IIA sPLA Research Articles

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.

Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: Regulation by Group IV cytosolic phospholipase A 2, but not by Group V and Group IIA secretory phospholipase A 2

Lipopolysaccharide (LPS) induces a delayed release (lag phase of 2–4 h) of arachidonic acid (AA) and prostaglandin (PG) D 2 in rat liver macrophages. Group IV cytosolic phospholipase A 2 (cPLA 2) becomes phosphorylated within minutes after the addition of LPS. The phosphorylated form of cPLA 2 shows an enhanced in vitro activity. The Ca 2+ dependence of cPLA 2 activity is not affected by phosphorylation of the enzyme. In addition, LPS induces an enhanced expression of cPLA 2 mRNA (after 2–4 h) and an enhanced expression of cPLA 2 protein (after 8 h). The cellular cPLA 2 activity is enhanced about twofold 24 h after LPS treatment. Liver macrophages constitutively express mRNAs encoding Groups V and IIA secretory PLA 2 (sPLA 2). LPS has no effect on the levels of Groups V and IIA sPLA 2 mRNA expression. Despite mRNA expression, Groups V and IIA sPLA 2 protein and sPLA 2 activity are not detectable in unstimulated or LPS-stimulated liver macrophages. Collectively, these and earlier [Mediators Inflammation 8 (1999) 295.] results suggest that in liver macrophages the LPS-induced delayed release of AA and prostanoids is mediated by phosphorylation and an enhanced expression of cPLA 2, a de novo expression of cyclooxygenase (COX)-2, but not by the actions of Group V or Group IIA sPLA 2.

Secretory phospholipases A2 induce cytokine release from blood and synovial fluid monocytes

Secretory phospholipases A2 (sPLA2) are released in the blood of patients with various inflammatory diseases and exert proinflammatory activities by releasing arachidonic acid (AA), the precursor of eicosanoids. We examined the ability of four sPLA2 to activate blood and synovial fluid monocytes in vitro. Monocytes were purified from blood of healthy donors or from synovial fluid of patients with rheumatoid arthritis by negative immunoselection and by adherence to plastic dishes, respectively. The cells were incubated with group IA, IB, IIA and III sPLA2 and the release of TNF-alpha, IL-6 and IL-12 was determined by ELISA. Group IA, IB and IIA sPLA2 induced a concentration-dependent release of TNF-alpha and IL-6 from blood monocytes. These sPLA2 activated IL-12 production only in monocytes preincubated with IFN-gamma. Group IA and IIA sPLA2 also induced TNF-alpha and IL-6 release from synovial fluid monocytes. TNF-alpha and IL-6 release paralleled an increase in their mRNA expression and was independent from the capacity of sPLA2 to mobilize AA. These results indicate that sPLA2 stimulate cytokine release from blood and synovial fluid monocytes by a mechanism at least partially unrelated to their enzymatic activity. This effect may concur with the generation of AA in the proinflammatory activity of sPLA2 released during inflammatory diseases.

VEGF stimulation of endothelial cell PAF synthesis is mediated by group V 14 kDa secretory phospholipase A2.

1. Vascular endothelial growth factor (VEGF) is a potent inducer of inflammation, and we have shown that this latter effect is mediated through endothelial cell (EC) PAF synthesis. Since the phospholipid remodelling pathway enzymes (CoA-independent transacylase, CoA-IT; phospholipase A2, PLA2; and lyso-PAF acetyltransferase, lyso-PAF-AT) may participate in PAF synthesis, we assessed their contribution to VEGF-induced PAF synthesis in bovine aortic EC (BAEC) and human umbilical vein EC (HUVEC). 2. VEGF enhanced BAEC and HUVEC PAF synthesis by up to 28 and 4 fold above basal levels respectively. 3. A pretreatment with a CoA-IT and lyso-PAF-AT inhibitor (Sanguinarin; 500 nM) blocked VEGF-induced PAF synthesis by 95%, a specific CoA-IT inhibitor (SKF45905; 10 - 50 microM) was without effect, confirming the crucial role of the PLA2 and lyso-PAF-AT. 4. Treatment with secreted PLA2 (sPLA2) inhibitors which have been shown to inhibit both groups IIA and V sPLA2 (SB203347; 10 microM and LY311727; 100 microM) blocked EC PAF synthesis by up to 90%, whereas selective inhibition of group IIA sPLA2 (LY311727; 1 microM) had no significant effect. 5. RT - PCR and Western blot analyses demonstrated the presence of group V sPLA2 whereas group IIA sPLA2 was undetected in EC. 6. Treatment with cytosolic and calcium-independent PLA2 inhibitors (Arachidonyl trifluoromethyl ketone, Bromoenol lactone, Methyl arachydonyl fluorophosphate, up to 50 microM) did not prevent but rather potentiated the VEGF effect on EC PAF synthesis. 7. These results provide evidence that with VEGF activation of EC cells, the group V sPLA2 provides substrate for EC PAF formation.

