Distinct arachidonate-releasing functions of mammalian secreted phospholipase A2s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms

We analyzed the ability of a diverse set of mammalian secreted phospholipase A(2) (sPLA(2)) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA(2)-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E(2) production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA(2)-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA(2)-IIC allows it to release arachidonate. Heparin nonbinding sPLA(2)-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA(2)-V and -X, which are unique among sPLA(2)s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C(4) and platelet-activating factor. sPLA(2)-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA(2) mutants and immunocytostaining and reveal that sPLA(2)-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells.     

Presence of the M-type sPLA(2) receptor on neutrophils and its role in elastase release and adhesion

Secretory phospholipase A(2) (sPLA(2)) produces lipids that stimulate polymorphonuclear neutrophils (PMNs). With the discovery of sPLA(2) receptors (sPLA(2)-R), we hypothesize that sPLA(2) stimulates PMNs through a receptor. Scatchard analysis was used to determine the presence of a sPLA(2) ligand. Lysates were probed with an antibody to the M-type sPLA(2)-R, and the immunoreactivity was localized. PMNs were treated with active and inactive (+EGTA) sPLA(2) (1-100 units of enzyme activity/ml, types IA, IB, and IIA), and elastase release and PMN adhesion were measured. PMNs incubated with inactive, FITC-linked sPLA(2)-IB, but not sPLA(2)-IA, demonstrated the presence of a sPLA(2)-R with saturation at 2.77 fM and a K(d) of 167 pM. sPLA(2)-R immunoreactivity was present at 185 kDa and localized to the membrane. Inactive sPLA(2)-IB activated p38 MAPK, and p38 MAPK inhibition attenuated elastase release. Active sPLA(2)-IA caused elastase release, but inactive type IA did not. sPLA(2)-IB stimulated elastase release independent of activity; inactive sPLA(2)-IIA partially stimulated PMNs. sPLA(2)-IB and sPLA(2)-IIA caused PMN adhesion. We conclude that PMNs contain a membrane M-type sPLA(2)-R that activates p38 MAPK.     

Polyunsaturated fatty acids potentiate interleukin-1-stimulated arachidonic acid release by cells overexpressing type IIA secretory phospholipase A2.

By analyzing human embryonic kidney 293 cell transfectants stably overexpressing various types of phospholipase A2 (PLA2), we have shown that polyunsaturated fatty acids (PUFAs) preferentially activate type IIA secretory PLA2 (sPLA2-IIA)-mediated arachidonic acid (AA) release from interleukin-1 (IL-1)-stimulated cells. When 293 cells prelabeled with 13H]AA were incubated with exogenous PUFAs in the presence of IL-1 and serum, there was a significant increase in [3H]AA release (in the order AA > linoleic acid > oleic acid), which was augmented markedly by sPLA2-IIA and modestly by type IV cytosolic PLA2 (cPLA2), but only minimally by type VI Ca2(+)-independent PLA2, overexpression. Transfection of cPLA2 into sPLA2-IIA-expressing cells produced a synergistic increase in IL-1-dependent [3H]AA release and subsequent prostaglandin production. Our results support the proposal that prior production of AA by cPLA2 in cytokine-stimulated cells destabilizes the cellular membranes, thereby rendering them more susceptible to subsequent hydrolysis by sPLA2-IIA.     

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.     

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.     

Potential role of group X secretory phospholipase A(2) in cyclooxygenase-2-dependent PGE(2) formation during colon tumorigenesis

Although the cyclooxygenase-2 (COX-2) pathway of the arachidonic acid cascade has been suggested to play an important role in colon carcinogenesis, there is little information concerning the identity of phospholipase A(2) (PLA(2)) involved in the arachidonic acid release in colon tumors. Here, we compared the potencies of three types of secretory PLA(2)s (group IB, IIA and X sPLA(2)s) for the arachidonic acid release from cultured human colon adenocarcinoma cells, and found that group X sPLA(2) has the most powerful potency in the release of arachidonic acid leading to COX-2-dependent prostaglandin E(2) (PGE(2)) formation. Furthermore, immunohistological analysis revealed the elevated expression of group X sPLA(2) in human colon adenocarcinoma neoplastic cells in concert with augmented expression of COX-2. These findings suggest a critical role of group X sPLA(2) in the PGE(2) biosynthesis during colon tumorigenesis.     

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.     

Biological effects of group IIA secreted phosholipase A(2)

Group IIA secreted phospholipase A(2) (sPLA(2)-IIA) is the most abundant element in human tissues of a large family of low molecular weight phospholipases A(2), which shows properties different from those displayed by the cytosolic phospholipase A(2) involved in the release of arachidonic acid. sPLA(2)-IIA behaves as a ligand for a group of receptors inside the C-type multilectin mannose receptor family and also interacts with heparan sulfate proteoglycans such as glypican, the dermatan/chondroitin sulfate-rich decorin, and the chondroitin sulfate-rich versican, thus being able to internalize to specific compartments within the cell and producing biological responses. This review provides a short summary of the biological actions of sPLA(2)-IIA on intracellular signaling pathways.     

Functional association of type IIA secretory phospholipase A(2) with the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan in the cyclooxygenase-2-mediated delayed prostanoid-biosynthetic pathway.

An emerging body of evidence suggests that type IIA secretory phospholipase A(2) (sPLA(2)-IIA) participates in the amplification of the stimulus-induced cyclooxygenase (COX)-2-dependent delayed prostaglandin (PG)-biosynthetic response in several cell types. However, the biological importance of the ability of sPLA(2)-IIA to bind to heparan sulfate proteoglycan (HSPG) on cell surfaces has remained controversial. Here we show that glypican, a glycosylphosphatidylinositol (GPI)-anchored HSPG, acts as a physical and functional adaptor for sPLA(2)-IIA. sPLA(2)-IIA-dependent PGE(2) generation by interleukin-1-stimulated cells was markedly attenuated by treatment of the cells with heparin, heparinase or GPI-specific phospholipase C, which solubilized the cell surface-associated sPLA(2)-IIA. Overexpression of glypican-1 increased the association of sPLA(2)-IIA with the cell membrane, and glypican-1 was coimmunoprecipitated by the antibody against sPLA(2)-IIA. Glypican-1 overexpression led to marked augmentation of sPLA(2)-IIA-mediated arachidonic acid release, PGE(2) generation, and COX-2 induction in interleukin-1-stimulated cells, particularly when the sPLA(2)-IIA expression level was suboptimal. Immunofluorescent microscopic analyses of cytokine-stimulated cells revealed that sPLA(2)-IIA was present in the caveolae, a microdomain in which GPI-anchored proteins reside, and also appeared in the perinuclear area in proximity to COX-2. We therefore propose that a GPI-anchored HSPG glypican facilitates the trafficking of sPLA(2)-IIA into particular subcellular compartments, and arachidonic acid thus released from the compartments may link efficiently to the downstream COX-2-mediated PG biosynthesis.     

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.     

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.     

Pro-inflammatory secretory phospholipase A2 type IIA binds to integrins alphavbeta3 and alpha4beta1 and induces proliferation of monocytic cells in an integrin-dependent manner

Secretory phospholipase A2 group IIA (sPLA2-IIA) plays an important role in the pathogenesis of inflammatory diseases. Catalytic activity of this enzyme that generates arachidonic acid is a major target for development of anti-inflammatory agents. Independent of its catalytic activity, sPLA2-IIA induces pro-inflammatory signals in a receptor-mediated mechanism (e.g. through the M-type receptor). However, the M-type receptor is species-specific: sPLA2-IIA binds to the M-type receptor in rodents and rabbits, but not in human. Thus sPLA2-IIA receptors in human have not been established. Here we demonstrated that sPLA2-IIA bound to integrin alphavbeta3 at a high affinity (K(D) = 2 x 10(-7) m). We identified amino acid residues in sPLA2-IIA (Arg-74 and Arg-100) that are critical for integrin binding using docking simulation and mutagenesis. The integrin-binding site did not include the catalytic center or the M-type receptor-binding site. sPLA2-IIA also bound to alpha4beta1. We showed that sPLA2-IIA competed with VCAM-1 for binding to alpha4beta1, and bound to a site close to those for VCAM-1 and CS-1 in the alpha4 subunit. Wild type and the catalytically inactive H47Q mutant of sPLA2-IIA induced cell proliferation and ERK1/2 activation in monocytic cells, but the integrin binding-defective R74E/R100E mutant did not. This indicates that integrin binding is required, but catalytic activity is not required, for sPLA2-IIA-induced proliferative signaling. These results suggest that integrins alphavbeta3 and alpha4beta1 may serve as receptors for sPLA2-IIA and mediate pro-inflammatory action of sPLA2-IIA, and that integrin-sPLA2-IIA interaction is a novel therapeutic target.     

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.     

A novel approach to the design of inhibitors of human secreted phospholipase A2 based on native peptide inhibition.

Human Type IIA secreted phospholipase A(2) (sPLA(2)-IIA) is an important modulator of cytokine-dependent inflammatory responses and a member of a growing superfamily of structurally related phospholipases. We have previously shown that sPLA(2)-IIA is inhibited by a pentapeptide sequence comprising residues 70-74 of the native sPLA(2)-IIA protein and that peptides derived from the equivalent region of different sPLA(2)-IIA species specifically inhibit the enzyme from which they are derived. We have now used an analogue screen of the human pentapeptide (70)FLSYK(74) in which side-chain residues were substituted, together with molecular docking approaches that modeled low-energy conformations of (70)FLSYK(74) bound to human sPLA(2)-IIA, to generate inhibitors with improved potency. Importantly, the modeling studies showed a close association between the NH(2) and COOH termini of the peptide, predicting significant enhancement of the potency of inhibition by cyclization. Cyclic compounds were synthesized and indeed showed 5-50-fold increased potency over the linear peptide in an Escherichia coli membrane assay. Furthermore, the potency of inhibition correlated with steady-state binding of the cyclic peptides to sPLA(2)-IIA as determined by surface plasmon resonance studies. Two potential peptide interaction sites were identified on sPLA(2)-IIA from the modeling studies, one in the NH(2)-terminal helix and the other in the beta-wing region, and in vitro association assays support the potential for interaction of the peptides with these sites. The inhibitors were effective at nanomolar concentrations in blocking sPLA(2)-IIA-mediated amplification of cytokine-induced prostaglandin synthesis in human rheumatoid synoviocytes in culture. These studies provide an example where native peptide sequences can be used for the development of potent and selective inhibitors of enzyme function.     

Delayed production of arachidonic acid contributes to the delay of proteinase-activated receptor-1 (PAR1)-triggered prostaglandin E2 release in rat gastric epithelial RGM1 cells

Proteinase-activated receptor-1 (PAR1), upon activation, exerts prostanoid-dependent gastroprotection, and increases prostaglandin E(2) (PGE(2)) release through cyclooxygenase-2 (COX-2) upregulation in rat gastric mucosal epithelial RGM1 cells. However, there is a big time lag between the PAR1-triggered PGE(2) release and COX-2 upregulation in RGM1 cells; that is, the former event takes 18 h to occur, while the latter rapidly develops and reaches a plateau in 6 h. The present study thus aimed at clarifying mechanisms for the delay of PGE(2) release after PAR1 activation in RGM1 cells. Although a PAR1-activating peptide, TFLLR-NH(2), alone caused PGE(2) release at 18 h, but not 6 h, TFLLR-NH(2) in combination with arachidonic acid dramatically enhanced PGE(2) release even for 1-6 h. TFLLR-NH(2) plus linoleic acid caused a similar rapid response. CP-24879, a Δ(5)/Δ(6)-desaturase inhibitor, abolished the PGE(2) release induced by TFLLR-NH(2) plus linoleic acid, but not by TFLLR-NH(2) alone. The TFLLR-NH(2)-induced PGE(2) release was not affected by inhibitors of cytosolic phospholipase A(2) (cPLA(2)), Ca(2+)-independent PLA(2) (cPLA(2)) or secretory PLA(2) (sPLA(2)), but was abolished by their mixture or a pan-PLA(2) inhibitor. Among PLA(2) isozymes, mRNA of group IIA sPLA(2) (sPLA(2)-IIA) was upregulated following PAR1 stimulation for 6-18 h, whereas protein levels of PGE synthases were unchanged. These data suggest that the delay of PGE(2) release after COX-2 upregulation triggered by PAR1 is due to the poor supply of free arachidonic acid at the early stage in RGM1 cells, and that plural isozymes of PLA(2) including sPLA(2)-IIA may complementarily contribute to the liberation of free arachidonic acid.