzymes, phospholipase A2 (PLA2) and inducible cyclooxygenase-2 (COX-2). PLA2 acts to generate AA, which serves as the precursor substrate for COX-2 in the metabolic pathway leading to prostaglandin production. Amongst more than 30 members of the PLA2 family, cytosolic PLA2α (cPLA2α, group IVA) plays a major role in releasing AA from cellular membranes. Sphingolipids are a novel class of bioactive lipids that play key roles in the regulation of several cellular processes including growth, differentiation, inflammatory responses, and apoptosis. Recent studies implicated a regulatory function of sphingolipids in prostaglandin production. Whereas ceramide-1-phosphate and lactosylceramide activate cPLA2α directly, sphingosine-1-phosphate induces COX-2 expression. Sphingomyelin has been shown to inhibit the activity of cPLA2α. In addition, several sphingolipid analogs including a therapeutic agent currently used clinically are also reported to be inhibitors of cPLA2α. This review explores the role of sphingolipids in the regulation of cPLA2α and COX-2.

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE2, PGD2, and PGF2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A2 (PLA2) isozymes revealed that cytosolic PLA2α, but not calcium-independent PLA2s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.

A novel C(28)-hydroxylated lupeolic acid suppresses the biosynthesis of eicosanoids through inhibition of cytosolic phospholipase A2

Eicosanoids are potent lipid mediators derived from phospholipase (PL)-released arachidonic acid (AA) coupled to subsequent metabolism by cyclooxygenase (COX)-1/2 or lipoxygenases (LO) which are involved in a variety of homeostatic biological functions and inflammation. We have investigated three lupeolic acids (LA) from the gum resin of Boswellia carterii for their ability to interfere with eicosanoid biosynthesis in human blood cells. A novel, yet unknown C(28)-hydroxylated LA, that is, 3α-acetoxy-28-hydroxylup-20(29)-en-4β-oic acid (Ac-OH-LA) was found to inhibit the biosynthesis of COX-, 5-LO- and 12-LO-derived eicosanoids from endogenous AA in activated platelets, neutrophils, and monocytes from human blood with consistent IC50 values of 2.3–6.9μM. In contrast, two other LAs lacking the C(28)-OH moiety were essentially inactive in this respect. Inhibition of eicosanoids by Ac-OH-LA correlated with reduced release of AA in intact cells. When AA was exogenously provided as substrate for cellular eicosanoid biosynthesis the inhibitory effects of Ac-OH-LA were essentially reversed, even though some inhibition of 5-LO and COX-1 product formation still remained. Finally, by means of a cell-free phospholipid hydrolysis assay using human recombinant cytosolic PLA2α, we show that Ac-OH-LA may directly interfere with cPLA2α activity (IC50=3.6μM). Together, we identified a novel, naturally occuring C(28)-hydroxylated LA which acts as efficient inhibitor of cPLA2α and consequently suppresses eicosanoid biosynthesis in intact cells.

Analysis of Two Major Intracellular Phospholipases A2 (PLA2) in Mast Cells Reveals Crucial Contribution of Cytosolic PLA2α, Not Ca2+-independent PLA2β, to Lipid Mobilization in Proximal Mast Cells and Distal Fibroblasts

Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)α) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)β), and the role of cPLA(2)α in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)α (Pla2g4a(-/-)) or iPLA(2)β (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)α, not by iPLA(2)β, during FcεRI-mediated activation and even during fibroblast-dependent maturation. Neither FcεRI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)α from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)α in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)β is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.

Towards Novel Radiosensitizing Agents: The Role of Cytosolic PLA2α in Combined Modality Cancer Therapy

The radioresistant nature of some tumors serves as an obstacle to curative therapy for several poor-prognosis malignancies. The radiosensitivity of a cancer is dependent not only on the intrinsic ability of tumor cells to recover from radiation-induced damage, but also the ability of stromal elements (e.g., vasculature) in the tumor microenvironment to survive and continue proliferating in the face of ionizing radiation. In this regard, it is important to understand the initial events activating radiation-induced signal transduction pathways. Among these events is the activation of cytosolic phospholipase A2 α and the subsequent production of the lipid second messengers. These events occur within minutes following exposure to ionizing radiation, and have been shown to enhance cell viability through a number of prosurvival signaling pathways. Furthermore, inhibition of cytosolic phospholipase A2 α has now been shown to reduce the viability of endothelial cells in culture after exposure to ionizing radiation, as well as slowing the growth of tumors in animal models of cancer.

