Pseudomonas aeruginosa and sPLA2 IB stimulate ABCA1-mediated phospholipid efflux via ERK-activation of PPARalpha-RXR

Bacterial infection triggers an acute inflammatory response that might alter phospholipid metabolism. We have investigated the acute-phase response of murine lung epithelia to Pseudomonas aeruginosa infection. Ps. aeruginosa triggered secretion of the pro-inflammatory lipase, sPLA2 IB (phospholipase A2 IB), from lung epithelium. Ps. aeruginosa and sPLA2 IB each stimulated basolateral PtdCho (phosphatidylcholine) efflux in lung epithelial cells. Pre-treatment of cells with glyburide, an inhibitor of the lipid-export pump, ABCA1 (ATP-binding cassette transporter A1), attenuated Ps. aeruginosa and sPLA2 IB stimulation of PtdCho efflux. Effects of Ps. aeruginosa and sPLA2 IB were completely abolished in human Tangier disease fibroblasts, cells that harbour an ABCA1 genetic defect. Ps. aeruginosa and sPLA2 IB induced the heterodimeric receptors, PPARa (peroxisome-proliferator-activated receptor-a) and RXR (retinoid X receptor), factors known to modulate ABCA1 gene expression. Ps. aeruginosa and sPLA2 IB stimulation of PtdCho efflux was blocked with PD98059, a p44/42 kinase inhibitor. Transfection with MEK1 (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase 1), a kinase upstream of p44/42, increased PPARa and RXR expression co-ordinately with increased ABCA1 protein. These results suggest that pro-inflammatory effects of Ps. aeruginosa involve release of an sPLA2 of epithelial origin that, in part, via distinct signalling molecules, transactivates the ABCA1 gene, leading to export of phospholipid.     

Both group IB and group IIA secreted phospholipases A2 are natural ligands of the mouse 180-kDa M-type receptor

Snake venom and mammalian secreted phospholipases A2 (sPLA2s) have been associated with toxic (neurotoxicity, myotoxicity, etc.), pathological (inflammation, cancer, etc.), and physiological (proliferation, contraction, secretion, etc.) processes. Specific membrane receptors (M and N types) for sPLA2s have been initially identified with snake venom sPLA2s as ligands, and the M-type 180-kDa receptor was cloned from different animal species. This paper addresses the problem of the endogenous ligands of the M-type receptor. Recombinant group IB and group IIA sPLA2s from human and mouse species have been prepared and analyzed for their binding properties to M-type receptors from different animal species. Both mouse group IB and group IIA sPLA2s are high affinity ligands (in the 1-10 nM range) for the mouse M-type receptor. These two sPLA2s are expressed in the mouse tissues where the M-type receptor is also expressed, making it likely that both types of sPLA2s are physiological ligands of the mouse M-type receptor. This conclusion does not hold for human group IB and IIA sPLA2s and the cloned human M-type receptor. The two mouse sPLA2s have relatively high affinities for the mouse M-type receptor, but they can have much lower affinities for receptors from other animal species, indicating that species specificity exists for sPLA2 binding to M-type receptors. Caution should thus be exerted in avoiding mixing sPLA2s, cells, or tissues from different animal species in studies of the biological roles of mammalian sPLA2s associated with an action through their membrane receptors.     

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(2)s. 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.     

Identification of group X secretory phospholipase A(2) as a natural ligand for mouse phospholipase A(2) receptor

Phospholipase A(2) receptor (PLA(2)R) mediates various biological responses elicited by group IB secretory phospholipase A(2) (sPLA(2)-IB). The recently cloned group X sPLA(2) (sPLA(2)-X) possesses several structural features characteristic of sPLA(2)-IB. Here, we detected a specific binding site of sPLA(2)-X in mouse osteoblastic MC3T3-E(1) cells. Cross-linking experiments demonstrated its molecular weight (180 kDa) to be similar to that of PLA(2)R. In fact, sPLA(2)-X was found to bind the recombinant PLA(2)R expressed in COS-7 cells, and its specific binding detected in mouse lung membranes was abolished by the deficiency of PLA(2)R. These findings demonstrate sPLA(2)-X to be one of the high-affinity ligands for mouse PLA(2)R.     