Group IIA secretory phospholipase A(2) stimulates inducible nitric oxide synthase expression via ERK and NF-kappaB in macrophages.

The mammalian group IIA secretory phospholipase A(2) (sPLA(2)) is believed to play an important role in inflammation and cell injury. The present study underlines the importance of group IIA sPLA(2) in the regulation of iNOS. Treatment of cells with sPLA(2) induced protein expression and mRNA accumulation of iNOS in a dose-dependent manner. The pretreatment of cells with rho-BPB or SCA, selective sPLA(2) inhibitors, inhibited sPLA(2)-induced iNOS expression. sPLA(2) stimulated the simultaneous activation of two classes of mitogen-activated protein kinases ERK and JNK, but did not stimulate p38 MAPK. PD98059, a selective MEK inhibitor, inhibited sPLA(2)-induced nitrite production and iNOS expression as well as ERK phosphorylation. In addition, pretreatment of rho-BPB or SCA also resulted in inhibition of sPLA(2)-induced ERK phosphorylation. The sPLA(2) signaling mechanisms involving the activation of transcription factor NF-kappaB were studied in the same cells. That stimulation of cells with sPLA(2) caused NF-kappaB activation in a time-dependent manner was shown by the detection of NF-kappaB-specific DNA-protein binding and by IkappaBalpha degradation. sPLA(2)-induced NF-kappaB activation was prevented in the presence of rho-BPB. Furthermore, the NF-kappaB inhibitor PDTC suppressed sPLA(2)-induced nitrite production and iNOS expression as well as IkappaBalpha degradation. The results strongly suggest that group IIA sPLA(2) induces iNOS in macrophages and that this induction occurs through ERK and NF-kappaB.

Ischemic-reperfused isolated working mouse hearts: membrane damage and type IIA phospholipase A2.

For the murine heart the relationships between ischemia-reperfusion-induced loss of cardiac function, enzyme release, high-energy phosphate (HEP), and membrane phospholipid metabolism are ill-defined. Accordingly, isolated ejecting murine hearts were subjected to varying periods of ischemia, whether or not followed by reperfusion. On reperfusion, hemodynamic function was almost completely restored after 10 min of ischemia [83 +/- 14% recovery of cardiac output (CO)], but was severely depressed after 15 and 20 min of ischemia (40 +/- 24 and 31 +/- 24% recovery of CO, respectively). Reperfusion was associated with partial recovery of HEP stores and enhanced degradation of phospholipids as indicated by the accumulation of fatty acids (FA). Myocardial FA content and enzyme release during reperfusion were correlated (r = 0.70), suggesting that membrane phospholipid degradation and cellular damage are closely related phenomena. To investigate the role of type IIA secretory phospholipase A2 (sPLA2) in this process, hearts from wild-type and sPLA2-deficient mice were subjected to ischemia-reperfusion. Postischemic functional recovery, ATP depletion, enzyme release, and FA accumulation were not significantly different between wild-type and sPLA2- deficient hearts. These findings argue against a prominent role of type IIA sPLA2 in the development of irreversible cell damage in the ischemic-reperfused murine myocardium.

