Hydrolysis of minor glycerophospholipids of plasma lipoproteins by human group IIA, V and X secretory phospholipases A2

We investigated the hydrolysis of the minor glycerophospholipids of human HDL(3), total HDL and LDL using human group IIA, V and X secretory phospholipases A(2) (sPLA(2)s). For this purpose we employed the enzyme and substrate concentrations and incubation times optimized for hydrolysis of phosphatidylcholine (PtdCho), the major glycerophospholipid of plasma lipoproteins. In contrast to PtdCho, which was readily hydrolyzed by group V and X sPLA(2)s, and to a lesser extent by group IIA sPLA(2), the minor ethanolamine, inositol and serine glycerophospholipids exhibited marked resistance to hydrolysis by all three sPLA(2)s. Thus, when PtdCho was hydrolyzed about 80%, the ethanolamine and inositol glycerophospholipids reached a maximum of 40% hydrolysis. The hydrolysis of phosphatidylserine (PtdSer), which was examined to a more limited extent, showed similar resistance to group IIA, V and X sPLA(2)s, although the group V sPLA(2) attacked it more readily than group X sPLA(2) (52% versus 39% hydrolysis, respectively). Surprisingly, the group IIA sPLA(2) hydrolysis remained minimal at 10-15% for all minor glycerophospholipids, and was of the order seen for the PtdCho hydrolysis by group IIA sPLA(2) at the 4-h digestion time. All three enzymes attacked the oligo- and polyenoic species in proportion to their mole percentage in the lipoproteins, although there were exceptions. There was evidence of a more rapid destruction of the palmitoyl compared to the stearoyl arachidonoyl glycerophospholipids. Overall, the characteristics of hydrolysis of the molecular species of the lipoprotein-bound diradyl GroPEtn, GroPIns and GroPSer by group V and X sPLA(2)s differed significantly from those observed with lipoprotein-bound PtdCho. As a result, the acidic inositol and serine glycerophospholipids accumulated in the digestion residues of both LDL and HDL, and presumably increased the acidity of the residual particles. An accumulation of the ethanolamine glycerophospholipids in the sPLA(2) digestion residues also had not been previously reported. These results further emphasize the diversity in the enzymatic activity of the group IIA, V and X sPLA(2)s. Since these sPLA(2)s possess comparable tissue distribution, their combined activity may exacerbate their known proinflammatory and proatherosclerotic function.     

Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase

LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A₂ (sPLA₂-IIa and sPLA₂-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA₂-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA₂-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA₂-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis.     

Clearance of group X secretory phospholipase A(2) via mouse phospholipase A(2) receptor.

Given the potent hydrolyzing activity toward phosphatidylcholine, group X secretory phospholipase A(2) (sPLA(2)-X) elicits a marked release of arachidonic acid linked to the potent production of lipid mediators in various cell types. We have recently shown that sPLA(2)-X can also act as a ligand for mouse phospholipase A(2) receptor (PLA(2)R). Here, we found that sPLA(2)-X was internalized and degraded via binding to PLA(2)R associated with the diminished prostaglandin E(2) (PGE(2)) formation in PLA(2)R-expressing Chinese hamster ovary (CHO) cells compared to CHO cells. Indirect immunocytochemical analysis revealed that internalized sPLA(2)-X was co-localized with PLA(2)R in the punctate structures in PLA(2)R-expressing CHO cells. Moreover, in mouse osteoblastic MC3T3-E(1) cells that endogenously express the PLA(2)R, the internalized sPLA(2)-X was localized in lysosomes. These findings demonstrate that PLA(2)R acts as a clearance receptor for sPLA(2)-X to suppress its strong enzymatic activity.     