Activation of cytokine production by secreted phospholipase A2 in human lung macrophages expressing the M-type receptor

Secreted phospholipases A(2) (sPLA(2)) are enzymes released in plasma and extracellular fluids during inflammatory diseases. Because human group IB and X sPLA(2)s are expressed in the lung, we examined their effects on primary human lung macrophages (HLM). Both sPLA(2)s induced TNF-alpha and IL-6 release in a concentration-dependent manner by increasing their mRNA expression. This effect was independent of their enzymatic activity because 1) the capacity of sPLA(2)s to mobilize arachidonic acid from HLM was unrelated to their ability to induce cytokine production; and 2) two catalytically inactive isoforms of group IB sPLA(2) (bromophenacyl bromide-inactivated human sPLA(2) and the H48Q mutant of the porcine sPLA(2)) were as effective as the catalytically active sPLA(2)s in inducing cytokine production. HLM expressed the M-type receptor for sPLA(2)s at both mRNA and protein levels, as determined by RT-PCR, immunoblotting, immunoprecipitation, and flow cytometry. Me-indoxam, which decreases sPLA(2) activity as well as binding to the M-type receptor, suppressed sPLA(2)-induced cytokine production. Incubation of HLM with the sPLA(2)s was associated with phosphorylation of ERK1/2, and a specific inhibitor of this pathway, PD98059, significantly reduced the production of IL-6 elicited by sPLA(2)s. In conclusion, two distinct sPLA(2)s produced in the human lung stimulate cytokine production by HLM via a mechanism that is independent of their enzymatic activity and involves activation of the ERK1/2 pathway. HLM express the M-type receptor, but its involvement in eliciting cytokine production deserves further investigation.     

Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A(2).

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.     

Exogenously added human group X secreted phospholipase A(2) but not the group IB, IIA, and V enzymes efficiently release arachidonic acid from adherent mammalian cells

Mammalian secreted phospholipases A(2) (sPLA2s) comprise a group of at least eight enzymes, including the recently identified group X sPLA2. A bacterial expression system was developed to produce human group X sPLA2 (hGX). Inhibition studies show that the sPLA2 inhibitor LY311727 binds modestly more tightly to human group IIA sPLA2 than to hGX and that a pyrazole-based inhibitor of group IIA sPLA2 is much less active against hGX. The phospholipid head group preference of vesicle-bound hGX was determined. hGX binds tightly to phosphatidylcholine vesicles, which is thought to be required to act efficiently on cells. Tryptophan 67 hGX makes a significant contribution to interfacial binding to zwitterionic vesicles. As little as 10 ng/ml hGX releases arachidonic acid for cyclooxygenase-2- dependent prostaglandin E(2) generation when added exogenously to adherent mammalian cells. In contrast, human group IIA, rat group V, and mouse group IB sPLA2s are virtually inactive at releasing arachidonate when added exogenously to adherent cells. Dislodging cells from the growth surface enhances the ability of all the sPLA2s to release fatty acids. Studies with CHO-K1 cell mutants show that binding of sPLA2s to glycosaminoglycans is not the basis for poor plasma membrane hydrolysis by group IB, IIA, and V sPLA2s.     

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(2)s 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(2)s can participate in inflammatory diseases according to tissues, animal species, and types of inflammation.     

Novel mammalian group XII secreted phospholipase A2 lacking enzymatic activity

An increasing number of mammalian secreted phospholipases A(2) (sPLA(2)s) has been identified over the past few years. Here, we report the identification and recombinant expression of a novel sPLA(2)-like protein in mouse and human species that has been called group XIIB (GXIIB). The mature protein has a molecular mass of 19.7 kDa and structural features similar to those of the previously identified GXII sPLA(2), now called GXIIA. Strikingly, the GXIIB sPLA(2) has a mutation in the active site, replacing the canonical histidine by a leucine, suggesting that this sPLA(2) is catalytically inactive. Recombinant expression of human (hGXIIB) and mouse (mGXIIB) sPLA(2)s in Escherichia coli indicates that GXIIB sPLA(2)s display no measurable lipolytic activity on various types of phospholipid substrates. Furthermore, these sPLA(2)-like proteins display relatively weak affinity to phospholipid vesicles. Binding experiments indicate that these proteins are also unable to bind to the well-known M-type sPLA(2) receptor. The RNA tissue distribution of GXIIB sPLA(2)s is distinct from that of other sPLA(2)s including the homologous GXIIA. Strong expression was observed in liver, small intestine, and kidney in both human and mouse species. Interestingly, the expression of the novel sPLA(2) is dramatically decreased in human tumors from the same tissues. The absence of enzymatic activity suggests that the GXIIB sPLA(2)-like proteins probably exert their biological roles by acting as ligands for as yet unidentified receptors.     

