Group IIA sPLA Research Articles

A novel neurotrophic role of secretory phospholipases A 2 for cerebellar granule neurons

Cultured cerebellar granule neurons (CGNs) require membrane depolarization or neurotrophic factors for their survival in vitro and undergo apoptosis when deprived of these survival-promoting stimuli. Here, we show that secretory phospholipases A 2s (sPLA 2s) rescue CGNs from apoptosis after potassium deprivation. The neurotrophic effect required the enzymatic activity of sPLA 2s, since catalytically inactive mutants of sPLA 2s failed to protect CGNs from apoptosis. Consistently, the ability of sPLA 2s to protect CGNs from apoptosis correlated with the extent of sPLA 2-induced arachidonic acid release from live CGNs. The survival-promoting effect of sPLA 2 was inhibited by depletion of extracellular Ca 2+ or by the presence of L-type Ca 2+ channel blocker nicardipine, suggesting that Ca 2+ influx occurs upon sPLA 2 treatment. Among the mammalian sPLA 2s tested, only group X sPLA 2, but not group IB nor IIA sPLA 2s, displayed neurotrophic activity. These results suggest a novel, unexpected neurotrophin-like role of sPLA 2 in the nervous system.

Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells

Although a number of sPLA2 (secretory phospholipase A2) enzymes have been identified in mammals, the localization and functions of individual enzymes in human pathologic tissues still remain obscure. In the present study, we have examined the expression and function of sPLA2s in human lung-derived cells and in human lungs with pneumonia. Group IID, V and X sPLA2s were expressed in cultured human bronchial epithelial cells (BEAS-2B) and normal human pulmonary fibroblasts with distinct requirement for cytokines (interleukin-1b, tumour necrosis factor a and interferon-g). Lentivirus- or adenovirus-mediated transfection of various sPLA2s into BEAS-2B or normal human pulmonary fibroblast cells revealed that group V and X sPLA2s increased arachidonate release and prostaglandin production in both cell types, whereas group IIA and IID sPLA2s failed to do so. Immunohistochemistry of human lungs with pneumonia demonstrated that group V and X sPLA2s were widely expressed in the airway epithelium, interstitium and alveolar macrophages, in which group IID sPLA2 was also positive, whereas group IIA sPLA2 was restricted to the pulmonary arterial smooth muscle layers and bronchial chondrocytes, and group IIE and IIF sPLA2s were minimally detected. These results suggest that group V and X sPLA2s affect lung pathogenesis by facilitating arachidonate metabolism or possibly through other functions.

A potent and selective inhibitor of group IIa secretory phospholipase A 2 protects rats from TNBS-induced colitis

Secretory phospholipase A 2 (sPLA 2) enzymes have been implicated in the pathogenesis of human inflammatory bowel disease (IBD). In this study we compared the efficacy of a potent, new and highly selective inhibitor of group IIa human sPLA 2 enzyme (5-(4-benzyloxyphenyl)-4 S-(7-phenylheptanoylamino)-pentanoic acid; sPLA 2I), with that of sulfasalazine, in a rat model of trinitrobenzene sulfonic acid (TNBS)-induced colitis. Following a single oral dose of sPLA 2I (5 mg/kg), pharmacoactive levels of drug were detected in the serum within 15 min and for up to 24 h by liquid chromatography mass spectrometry analysis. Rats treated with sPLA 2I (5 mg/kg/day) prior to induction of colitis were significantly healthier than TNBS-alone rats, as shown by reduced mortality, improved food intake and increased body weight, and significantly reduced colon myeloperoxidase levels, edema, tumour necrosis factor-α levels, and colon macroscopic pathology scores after 8 days. Rats pretreated with sulfasalazine (100 mg/kg/day) also had reduced disease expression markers similar to the sPLA 2I, but exhibited no improvement in colon edema. This study supports a role for the group IIa sPLA 2 enzyme in pathology associated with the TNBS rat model of IBD, and suggests a possible therapeutic application for selective inhibitors of group IIa sPLA 2 inhibitors in the treatment of IBD.