Role of Calcium-independent Phospholipase A2β in High Glucose-induced Activation of RhoA, Rho Kinase, and CPI-17 in Cultured Vascular Smooth Muscle Cells and Vascular Smooth Muscle Hypercontractility in Diabetic Animals

Previous studies suggest that high glucose-induced RhoA/Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A(2)beta (iPLA(2)beta) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA(2)beta mRNA, protein, and iPLA(2) activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA(2)beta protein up-regulation. Inhibiting iPLA(2)beta activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA(2)beta in iPLA(2)beta-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA(2)beta inhibition. Moreover, increases in iPLA(2) activity and iPLA(2)beta protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA(2)beta, but not iPLA(2)gamma, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA(2)beta up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.

Cytosolic Phospholipase A2-α: A Potential Therapeutic Target for Prostate Cancer

Abstract Purpose: Cytosolic phospholipase A2-α (cPLA2-α) provides intracellular arachidonic acid to supply both cyclooxygenase and lipoxygenase pathways. We aim to determine the expression and activation of cPLA2-α in prostate cancer cell lines and tissue and the effect of targeting cPLA2-α in vitro and in vivo. Experimental Design: The expression of cPLA2-α was determined in prostate cancer cells by reverse transcription-PCR, Western blot, and immunocytochemistry. Growth inhibition, apoptosis, and cPLA2-α activity were determined after inhibition with cPLA2-α small interfering RNA or inhibitor (Wyeth-1). Cytosolic PLA2-α inhibitor or vehicle was also administered to prostate cancer xenograft mouse models. Finally, the expression of phosphorylated cPLA2-α was determined by immunohistochemistry in human normal, androgen-sensitive and androgen-insensitive prostate cancer specimens. Results: cPLA2-α is present in all prostate cancer cells lines, but increased in androgen-insensitive cells. Inhibition with small interfering RNA or Wyeth-1 results in significant reductions in prostate cancer cell numbers, as a result of reduced proliferation as well as increased apoptosis, and this was also associated with a reduction in cPLA2-α activity. Expression of cyclin D1 and phosphorylation of Akt were also observed to decrease. Wyeth-1 inhibited PC3 xenograft growth by ∼33% and again, also reduced cyclin D1. Immunohistochemistry of human prostate tissue revealed that phosphorylated cPLA2-α is increased when hormone refractory is reached. Conclusions: Expression and activation of cPLA2-α are increased in the androgen-insensitive cancer cell line and tissue. Inhibition of cPLA2-α results in cells and xenograft tumor growth inhibition and serves as a potentially effective therapy for hormone refractory prostate cancer.

Cytosolic PLA2α activation in Purkinje neurons and its role in AMPA-receptor trafficking

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) selectively releases arachidonic acid from membrane phospholipids and has been proposed to be involved in the induction of long-term depression (LTD), a form of synaptic plasticity in the cerebellum. This enzyme requires two events for its full activation: Ca(2+)-dependent translocation from the cytosol to organelle membranes in order to access phospholipids as substrates, and phosphorylation by several kinases. However, the subcellular distribution and activation of cPLA(2)alpha in Purkinje cells and the role of arachidonic acid in cerebellar LTD have not been fully elucidated. In cultured Purkinje cells, stimulation of AMPA receptors, but not metabotropic glutamate receptors, triggered translocation of cPLA(2)alpha to the somatic and dendritic Golgi compartments. This translocation required Ca(2+) influx through P-type Ca(2+) channels. AMPA plus PMA, a chemical method for inducing LTD, released arachidonic acid via phosphorylation of cPLA(2)alpha. AMPA plus PMA induced a decrease in surface GluR2 for more than 2 hours. Interestingly, this reduction was occluded by a cPLA(2)alpha-specific inhibitor. Furthermore, PMA plus arachidonic acid caused the prolonged internalization of GluR2 without activating AMPA receptors. These results suggest that cPLA(2)alpha regulates the persistent decrease in the expression of AMPA receptors, underscoring the role of cPLA(2)alpha in cerebellar LTD.