Secreted phospholipases A2 induce the expression of chemokines in microvascular endothelium

Acute respiratory distress syndrome (ARDS) is characterized by alterations in microvascular permeability. In ARDS secreted phospholipase A(2) (sPLA(2)) IB and IIA are found to be highly upregulated. In this study, we therefore investigated the influence of exogenously added sPLA(2)-IB and sPLA(2)-IIA on the production of chemokines and adhesion molecules in lung microvascular endothelial cells (LMVEC). Treatment of LMVEC with sPLA(2)s resulted in a significant increase in the production of chemokines and adhesion molecules due to an increased expression of their mRNA and in an enhanced release of oleic acid. The upregulation of chemokines and adhesion molecules by LPS was stronger in the presence of sPLA(2). Activation of NF-kappaB occurred upon stimulation with sPLA(2). Moreover the MAPkinase pERK seems to be involved since a specific pERK inhibitor, e.g., U0126, but not a p38Kinase inhibitor, e.g., SB203580 prevented sPLA(2)-induced chemokine upregulation. Our data therefore suggest that LMVEC are a highly sensitive target for the direct action of extracellular sPLA(2)s.     

Suppression of murine endotoxic shock by sPLA2 inhibitor, indoxam, through group IIA sPLA2-independent mechanisms.

Endotoxic shock is a systemic inflammatory process, involving a variety of proinflammatory mediators. Two types of secretory phospholipase A2 (sPLA2) have been implicated in this process. Group IB sPLA2 (PLA2-IB) binds to the PLA2 receptor (PLA2R), and PLA2R-deficient mice exhibit resistance to endotoxin-induced lethality with reduced plasma levels of proinflammatory cytokines, such as TNF-alpha. Group IIA sPLA2 (PLA2-IIA) is found in many tissues and cell types, and local and systemic levels are elevated under numerous inflammatory conditions including sepsis. In this study, we investigated the effect of a specific sPLA2 inhibitor, indoxam, on murine endotoxic shock. Indoxam suppressed the elevation of plasma TNF-alpha with a similar potency in PLA2-IIA-expressing and PLA2-IIA-deficient mice after LPS challenge. In PLA2-IIA-deficient mice, indoxam also suppressed the elevation of plasma IL-1beta, IL-6 and NO, and prolonged survival after LPS challenge. Indoxam was found to block the PLA2-IB binding to murine PLA2R with a high potency (Ki=30 nM). The inhibitory effects of indoxam on the LPS-induced elevation of plasma TNF-alpha levels could not be observed in mice deficient in PLA2R. These findings suggest that indoxam blocks the production of proinflammatory cytokines during endotoxemia through PLA2-IIA-independent mechanisms, possibly via blockade of the PLA2R function.     

Regulation of the expression of group IIA and group V secretory phospholipases A(2) in rat mesangial cells

Rat mesangial cells synthesize and secrete a secretory phospholipase A(2) upon stimulation of the cells with cytokines, like IL-1beta and TNF and with cAMP elevating agents like forskolin. This enzyme was previously characterized to belong to group IIA sPLA(2). The discovery of several other low molecular weight phospholipases, like group IIC in murine testis and group V in human and rat heart, prompted investigations on the presence of group IIC and group V sPLA(2) in rat mesangial cells. This was done by isolating the RNA from stimulated cells and performing RT-PCR, using primers specific for group IIC and V sPLA(2). The results indicate that rat mesangial cells upon stimulation express next to group IIA also group V sPLA(2). No indications were obtained for the expression of group IIC sPLA(2). The regulation of the expression of group V sPLA(2) at the mRNA level was further investigated by examining the time-dependent expression, the influence of dexamethasone and the signaling route of the IL-1beta stimulation. The results show that the IL-1beta induced expression of group V sPLA(2) mRNA was time dependent and, similar to that of group IIA sPLA(2) mRNA, involves activation of NF-kappaB. However, in contrast to the group IIA sPLA(2), the expression of group V sPLA(2) was not influenced by the presence of dexamethasone. The expression of both phospholipases was also examined at the protein level in stimulated mesangial cells. Western blot analysis shows that stimulated mesangial cells synthesize both group IIA and group V sPLA(2) protein but the expression of group V is lower compared to that of group IIA sPLA(2). In addition, the extent of secretion into the medium appears to be considerably higher for group IIA than for group V sPLA(2).     

Murine ortholog of the novel glycosyltransferase, B3GTL: primary structure, characterization of the gene and transcripts, and expression in tissues

Glycosylation of proteins and lipids is important in cellular communication and maintenance of tissues. B3GTL (beta3-glycosyltransferase-like) is a novel glycosyltransferase that is found in multicellular animals ranging from mammals to insects and nematodes. The aim of this work was to identify and characterize the B3GTL gene in the mouse and to study its expression in various tissues. The murine gene codes for a protein which shares 84% amino acid sequence identity with its human ortholog, and contains all the primary structural features that characterize B3GTL proteins. The murine and human B3GTL genes share an identical exon/intron organization, and both genes utilize multiple polyadenylation signals. Their promoter regions show extensive conservation, implying that the two genes also share regulatory similarities. This notion was reinforced by Northern hybridization analysis of mouse tissues, which showed the tissue distribution of B3GTL mRNA to be similar to that previously found in human tissues, with the heart, kidney, and brain being major sites of expression in both species. The localization of B3GTL mRNA was studied by in situ hybridization in an extensive collection of mouse tissues, of which the granular cells of the olfactory bulb and the epithelium of the seminal vesicle displayed particularly strong signals. Together, these analyses indicate that the B3GTL mRNA is subject to strong tissue-specific and developmental regulation. The findings reported here make possible the design of a B3GTL "knock-out" mouse, provide a framework for analyzing the regulation of the gene, and provide an extensive catalog of tissues in which this novel protein acts.     

Murine glutamine synthetase: Cloning, developmental regulation, and glucocorticoid inducibility

We have cloned the murine glutamine synthetase (GS) gene and measured GS enzyme activity and mRNA in five tissues (retina, brain, liver, kidney, and skeletal muscle) during perinatal development. Retinal GS enzyme activity increases 200-fold between Day 1 and Day 21 and is accompanied by an increase in the level of GS mRNA; developmental regulation in other tissues is much less dramatic. Based on Southern blotting analysis, a single GS gene gives rise to the tissue-specific patterns of GS mRNA expression. The increase in murine retinal GS observed during perinatal development is similar in magnitude to that observed in the chicken retina just prior to hatching. In the embryonic chicken retina, glucocorticoid hormones mediate a large increase in the level of GS mRNA. However, although glucocorticoids induce a 12-fold increase in GS mRNA in murine skeletal muscle, expression of the retinal enzyme and mRNA is only modestly glucocorticoid-inducible in the mouse. Therefore, despite the hormonal responsiveness of the murine GS gene, it is not likely that glucocorticoids are important physiological modulators of the developmental rise in murine retinal GS.     

Internalization and degradation of type IIA phospholipase A(2) in mast cells

Whereas exogenous types IB and X secretory phospholipase A(2) (sPLA(2)) elicited prostaglandin D(2) (PGD(2)) production in mouse bone marrow-derived mast cells (BMMC), sPLA(2)-IIA was unable to do so. In search of a mechanism underlying this cellular refractoriness to exogenous sPLA(2)-IIA, we now report that this isozyme is promptly associated with cell surfaces, internalized, and then degraded in BMMC. Adsorption of sPLA(2)-IIA to BMMC was prevented by addition of heparin to the medium. Moreover, a heparin-nonbinding sPLA(2)-IIA mutant did not bind to BMMC. These results indicate that this sPLA(2)-IIA inactivation process depends on its rapid binding to heparan sulfate proteoglycan (HSPG) on BMMC surfaces. Thus, the present observations represent a particular situation in which cell surface HSPG exhibit a negative regulatory effect on cellular function of sPLA(2)-IIA, and argue that HSPG does not always act as a functional adapter for heparin-binding sPLA(2)s in mammalian cells as has been demonstrated before.     

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.