Glycogen synthase kinase-3β negatively regulates group IIA phospholipase A 2 expression in human aortic smooth muscle and HepG2 hepatoma cells

The present study shows that the IFN-γ-mediated upregulation of secretory phospholipase A 2 of group IIA (sPLA 2-IIA) in HASMC and HepG2 cells is synergistically increased after simultaneous inhibition of glycogen synthase kinase-3β (GSK-3β) by indirubin-3 ′-monoxime, 5-iodo or AR-A014418. The effect of GSK-3β inhibition was dose- and time-dependent and can be further augmented by its concomitant incubation with Clostridium difficile toxin B, an inhibitor of small Rho proteins, or H-1152, an inhibitor of Rho-associated kinase. Using AG-490 and caffeic acid phenethyl ester (CAPE), it is further demonstrated that the effect of GSK-3β inhibition on sPLA 2-IIA expression depends on Janus kinase-2 and NF-κB-signaling.

Increased hepatic cholesterol accumulation in transgenic mice overexpressing human secretory phospholipase A2 group IIA.

It has been demonstrated in transgenic mice that the overexpression of human phospholipase A2 group IIA (sPLA2), an acute-phase reactant, is associated with depressed plasma cholesterol levels, altered lipoprotein compositions, and increased lipid depositions in aortic walls. It was the aim of the present study to investigate whether the reduced plasma cholesterol levels in sPLA2-transgenic mice may be due to an increased transfer of lipids from sPLA2-modified lipoproteins to the liver and/or other nonvascular tissues. Ten sPLA2-transgenic mice and an equal number of nontransgenic littermates were fed a cholesterol-enriched (1%) diet for 13 weeks. After autopsy, cholesterol and triglyceride concentrations were measured in homogenates of liver, spleen, kidney, and myocardial tissues. Compared to the nontransgenic controls, the sPLA2-transgenic mice exhibited significantly lower plasma cholesterol levels, which was due to a reduction in both HDL and beta-lipoprotein (LDL + beta-VLDL) cholesterol. Liver tissues from the transgenic mice were found to contain significantly increased concentrations of free and esterified cholesterol, which was not associated with increased triglyceride concentrations. Spleen, kidney, and heart tissues of the two animal groups showed no significant differences in cholesterol or triglyceride concentrations. The findings suggest that the overexpression of human secretory phospholipase A2 group IIA leads to an enhanced delivery of cholesterol from phospholipolysed lipoproteins to the liver. This mechanism is likely to contribute to the development of hypocholesterolemia observed in patients with inflammatory diseases.

Secretory phospholipases A2 induce neurite outgrowth in PC12 cells.

sPLA(2)s (secretory phospholipases A(2)) belong to a broad and structurally diverse family of enzymes that hydrolyse the sn -2 ester bond of glycerophospholipids. We previously showed that a secreted fungal 15 kDa protein, named p15, as well as its orthologue from Streptomyces coelicolor (named Scp15) induce neurite outgrowth in PC12 cells at nanomolar concentrations. We report here that both p15 and Scp15 are members of a newly identified group of fungal/bacterial sPLA(2)s. The phospholipid-hydrolysing activity of p15 is absolutely required for neurite outgrowth induction. Mutants with a reduced PLA(2) activity exhibited a comparable reduction in neurite-inducing activity, and the ability to induce neurites closely matched the capacity of various p15 forms to promote fatty acid release from live PC12 cells. A structurally divergent member of the sPLA(2) family, bee venom sPLA(2), also induced neurites in a phospholipase activity-dependent manner, and the same effect was elicited by mouse group V and X sPLA(2)s, but not by group IB and IIA sPLA(2)s. Lysophosphatidylcholine, but not other lysophospholipids, nor arachidonic acid, elicited neurite outgrowth in an L-type Ca(2+) channel activity-dependent manner. In addition, p15-induced neuritogenesis was unaffected by various inhibitors that block arachidonic acid conversion into bioactive eicosanoids. Altogether, these results delineate a novel, Ca(2+)- and lysophosphatidylcholine-dependent neurotrophin-like role of sPLA(2)s in the nervous system.