Group V secretory PLA2 regulates TLR2-dependent eicosanoid generation in mouse mast cells through amplification of ERK and cPLA2α activation

AbstractMast cells may be activated through Toll-like receptors (TLRs) for the dose- and time-dependent release of eicosanoids. However, the signaling mechanisms of TLR-dependent rapid eicosanoid generation are not known. We previously reported a role for group V secretory phospholipase A2 (PLA2) in regulating phagocytosis of zymosan and the ensuing eicosanoid generation in mouse resident peritoneal macrophages, suggesting a role for the enzyme in innate immunity. In the present study, we have used gene knockout mice to define an essential role for MyD88 and cytosolic PLA2α in TLR2-dependent eicosanoid generation. Furthermore, in mast cells lacking group V secretory PLA2, the time course of phosphorylation of ERK1/2 and of cPLA2α was markedly truncated, leading to attenuation of eicosanoid generation in response to stimulation through TLR2, but not through c-kit or FcεRI. These findings provide the first dissection of the mechanisms of TLR-dependent rapid eicosanoid generation, which is MyD88-dependent, requires cPLA2α, and is amplified by group V sPLA2 through its regulation of the sequential phosphorylation and activation of ERK1/2 and cPLA2α. The findings support the suggestion that group V sPLA2 regulates innate immune responses.

Importance of group X–secreted phospholipase A2 in allergen-induced airway inflammation and remodeling in a mouse asthma model

Arachidonic acid metabolites, the eicosanoids, are key mediators of allergen-induced airway inflammation and remodeling in asthma. The availability of free arachidonate in cells for subsequent eicosanoid biosynthesis is controlled by phospholipase A2s (PLA2s), most notably cytosolic PLA2-α. 10 secreted PLA2s (sPLA2s) have also been identified, but their function in eicosanoid generation is poorly understood. We investigated the role of group X sPLA2 (sPLA2-X), the sPLA2 with the highest in vitro cellular phospholipolysis activity, in acute and chronic mouse asthma models in vivo. The lungs of sPLA2-X−/− mice, compared with those of sPLA2-X+/+ littermates, had significant reduction in ovalbumin-induced infiltration by CD4+ and CD8+ T cells and eosinophils, goblet cell metaplasia, smooth muscle cell layer thickening, subepithelial fibrosis, and levels of T helper type 2 cell cytokines and eicosanoids. These data direct attention to sPLA2-X as a novel therapeutic target for asthma.

Group VIA Calcium-independent Phospholipase A2 Mediates Endothelial Cell S Phase Progression

Arachidonic acid and its metabolites have been previously implicated in the regulation of endothelial cell proliferation. Arachidonic acid may be liberated from cellular phospholipids by the action of group VIA calcium-independent phospholipase A2 (iPLA2-VIA). Consequently, we tested the hypothesis that iPLA2-VIA activity is linked to the regulation of endothelial cell proliferation. Inhibition of iPLA2 activity by bromoenol lactone (BEL) was sufficient to entirely block endothelial cell growth. BEL dose-dependently inhibited endothelial cell DNA synthesis in a manner that was reversed upon the exogenous addition of arachidonic acid. DNA synthesis was inhibited by the S-isomer and not by the R-isomer of BEL, demonstrating that endothelial cell proliferation is mediated specifically by iPLA2-VIA. iPLA2-VIA activity was critical to the progression of endothelial cells through S phase and is required for the expression of the cyclin A/cdk2 complex. Thus, inhibition of iPLA2-VIA blocks S phase progression and results in exit from the cell cycle. Inhibition of iPLA2-VIA-mediated endothelial cell proliferation is sufficient to block angiogenic tubule formation in co-culture assays. Consequently, iPLA2-VIA is a novel regulator of endothelial cell S phase progression, cell cycle residence, and angiogenesis.

Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D.

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2a were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.