Distinctiveness of secretory phospholipase A2 group IIA and V suggesting unique roles in atherosclerosis

Clinical observations strongly support an association of circulating levels of secretory phospholipases A(2) (sPLA(2)) in atherosclerotic cardiovascular disease (ACVD). Two modes of action can provide causal support for these statistical correlations. One is the action of the enzymes on circulating lipoproteins and the other is direct action on the lipoproteins once in the arterial extracellular intima. In this review we discuss results suggesting a distinct profile of characteristics related to localization, action on plasma lipoproteins and interaction with arterial proteoglycans for sPLA(2)-IIA and sPLA(2)-V. The differences observed indicate that these enzymes may contribute to atherosclerosis through dissimilar pathways. Furthermore, we comment on recent animal studies from our laboratory indicating that the expression of type V enzyme is up-regulated by genetically and nutritionally-induced dyslipidemias but not the group type IIA enzyme, which is well known to be up-regulated by acute inflammation. The results suggest that if similar up-regulation occurs in humans in response to hyperlipidemia, it may create a distinctive link between the group V enzyme and the disease.     

Potent modification of low density lipoprotein by group X secretory phospholipase A2 is linked to macrophage foam cell formation

The deposition of cholesterol ester within foam cells of the artery wall is fundamental to the pathogenesis of atherosclerosis. Modifications of low density lipoprotein (LDL), such as oxidation, are prerequisite events for the formation of foam cells. We demonstrate here that group X secretory phospholipase A2 (sPLA2-X) may be involved in this process. sPLA2-X was found to induce potent hydrolysis of phosphatidylcholine in LDL leading to the production of large amounts of unsaturated fatty acids and lysophosphatidylcholine (lyso-PC), which contrasted with little, if any, lipolytic modification of LDL by the classic types of group IB and IIA secretory PLA2s. Treatment with sPLA2-X caused an increase in the negative charge of LDL with little modification of apolipoprotein B (apoB) in contrast to the excessive aggregation and fragmentation of apoB in oxidized LDL. The sPLA2-X-modified LDL was efficiently incorporated into macrophages to induce the accumulation of cellular cholesterol ester and the formation of non-membrane-bound lipid droplets in the cytoplasm, whereas the extensive accumulation of multilayered structures was found in the cytoplasm in oxidized LDL-treated macrophages. Immunohistochemical analysis revealed marked expression of sPLA2-X in foam cell lesions in the arterial intima of high fat-fed apolipoprotein E-deficient mice. These findings suggest that modification of LDL by sPLA2-X in the arterial vessels is one of the mechanisms responsible for the generation of atherogenic lipoprotein particles as well as the production of various lipid mediators, including unsaturated fatty acids and lyso-PC.     

Internalized group V secretory phospholipase A2 acts on the perinuclear membranes.

Mammalian secretory phospholipases A(2) (sPLA(2)) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA(2) mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA(2) acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA(2)s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA(2) hydrolysis products or cytokines, are necessary for the internalization of sPLA(2) into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA(2) internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA(2) induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA(2) acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis.     

Immunochemical relatedness between secretory phospholipase A2 and intracellular phospholipase A2

The immunochemical relationship between rat pancreatic phospholipase A2 and rat splenic phospholipase A2 was examined with the use of anti-rat pancreatic phospholipase A2 antibody as a probe. The immunoelectrophoretic patterns showed that the antibody cross-reacted with the splenic enzyme. The immuno-crossreactivity was also shown by counter immunoelectrophoresis. The splenic phospholipase A2, whether it was purified from the cytosolic fraction or the microsomal fraction, formed an immunoprecipitin band with the anti-pancreatic phospholipase A2 antibody. The antibody was shown to inhibit the activity of the pancreatic phospholipase A2 as well as that of the splenic phospholipase A2.     

Group V and X secretory phospholipase A(2)s-induced modification of high-density lipoprotein linked to the reduction of its antiatherogenic functions