Induction of distinct sets of secretory phospholipase A 2 in rodents during inflammation

Although the expression of the prototypic secretory phospholipase A 2 (sPLA 2), group IIA (sPLA 2-IIA), is known to be up-regulated during inflammation, it remains uncertain if other sPLA 2 enzymes display similar or distinct profiles of induction under pathological conditions. In this study, we investigated the expression of several sPLA 2s in rodent inflammation models. In lipopolysaccharide (LPS)-treated mice, the expression of sPLA 2-V, and to a lesser extent that of sPLA 2-IID, -IIE, and -IIF, were increased, whereas that of sPLA 2-X was rather constant, in distinct tissues. 12- O-Tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, in which the expression of sPLA 2-IID, -IIF and -V was increased, was significantly reduced by YM-26734, a competitive sPLA 2-IIA inhibitor that turned out to inhibit sPLA 2-IID, -IIE, -V and -X as well. In contrast, sPLA 2-IIA was dominant in carageenin-induced pleurisy in rats, where the accumulation of exudate fluids and leukocytes was significantly ameliorated by YM-26734. These results indicate that distinct sPLA 2s can participate in inflammatory diseases according to tissues, animal species, and types of inflammation.

Identification of an autoantigen on the surface of apoptotic human T cells as a new protein interacting with inflammatory group IIA phospholipase A2

AbstractOne of the most studied secreted phospholipases A2 (sPLA2), the group IIA sPLA2, is found at high levels in inflammatory fluids of patients with autoimmune diseases. A characteristic of group IIA sPLA2 is its preference for negatively charged phospholipids, which become exposed on the extracellular leaflet of apoptotic cell membranes. We recently showed that low molecular weight heparan sulfate proteoglycans (HSPGs) and uncharacterized detergent-insoluble binding site(s) contribute to the enhanced binding of human group IIA PLA2 (hGIIA) to apoptotic human T cells. Using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry we now identify vimentin as the major HSPG-independent binding protein of hGIIA on apoptotic primary T lymphocytes. Vimentin is partially exposed on the surface of apoptotic T cells and binds hGIIA via its rod domain in a calcium-independent manner. Studies with hGIIA mutants showed that specific motifs in the interfacial binding surface are involved in the interaction with vimentin. The sPLA2 inhibitor LY311727, but not heparin, inhibited this interaction. In contrast, heparin but not LY311727 abrogated the binding of hGIIA to cellular HSPGs. Importantly, vimentin does not inhibit the catalytic activity of hGIIA. Altogether, the results show that vimentin, in conjunction with HSPGs, contributes to the enhanced binding of hGIIA to apoptotic T cells.

Group IIA secretory phospholipase A 2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons

Recent evidence shows that secretory phospholipase A 2 (sPLA 2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA 2 on vesicle exocytosis. External application of group IIA sPLA 2 (purified crotoxin subunit B or purified human synovial sPLA 2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA 2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA 2 was dependent on calcium and sPLA 2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA 2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA 2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA 2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA 2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA 2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.

Secretory phospholipases A2 activate selective functions in human eosinophils.

Secretory phospholipases A(2) (sPLA(2)s) are released in large amounts in the blood of patients with systemic inflammatory diseases and accumulate at sites of chronic inflammation, such as the airways of patients with bronchial asthma. Blood eosinophils or eosinophils recruited in inflammatory areas therefore can be exposed in vivo to high concentrations of sPLA(2). We have examined the effects of two structurally different sPLA(2)s (group IA and group IIA) on several functions of eosinophils isolated from normal donors and patients with hypereosinophilia. Both group IA and IIA sPLA(2) induced a concentration-dependent release of beta-glucuronidase, IL-6, and IL-8. Release of the two cytokines was associated with the accumulation of their specific mRNA. In addition, sPLA(2)s induced the surface expression of CD44 and CD69, two major activation markers of eosinophils. In contrast, none of the sPLA(2)s examined induced the production of IL-5, the de novo synthesis of leukotriene C(4) and platelet-activating factor, or the generation of superoxide anion from human eosinophils. Incubation of eosinophils with the major enzymatic products of the sPLA(2)s (arachidonic acid, lysophosphatidylcholine, or lysophosphatidic acid) did not reproduce any of the enzymes' effects. In addition, inactivation of sPLA(2) enzymatic activity by bromophenacyl bromide did not influence the release of beta-glucuronidase or of cytokines. Stimulation of eosinophils by sPLA(2)s was associated with activation of extracellular signal-regulated kinases 1/2. These results indicate that sPLA(2)s selectively activate certain proinflammatory and immunoregulatory functions of human eosinophils through mechanism(s) independent from enzymatic activity and from the generation of arachidonic acid.

Macrophage-expressed group IIA secretory phospholipase A2 increases atherosclerotic lesion formation in LDL receptor-deficient mice.

Transgenic mice expressing human group IIA secretory phospholipase A(2) (group IIA sPLA(2)) spontaneously develop atherosclerotic lesions. The mechanism for this proatherogenic effect is likely multifactorial, because HDL-cholesterol is significantly lower and LDL/VLDL cholesterol is slightly higher in transgenic mice compared with nontransgenic littermates. In the present study, we show for the first time that elicited peritoneal macrophages from transgenic mice express human group IIA sPLA(2). This study tested whether macrophage-expressed sPLA(2) contributes to atherogenesis. Bone marrow cells from either sPLA(2) transgenic mice or control C57BL/6 mice were transplanted into LDL receptor-deficient mice. After hematopoietic engraftment, animals were fed a diet enriched with saturated fat and cholesterol for 12 weeks. Despite a lack of effect on serum lipoprotein concentrations, the presence of bone marrow-derived cells expressing human group IIA sPLA(2) resulted in a significant increase in the extent of atherosclerosis in the aortic arch (12.8+/-1.4% versus 7.4+/-0.9%; P<0.005) and aortic sinus (0.3+/-0.03 mm(2) versus 0.2+/-0.04 mm(2); P<0.05). Group IIA sPLA(2) can contribute to atherosclerotic lesion development through a mechanism that is independent of systemic lipoprotein metabolism.

On the Binding Preference of Human Groups IIA and X Phospholipases A2 for Membranes with Anionic Phospholipids

Mammals contain 9-10 secreted phospholipases A(2) (sPLA(2)s) that display widely different affinities for membranes, depending on the phospholipid composition. The much higher enzymatic activity of human group X sPLA(2) (hGX) compared with human group IIA sPLA(2) (hGIIA) on phosphatidylcholine (PC)-rich vesicles is due in large part to the higher affinity of the former enzyme for such vesicles; this result also holds when vesicles contain cholesterol and sphingomyelin. The inclusion of anionic phosphatidylserine in PC vesicles dramatically enhances interfacial binding and catalysis of hGIIA but not of hGX. This is the result of the large number of lysine and arginine residues scattered over the entire surface of hGIIA, which cause the enzyme to form a supramolecular aggregate with multiple vesicles. Thus, high affinity binding of hGIIA to anionic vesicles is a complex process and cannot be attributed to a few basic residues on its interfacial binding surface, as is also evident from mutagenesis studies. The main reason hGIIA binds poorly to PC-rich vesicles is that it lacks a tryptophan residue on its interfacial binding surface, a residue that contributes to the high affinity binding of hGX to PC-rich vesicles. Results show that the lag in the onset of hydrolysis of PC vesicles by hGIIA is due in part to the poor affinity of this enzyme for these vesicles. Binding affinity of hGIIA, hGX, and their mutants to PC-rich vesicles is well correlated to the ability of these enzymes to act on the PC-rich outer plasma membrane of mammalian cells.

High-performance liquid chromatographic assay with ultraviolet spectrometric detection for the evaluation of inhibitors of secretory phospholipase A 2

A non-radioactive spectrometric assay for the evaluation of inhibitors of pancreatic group IB and non-pancreatic group IIA secretory phospholipase A 2 (sPLA 2) is described. Mixed-micelles consisting of 1 m M of a 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol and 6 m M of sodium deoxycholate were used as substrate. The enzyme activity was determined directly without any sample clean-up by measuring the sPLA 2-mediated oleic acid release with reversed-phase HPLC and UV-detection at 200 nm. The known sPLA 2 inhibitors MJ33 and AR-C 67047MI were analyzed in this assay for their inhibitory potency. While MJ33 revealed only a very weak inhibition of group IB and IIA sPLA 2 at the highest test concentration (33 μ M), AR-C 67047MI proved to be a potent inhibitor of both enzymes with IC 50-values of 0.36 and 0.14 μ M, respectively.

85-kDa Cytosolic Phospholipase A2 Mediates Peroxisome Proliferator-activated Receptor γ Activation in Human Lung Epithelial Cells

The 85-kDa cytosolic phospholipase A(2) (cPLA(2)) plays an important role in the control of arachidonic acid metabolism. This study was designed to investigate the possible contributions of cPLA(2) and group IIA secretory phospholipase A(2) (sPLA(2)) in the regulation of peroxisome proliferator-activated receptor (PPAR)-mediated gene transcription in human airway epithelial cells. Primary normal human bronchial epithelial cells and human lung epithelial cell lines BEAS 2B, A549, and NCI-H292 all express PPARgamma and -beta. Overexpression of cPLA(2) in BEAS 2B cells and primary bronchial epithelial cells resulted in a significant increase of PPARgamma-mediated reporter activity. In contrast, overexpression of group IIA sPLA(2) had no effect on PPARgamma activation. The PPARgamma activity in A549 cells was significantly inhibited by the cPLA(2) inhibitor arachidonyltrifluoromethyl ketone but not by the sPLA(2) inhibitor LY311727 and the iPLA(2) inhibitor HELSS. Activation of cPLA(2) by the calcium ionophore, induced a dose-dependent increase of PPAR activity in normal human bronchial epithelial cells and in the A549 cells. Electrophoretic mobility shift assays show that the binding between PPAR isolated from A549 cells and peroxisome proliferator response element (PPRE) is enhanced by but partially blocked by the cPLA(2) inhibitors arachidonyltrifluoromethyl ketone and methyl arachidonyl fluorophosphate. Finally, NS 398, a COX-2 inhibitor, partially blocked the effect on PPAR activity and binding to the PPRE suggesting involvement of COX-2 metabolites in PPRE activation. The above results demonstrate a novel function of cPLA(2) in the control of PPARgamma activation in human lung epithelial cells.

Crystal Structure of Human Group X Secreted Phospholipase A2: ELECTROSTATICALLY NEUTRAL INTERFACIAL BINDING SURFACE TARGETS ZWITTERIONIC MEMBRANES

The crystal structure of human group X (hGX) secreted phospholipase A2 (sPLA2) has been solved to a resolution of 1.97 A. As expected the protein fold is similar to previously reported sPLA2 structures. The active site architecture, including the positions of the catalytic residues and the first and second shell water around the Ca2+ cofactor, are highly conserved and remarkably similar to the group IB and group IIA enzymes. Differences are seen in the structures following the (1-12)-N-terminal helix and at the C terminus. These regions are proposed to interact with the substrate membrane surface. The opening to the active site slot is considerably larger in hGX than in human group IIA sPLA2. Furthermore, the electrostatic surface potential of the hGX interfacial-binding surface does not resemble that of the human group IIA sPLA2; the former is highly neutral, whereas the latter is highly cationic. The cationic residues on this face of group IB and IIA enzymes have been implicated in membrane binding and in k(cat*) allostery. In contrast, hGX does not show activation by the anionic charge at the lipid interface when acting on phospholipid vesicles or short-chain phospholipid micelles. Together, the crystal structure and kinetic results of hGX supports the conclusion that it is as active on zwitterionic as on anionic interfaces, and thus it is predicted to target the zwitterionic membrane surfaces of mammalian cells.

Biological effects of secretory phospholipase A(2) group IIA on lipoproteins and in atherogenesis.

Secretory phospholipase A(2) group IIA(sPLA(2) IIA) can be produced and secreted by various cell types either constitutionally or as an acute-phase reactant upon stimulation by proinflammatory cytokines. The enzyme prefers phosphatidylethanolamine and phosphatidylserine as substrates. One important biological function may be the hydrolytic destruction of bacterial membranes. It has been demonstrated, however, that sPLA(2) can also hydrolyse the phospholipid monolayers of high density lipoprotein (HDL) and low density lipoprotein (LDL) in vitro. Secretory phospholipase A(2)-modified LDL show increased affinity to glycosaminoglycans and proteoglycans, a tendency to aggregate, and an enhanced ability to deliver cholesterol to cells. Incubation of cultured macrophages with PLA(2)-treated LDL and HDL is associated with increased intracellular lipid accumulation, resulting in the formation of foam cells. Elevated sPLA(2)(IIA) activity in blood serum leads to an increased clearance of serum cholesterol. Secretory phospholipase A(2)(IIA) can also be detected in the intima, adventitia and media of the atherosclerotic wall not only in developed lesions but also in very early stages of atherosclerosis. The presence of DNA of Chlamydia pneumoniae, herpes simplex virus, and cytomegalovirus was found to be associated with sPLA(2)(IIA) expression and other signs of local inflammation. Thus, sPLA(2)(IIA) appears to be one important link between the lipid and the inflammation hypothesis of atherosclerosis.

85-kDa cPLA(2) plays a critical role in PPAR-mediated gene transcription in human hepatoma cells.

In an effort to understand the role of key eicosanoid-forming enzymes in the activation of peroxisome proliferator-activated receptor (PPAR), this study was designed to evaluate the possible contributions of cytosolic phospholipase A(2) (cPLA(2)) and group IIA secretory phospholipase A(2) (sPLA(2)) in the regulation of PPAR-mediated gene transcription in a human hepatoma cell line (HepG2). The HepG2 cells express both PPAR-alpha and -gamma but not PPAR-beta. Overexpression of cPLA(2), but not group IIA sPLA(2) in the HepG2 cells, caused a significantly increased PPAR-alpha/gamma-mediated reporter activity. Antisense inhibition of cPLA(2) resulted in a significantly decreased PPAR-alpha/gamma activity. The PPAR-alpha/gamma-induced gene transcription in the HepG2 cells was inhibited by the cPLA(2) inhibitors methyl arachidonyl fluorophosphonate and arachidonyltrifluoromethyl ketone, but not by the sPLA(2) inhibitor LY311727. The expression of PPAR-alpha-mediated endogenous gene apolipoprotein A-II was increased in cells with overexpression of cPLA(2), decreased in cells with antisense inhibition of cPLA(2), but unaltered in cells with overexpression of group IIA sPLA(2). The above results demonstrated an important role of cPLA(2), but not group IIA sPLA(2) in the control of PPAR activation. The cPLA(2)-mediated PPAR activation was likely mediated by arachidonic acid and prostaglandin E(2). This study reveals a novel intracellular function of cPLA(2) in PPAR activation in HepG2 cells. The cPLA(2) thus may represent a potential therapeutic target for the control of PPAR-related liver and metabolic disorders such as obesity, lipid metabolic disorders, diabetes mellitus, and atherosclerosis.

Potentiation of TNF-alpha-stimulated group IIA phospholipase A(2) expression by peroxisome proliferator-activated receptor alpha activators in rat mesangial cells.

Natural activators of peroxisome proliferator-activated receptors (PPAR) are lipid metabolites, including those produced by phospholipases A(2) (PLA(2)). In glomerular mesangial cells, the secreted group IIA PLA(2) (sPLA(2)-IIA), which is thought to be a crucial factor in pathologic processes in the kidney, may provide free fatty acids and eicosanoids directly or indirectly, by activating a cytosolic PLA(2). The scope of this study was to investigate whether synthetic PPAR(alpha) activators have an effect on sPLA(2)-IIA mRNA expression in rat mesangial cells, thus constituting a feedback modulation of sPLA(2)-IIA transcription. In the presence of tumor necrosis factor-alpha (TNF-alpha), the PPAR(alpha) agonists WY14643 and LY171883 as well as the lipid-lowering compound clofibrate potentiated expression, secretion, and activity of group IIA sPLA(2) in mesangial cells. MK886, known as a noncompetitive inhibitor of PPAR(alpha), completely abolished the potentiation of sPLA(2)-IIA secretion and activity by WY14643, thus indicating that the effect of WY14643 is specifically mediated by PPAR(alpha). When cells were transfected with different constructs of the rat sPLA(2)-IIA promoter fused to a luciferase reporter gene, a stimulation with TNF-alpha in the presence of the PPAR(alpha) activators caused an enhanced promoter activity compared with that induced by TNF-alpha alone. Site-directed mutagenesis of a putative PPRE site in the sPLA(2)-IIA promoter abolished the potentiating effect of PPAR(alpha) agonists, thus strongly indicating its contribution to the enhanced promoter activity. In summary, this study shows that the rat sPLA(2)-IIA promoter is sensitive to PPAR(alpha) agonists, which act synergistically with cytokines, resulting in an enhanced expression of sPLA(2)-IIA in rat mesangial cells.

Bactericidal Properties of Human and Murine Groups I, II, V, X, and XII Secreted Phospholipases A2

Group IIA secreted phospholipase A(2) (sPLA2) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of recombinant murine and human groups I, II, V, X, and XII sPLA2s on Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. The rank order potency among human sPLA2s against Gram-positive bacteria is group IIA > X > V > XII > IIE > IB, IIF (for murine sPLA2s: IIA > IID > V > IIE > IIC, X > IB, IIF), and only human group XII displays detectable bactericidal activity against the Gram-negative bacterium E. coli. These studies show that highly basic sPLA2s display potent bactericidal activity with the exception of the ability of the acidic human group X sPLA2 to kill Gram-positive bacteria. By studying the Bacillus subtilis and S. aureus bactericidal potencies of a large panel of human group IIA mutants in which basic residues were mutated to acidic residues, it was found that: 1) the overall positive charge of the sPLA2 is the dominant factor in dictating bactericidal potency; 2) basic residues on the putative membrane binding surface of the sPLA2 are modestly more important for bactericidal activity than are other basic residues; 3) relative bactericidal potency tracks well with the ability of these mutants to degrade phospholipids in the bacterial membrane; and 4) exposure of the bacterial membrane of Gram-positive bacteria by disruption of the cell wall dramatically reduces the negative effect of charge reversal mutagenesis on bactericidal potency.

Expression of Group IIA Secretory Phospholipase A2 Is Elevated in Prostatic Intraepithelial Neoplasia and Adenocarcinoma

Phospholipase A2 (PLA2) enzymes release arachidonic acid from cellular phospholipids in a variety of mammalian tissues, including prostate. Group IIa secretory PLA2 (sPLA2) can generate arachidonate from cellular phospholipids. We examined the group IIa sPLA2 expression in benign prostatic tissues, prostatic intraepithelial neoplasia (PIN), and adenocarcinoma to determine whether sPLA2 expression is altered in the carcinogenesis of human prostatic cancer. Thirty-three of 74 total cases (45%) of benign prostatic tissue showed positive immunohistochemical staining for group IIA sPLA2, whereas 63 of 69 total cases (91%) of high-grade PINs and 70 of 78 total cases (90%) of adenocarcinomas gave positive results. Four of 10 cases of low-grade PIN showed positive immunoreactivity for sPLA2. The number of cells staining for sPLA2 was significantly less in benign epithelium (4%) and low-grade PIN (4%) compared to high-grade PIN (40%) or adenocarcinoma (38%) (P < 0.001). There was no significant difference between high-grade PIN and adenocarcinoma in the number of cells staining positively for sPLA2. The intensity of sPLA2 immunoreactivity was also different among benign prostatic tissue, low-grade PIN, high-grade PIN, and prostatic adenocarcinoma specimens. The malignant cells demonstrated more intense immunohistochemical staining (moderate to strong staining in 81% and 69% cases for high-grade PIN and adenocarcinoma, respectively) than benign glands (moderate staining in 11% of cases). No strong staining was observed in benign glands or low-grade PIN. Our data are consistent with the contention that group IIA sPLA2 expression is elevated in neoplastic prostatic tissue and support the hypothesis that dysregulation of sPLA2 may play a role in prostatic carcinogenesis.