Secretory Phospholipase A2 Mediates Cooperative Prostaglandin Generation by Growth Factor and Cytokine Independently of Preceding Cytosolic Phospholipase A2 Expression in Rat Gastric Epithelial Cells

Transforming growth factor (TGF)-alpha and interleukin (IL)-1beta are responsible for the healing of gastric lesions through, in part, prostaglandin (PG) generation. We examined the contribution of cytosolic and secretory phospholipase A(2)s (cPLA(2) and sPLA(2)) to the PG generation by rat gastric epithelial cells in response to both stimuli. Stimulation with TGF-alpha for 24 h increased cPLA(2) and cyclooxygenase (COX)-2 markedly, PGE(2) slightly, and type IIA sPLA(2) and COX-1 not at all, whereas IL-1beta increased sPLA(2) only. Both stimuli synergistically increased PGE(2), sPLA(2), and the two COXs but not cPLA(2). The onset of the PGE(2) generation paralleled the sPLA(2) release but was apparently preceded by increases in cPLA(2) and the two COXs. The increase in PGE(2) was impaired by inhibitors for sPLA(2) and COX-2 but not COX-1. cPLA(2) inhibitors suppressed PGE(2) generation by TGF-alpha alone but not augmentation of PGE(2) generation or sPLA(2) release by IL-1beta in combination with TGF-alpha. Furthermore, despite an increase in cPLA(2) including its phosphorylated form (phosphoserine), -induced arachidonic acid liberation was impaired in the TGF-alpha/IL-1beta-stimulated cells, in which p11, a putative cPLA(2) inhibitory molecule, was also increased and co-immunoprecipitated with cPLA(2). These results suggest that synergistic stimulation of sPLA(2) and COX-2 expression by TGF-alpha and IL-1beta results in an increase in PGE(2). Presumably, the preceding cPLA(2) expression is not involved in the PGE(2) generation, because of impairment of its hydrolytic activity in the stimulated cells.

Mitogenic Effect of Lipoproteins on Human Vascular Smooth Muscle Cells: The Impact of Hydrolysis by gr II A Phospholipase A2

Multifactorial interaction among lipoproteins, vascular wall cells, and inflammatory mediators has been recognized as the basis of atherogenesis. In the arterial wall high-density lipoprotein (HDL) and human secretory phospholipase A2 (sPLA2) colocalize with vascular smooth muscle cells and concentrate in the atherosclerotic lesions. It has been shown that gr IIA sPLA2 hydrolyzes lipoproteins, altering their structure and releasing active agents such as lyso-phosphatidylcholine (PtdCho) and free fatty acids. We investigated the impact of normal HDL3 (NHDL3), acute phase HDL3 (APHDL3), and low-density lipoprotein (LDL), both unhydrolyzed and sPLA2-hydrolyzed, and some products of hydrolysis, such as lyso-PtdCho, oleic and linoleic acid, on [3H] thymidine incorporation by DNA of cultured human vascular smooth muscle cells (VSMC). NHDL3 markedly enhanced mitogenic activity of VSMC in a dose- and time-dependent manner. Doubling of thymidine incorporation was usually achieved by 40 μg/ml of NHDL3 after 4 hours of incubation. APHDL3 had invariably a stronger inducing effect on the mitogenic activity than NHDL3; 40 μg/ml more than tripled [3H] thymidine incorporation after 4 hours of incubation. NHDL3 preincubated with human apo serum amyloid A apolipoprotein–induced higher mitogenic activity in VSMC than NHDL3 alone. Hydrolysis of NHDL3, APHDL3, or LDL by gr IIA sPLA2 markedly enhanced mitogenic activity of VSMC as compared with unhydrolyzed lipoproteins. sPLA2 concentrations that can be found in atherosclerotic vascular walls markedly enhanced lipoprotein-induced mitogenic activity of VSMC. sPLA2 per se did not affect thymidine incorporation and VSMC did not release sPLA2 into the medium. There was no evidence for hydrolysis of the wall of VSMC by gr IIA sPLA2. The presence of the products of hydrolysis of lipoproteins such as oleic and linoleic acids and lyso-PtdCho or their combinations with NHDL3 explains in part markedly enhanced mitogenic activity of VSMC. It is conceivable that sPLA2, which is known to colocalize with lipoproteins in the vascular wall in the domain of VSMC, is capable of induction of the mitogenic activity in these cells in vivo and should be considered as a proatherogenic enzyme.