The quantitative or qualitative decline of high-density lipoprotein (HDL) is linked to the pathogenesis of atherosclerosis because of its antiatherogenic functions, including the mediation of reverse cholesterol transport from the peripheral cells to the liver. We have recently shown that group X secretory phospholipase A(2) (sPLA(2)-X) is involved in the pathogenesis of atherosclerosis via potent lipolysis of low-density lipoprotein (LDL) leading to macrophage foam cell formation. We demonstrate here that sPLA(2)-X as well as group V secretory PLA(2) (sPLA(2)-V), another group of sPLA(2) that can potently hydrolyze phosphatidylcholine (PC), also possess potent hydrolytic potency for PC in HDL linked to the production of a large amount of unsaturated fatty acids and lysophosphatidylcholine (lysoPC). In contrast, the classical types of group IB and IIA secretory PLA(2)s evoked little, if any, lypolytic modification of HDL. Treatment with sPLA(2)-X or -V also caused an increase in the negative charge of HDL with no oxidation and little modification of apolipoprotein AI (apoAI). Modification with sPLA(2)-X or -V resulted in significant decrease in the capacity of HDL to cause cellular cholesterol efflux from lipid-loaded macrophages. Immunohistochemical analysis revealed significant expression of sPLA(2)-X in foam cell lesions in the arterial intima of Watanabe heritable hyperlipidemic (WHHL) rabbit. These findings suggest that lipolytic modification of HDL by sPLA(2)-X or -V causes drastic change of HDL in terms of the production of a large amount of unsaturated fatty acids and lysoPC linked to the reduction of its antiatherogenic functions. These sPLA(2)-mediated modifications of plasma lipoproteins might be relevant to the pathogenesis of atherosclerosis.     

Deletion of secretory group V phospholipase A2 attenuates cell migration and airway hyperresponsiveness in immunosensitized mice

We investigated the role of group V phospholipase A2 (gVPLA2) in OVA-induced inflammatory cell migration and airway hyperresponsiveness (AHR) in C57BL/6 mice. Repeated allergen challenge induced biosynthesis of gVPLA2 in airways. By aerosol, gVPLA2 caused dose-related increase in airway resistance in saline-treated mice; in allergic mice, gVPLA2 caused persistent airway narrowing. Neither group IIa phospholipase A2, a close homolog of gVPLA2, nor W31A, an inactive gVPLA2 mutant with reduced activity, caused airway narrowing in immune-sensitized mice. Pretreatment with MCL-3G1, a blocking Ab against gVPLA2, before OVA challenge blocked fully gVPLA2-induced cell migration and airway narrowing as marked by reduction of migrating leukocytes in bronchoalveolar lavage fluid and decreased airway resistance. We also assessed whether nonspecific AHR caused by methacholine challenge was elicited by gVPLA2 secreted from resident airway cells of immune-sensitized mice. MCL-3G1 also blocked methacholine-induced airway bronchoconstriction in allergic mice. Blockade of bronchoconstriction by MCL-3G1 was replicated in allergic pla2g5-/- mice, which lack the gene encoding gVPLA2. Bronchoconstriction caused by gVPLA2 in pla2g4-/- mice was comparable to that in pla2g4+/+ mice. Our data demonstrate that gVPLA2 is a critical messenger enzyme in the development of AHR and regulation of cell migration during immunosensitization by a pathway that is independent of group IVa phospholipase A2.     

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.     

Modulation of the activity and arachidonic acid selectivity of group X secretory phospholipase A2 by sphingolipids

To investigate the role of sphingomyelin (SM) in the regulation of inflammatory reactions, we studied its effect on the activity and fatty acid specificity of group X secretory phospholipase A(2) (sPLA(2)X). Compared with other phospholipases, recombinant sPLA(2)X released more arachidonate from HDL. Pretreatment of HDL with sphingomyelinase (SMase) C activated the sPLA(2)X activity, but the release of arachidonate was stimulated less than that of linoleate. In liposomes containing synthetic phosphatidylcholines (PCs), sPLA(2)X showed no clear selectivity among the various sn-2 unsaturated fatty acids. However, when SM was incorporated into liposomes at 30 mol%, the enzyme exhibited strong preference for arachidonate, although its overall activity was inhibited. Degradation of liposomal SM by SMase C resulted in sPLA(2)X activation and loss of its arachidonate preference. Incorporation of ceramide into HDL or PC liposomes activated the enzyme activity, the release of arachidonate being stimulated more than that of linoleate. SM-deficient cells released more arachidonate than normal cells in response to exogenous sPLA(2)X. SMase pretreatment of normal cells stimulated the release of arachidonate by the exogenous sPLA(2)X. These results show that SM not only inhibits sPLA(2)X activity but also contributes to its selectivity for arachidonate, whereas ceramide stimulates the hydrolysis of arachidonate-containing PCs.     

Group X secretory phospholipase A2 enhances TLR4 signaling in macrophages

Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.     

Human group V phospholipase A2 induces group IVA phospholipase A2-independent cysteinyl leukotriene synthesis in human eosinophils

We previously reported that exogenously added human group V phospholipase A2 (hVPLA2) could elicit leukotriene B4 biosynthesis in human neutrophils through the activation of group IVA phospholipase A2 (cPLA2) (Kim, Y. J., Kim, K. P., Han, S. K., Munoz, N. M., Zhu, X., Sano, H., Leff, A. R., and Cho, W. (2002) J. Biol. Chem. 277, 36479-36488). In this study, we determined the functional significance and mechanism of the exogenous hVPLA2-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) synthesis in isolated human peripheral blood eosinophils. As low a concentration as 10 nm exogenous hVPLA2 was able to elicit the significant release of AA and LTC4 from unstimulated eosinophils, which depended on its ability to act on phosphatidylcholine membranes. hVPLA2 also augmented the release of AA and LTC4 from eosinophils activated with formyl-Met-Leu-Phe + cytochalasin B. A cellular fluorescent PLA2 assay showed that hVPLA2 had a lipolytic action first on the outer plasma membrane and then on the perinuclear region. hVPLA2 also caused the translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and a 2-fold increase in 5-lipoxygenase activity. However, hVPLA2 induced neither the increase in intracellular calcium concentration nor cPLA2 phosphorylation; consequently, cPLA2 activity was not affected by hVPLA2. Pharmacological inhibition of cPLA2 and the hVPLA2-induced activation of eosinophils derived from the cPLA2-deficient mouse corroborated that hVPLA2 mediates the release of AA and leukotriene in a cPLA2-independent manner. As such, this study represents a unique example in which a secretory phospholipase induces the eicosanoid formation in inflammatory cells, completely independent of cPLA2 activation.     

Cellular arachidonate-releasing function and inflammation-associated expression of group IIF secretory phospholipase A2

Here we report the cellular arachidonate (AA)-releasing function of group IIF secretory phospholipase A(2) (sPLA(2)-IIF), a sPLA(2) enzyme uniquely containing a longer C-terminal extension. sPLA(2)-IIF increased spontaneous and stimulus-dependent release of AA, which was supplied to downstream cyclooxygenases and 5-lipoxygenase for eicosanoid production. sPLA(2)-IIF also enhanced interleukin 1-stimulated expression of cyclooxygenase-2 and microsomal prostaglandin E synthase. AA release by sPLA(2)-IIF was facilitated by oxidative modification of cellular membranes. Cellular actions of sPLA(2)-IIF occurred independently of the heparan sulfate proteoglycan glypican, which acts as a functional adaptor for other group II subfamily sPLA(2)s. Confocal microscopy revealed the location of sPLA(2)-IIF on the plasma membrane. The unique C-terminal extension was crucial for its plasma membrane localization and optimal cellular functions. sPLA(2)-IIF expression was increased in various tissues from lipopolysaccharide-treated mice and in ears of mice with experimental atopic dermatitis. In human rheumatoid arthritic joints, sPLA(2)-IIF was detected in synovial lining cells, capillary endothelial cells, and plasma cells. These results suggest that sPLA(2)-IIF is a potent regulator of AA metabolism and participates in the inflammatory process under certain conditions.     

Regulation of delayed prostaglandin production in activated P388D1 macrophages by group IV cytosolic and group V secretory phospholipase A2s

Group V secretory phospholipase A2 (sPLA2) rather than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin E2 (PGE2) production in the macrophage-like cell line P388D1. When a new clone of these cells, P388D1/MAB, selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6-24 h) in response to lipopolysaccharide alone occurred in parallel with the induction of Group V sPLA2 and cyclooxygenase-2 (COX-2). No changes in the level of cytosolic phospholipase A2 (cPLA2) or COX-1 were observed, and Group IIA sPLA2 was not detectable. Use of a potent and selective sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2 revealed that delayed PGE2 was largely dependent on the induction of Group V sPLA2. Also, COX-2, not COX-1, was found to mediate delayed PGE2 production because the response was completely blocked by the specific COX-2 inhibitor NS-398. Delayed PGE2 production and Group V sPLA2 expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca2+-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response as well. Collectively, the results suggest a model whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2 production via COX-2.     

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.     

Endothelial protein C receptor-associated invasiveness of rheumatoid synovial fibroblasts is likely driven by group V secretory phospholipase A2

Introduction

Rheumatoid synovial fibroblasts (RASFs) mediate joint inflammation and destruction in rheumatoid arthritis (RA). Endothelial protein C receptor (EPCR) is a specific receptor for the natural anticoagulant activated protein C (APC). It mediates the cytoprotective properties of APC and is expressed in rheumatoid synovial tissue. A recent report shows that group V secretory phospholipase A2 (sPLA₂V) prevents APC from binding to EPCR in endothelium and inhibits EPCR/APC function. The aim of this study was to investigate the expression and function of EPCR on RASFs.

Methods

Human synovial fibroblasts (SFs) were isolated from RA or osteoarthritis (OA) synovial tissues and treated with control, EPCR, or sPLA₂V small interfering RNA (siRNA); recombinant human APC, tumor necrosis factor-alpha (TNF-α), or sPLA₂V. RASF viability and migration/invasion were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and collagen gel migration/invasion assays, respectively, and cartilage degradation by 1,9-dimethylmethylene blue (DMMB) assay in the presence of human OA articular cartilage explants. The expression or activation of cytokines, EPCR, cadherin-11, mitogen-activated protein (MAP) kinases, and nuclear factor-kappa-B (NF-κB) or both were detected by enzyme-linked immunosorbent assay, Western blotting, or immunostaining.

Results

EPCR was expressed by both OASFs and RASFs but was markedly increased in RASFs. When EPCR was suppressed by siRNA or blocking antibody cell viability, cell invasion and cartilage degradation were reduced by more than 30%. Inflammatory mediators interleukin-1-beta (IL-1β), cadherin-11, and NF-κB were significantly reduced by EPCR suppression under control or TNF-α-stimulated conditions. The expression or activation (or both) of MAP kinases ERK, p38, and JNK were also markedly decreased in cells transfected with EPCR siRNA. Further analysis revealed that sPLA₂V co-localized with EPCR on RASFs. Suppression of sPLA₂V reduced cell viability and cartilage degradation and increased APC binding to RASFs. Conversely, recombinant sPLA₂V increased cartilage degradation, blocked APC binding to RASFs, and could not rescue the effects induced by EPCR suppression.

Conclusions

Our results demonstrate that EPCR is overexpressed by RASFs and mediates the aggressive behavior of RASFs. This function of EPCR is contrary to its cytoprotective role in other settings and is likely driven by sPLA₂V.     

Role of sphingomyelin and ceramide in the regulation of the activity and fatty acid specificity of group V secretory phospholipase A2

We previously showed that group V secretory phospholipase A(2) (sPLA(2)V) is inhibited by sphingomyelin (SM), but activated by ceramide. Here, we investigated the effect of sphingolipid structure on the activity and acyl specificity of sPLA(2)V. Degradation of HDL SM to ceramide, but not to ceramide phosphate, stimulated the activity by 6-fold, with the release of all unsaturated fatty acids being affected equally. Ceramide-enrichment of HDL similarly stimulated the release of unsaturated fatty acids. Incorporation of SM into phosphatidylcholine (PC) liposomes preferentially inhibited the hydrolysis of 16:0-20:4 PC. Conversely, SMase C treatment or ceramide incorporation resulted in preferential stimulation of hydrolysis of 16:0-20:4 PC. The presence of a long chain acyl group in ceramide was essential for the activation, and long chain diacylglycerols were also effective. However, ceramide phosphate was inhibitory. These studies show that SM and ceramide in the membranes and lipoproteins not only regulate the activity of phospholipases, but also the release of arachidonate, the precursor of eicosanoids.