Group X secretory phospholipase A 2 induces potent productions of various lipid mediators in mouse peritoneal macrophages

We have previously shown the expression of group X secretory phospholipase A 2 (sPLA 2-X) in mouse splenic macrophages and its powerful potency for releasing fatty acids from various intact cell membranes. Here, we examined the potency of sPLA 2-X in the production of lipid mediators in murine peritoneal macrophages. Mouse sPLA 2-X was found to induce a marked release of fatty acids including arachidonic acid and linoleic acid, which contrasted with little, if any, release by the action of group IB and IIA sPLA 2s. In resting macrophages, sPLA 2-X elicited a modest production of prostaglandin E 2 and thromboxane A 2. After the induction of cyclooxygenase-2 (COX-2) by pretreatment with lipopolysaccharide, a dramatic increase in the production of these eicosanoids was observed in sPLA 2-X-treated macrophages, which was completely blocked by the addition of either the specific sPLA 2 inhibitor indoxam or the COX inhibitor indomethacin. In accordance with its higher hydrolyzing activity toward phosphatidylcholine, mouse sPLA 2-X induced a potent production of lysophosphatidylcholine. These findings strongly suggest that sPLA 2-X plays a critical role in the production of various lipid mediators from macrophages. These events might be relevant to the progression of various pathological states, including chronic inflammation and atherosclerosis.

Group IIA and group V secretory phospholipase A 2: quantitative analysis of expression and secretion and determination of the localization and routing in rat mesangial cells

Mesangial cells can be induced to express group IIA and group V secretory phospholipase A 2 (sPLA 2) at the mRNA level and at the protein level. In this report we quantitatively analyze the expression of both proteins in stimulated cells by Western blot techniques. We found that 75–80% of the total amount of synthesized group IIA sPLA 2 was secreted. The synthesized group V sPLA 2, however, was present almost exclusively intracellularly. The amount of group V present in the cell was comparable to the intracellular amount of group IIA sPLA 2. We furthermore studied the localization and routing of both proteins. Using fusion proteins of the group IIA or group V pre-sPLA 2 with green fluorescent protein it was established that both presequences are able to direct the proteins to the Golgi system. In immunofluorescence studies group V sPLA 2 expressed by rat mesangial cells was located in a punctate pattern in the cytosol with an enrichment near the nucleus. Immunofluorescent confocal laser scanning microscopy revealed that the group V and IIA sPLA 2 show partial colocalization in a Golgi-like structure in the inner part in the cell, but no colocalization was seen in the vesicles in the cytoplasm. The images also showed that group IIA sPLA 2 was located throughout the cell while group V was mainly present in the inner part of the cell. After treatment of the cells with brefeldin A or monensin the group IIA enzyme could no longer be detected, while group V sPLA 2 was still present although its localization was somewhat dependent on the treatment. Collectively, these results indicate that the two enzymes differ in both localization and routing in the cell, which underscores the hypothesis that the enzymes might have different functions.

Mouse Group X Secretory Phospholipase A2 Induces a Potent Release of Arachidonic Acid from Spleen Cells and Acts as a Ligand for the Phospholipase A2 Receptor

Group X secretory phospholipase A2 (sPLA2-X) has recently been shown to possess a powerful potency for releasing arachidonic acid from cell membrane phospholipids. Here, we report the purification of mouse pro- and mature forms of sPLA2-X, as well as its expression and biological functions. Purified pro-sPLA2-X was found to possess a propeptide of 11 amino acid residues attached at the NH2-terminals of the mature protein, and showed as little as 8% of the PLA2 activity of the mature form. Limited proteolysis of pro-sPLA2-X with trypsin resulted in the appearance of the mature form with a concomitant increase in PLA2 activity, suggesting a requirement of proteolytic removal of the propeptide for the optimal activity. The expression of sPLA2-X mRNA was detected in various tissues including the lung, thymus, and spleen, and immunohistochemical analysis revealed its expression in splenic macrophages. In the spleen cells, mature sPLA2-X elicited a prompt release of arachidonic acid with significant production of prostaglandin E2 more efficiently than group IB and IIA sPLA2s. In addition, sPLA2-X was identified as a high-affinity ligand for both native and recombinant form of mouse PLA2 receptor (PLA2R). However, there was no significant difference in the sPLA2-X-induced arachidonic acid release responses in the spleen cells between wild-type and PLA2R-deficient mice. These findings strongly suggest that sPLA2-X possesses two distinct biological functions in mice: it elicits a marked release of arachidonic acid from membrane phospholipids leading to the production of lipid mediators based on its enzymatic potency, and it acts as a natural ligand for the PLA2R that has been shown to play a critical role in the production of inflammatory cytokines during endotoxic shock.

Inhibition of NFkappaB-mediated pro-inflammatory gene expression in rat mesangial cells by the enolized 1,3-dioxane-4, 6-dione-5-carboxamide, CGP-43182.

1. CGP-43182 has been described as a potent inhibitor of group IIA secreted phospholipase A(2) (group IIA sPLA(2)) activity in vitro. In rat mesangial cells, inhibition of group IIA sPLA(2) activity by CGP-43182 results in a 70% reduction of cytokine-stimulated prostaglandin E(2) biosynthesis, suggesting that group IIA sPLA(2) participates in arachidonic acid release and eicosanoid formation. Under these conditions the cytosolic phospholipase A(2) is not affected. 2. In mesangial cells, in addition to inhibition of catalytic activity, the membrane-permeant CGP-43182 completely blocked interleukin 1beta (IL1beta)-stimulated group IIA sPLA(2) gene expression. 3. A further action of CGP-43182 was a complete inhibition of cyclo-oxygenase-2 gene expression, resulting in a drastic reduction of prostaglandin formation in mesangial cells. 4. Moreover, CGP-43182 completely blocked IL1beta-induced gene expression of the inducible nitric oxide synthase, leading to an inhibition of cytokine-stimulated nitric oxide formation. 5. In contrast, the stimulatory effect of the cell-permeant cyclic AMP-analogue, dibutyryl-cAMP, on the induction of these enzymes was not inhibited by CGP-43182. These data indicate that CGP-43182 interferes with IL1beta- but not cyclic AMP-activated transcriptional regulation. 6. By studying components of the upstream transcription machinery, we observed an inhibition of NFkappaB activation by CGP-43182 in IL1beta-treated cells. Moreover, we observed that CGP-43182 prevented the phosphorylation and proteolytic degradation of the endogenous NFkappaB inhibitor, IkappaB, a process necessary for NFkappaB activation. 7. From our data, we propose that CGP-43182 is a potent anti-inflammatory drug useful for preventing the consequences of a concerted action of cytokine-stimulated pro-inflammatory genes mediated by NFkappaB.

Secretory phospholipases A2 induce beta-glucuronidase release and IL-6 production from human lung macrophages.

Secretory phospholipases A2 (sPLA2s) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 has been detected in inflammatory fluids, and its plasma level is increased in inflammatory diseases. To investigate a potential mechanism of sPLA2-induced inflammation we studied the effect of group IA (from cobra venom) and group IIA (human synovial) sPLA2s on human macrophages. Both sPLA2s induced a concentration- and Ca2+-dependent, noncytotoxic release of beta-glucuronidase (16.2 +/- 2.4% and 13.1 +/- 1.5% of the total content with groups IA and IIA, respectively). Both sPLA2s also increased the rate of secretion of IL-6 and enhanced the expression of IL-6 mRNA. Preincubation of macrophages with inhibitors of the hydrolytic activity of sPLA2 or cytosolic PLA2 did not influence the release of beta-glucuronidase. Incubation of macrophages with p-aminophenyl-mannopyranoside-BSA (mp-BSA), a ligand of the mannose receptor, also resulted in beta-glucuronidase release. However, while preincubation of macrophages with mp-BSA had no effect on beta-glucuronidase release induced by group IIA sPLA2, it enhanced that induced by group IA sPLA2. A blocking Ab anti-mannose receptor inhibited both mp-BSA- and group IIA-induced beta-glucuronidase release. Taken together, these data indicate that group IA and IIA sPLA2s activate macrophages with a mechanism independent from their enzymatic activities and probably related to the activation of the mannose receptor or sPLA2-specific receptors. The secretion of enzymes and cytokines induced by sPLA2s from human macrophages may play an important role in inflammation and tissue damage associated with the release of sPLA2s.

Bacterial cell membrane hydrolysis by secreted phospholipases A 2: a major physiological role of human group IIa sPLA 2 involving both bacterial cell wall penetration and interfacial catalysis

The ability of human group IIa secreted phospholipase A 2 (human sPLA 2) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA 2s. This specificity is due to selective penetration of the highly cationic human sPLA 2 through the highly anionic bacterial cell wall. Disruption of the peptidoglycan cell wall by treatment with lysozyme allows all three enzymes to express similar hydrolytic activity against the anionic bacterial cell membrane. Extensive (>50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA 2 activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA 2 as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA 2s. Overall, the results highlight the potential of the human sPLA 2 as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected.