Cellular distribution, post-translational modification, and tumorigenic potential of human group III secreted phospholipase A(2)

Human group III secreted phospholipase A(2) (sPLA(2)-III) consists of a central group III sPLA(2) domain flanked by unique N- and C-terminal domains. We found that the sPLA(2) domain alone was sufficient for its catalytic activity and for its prostaglandin E(2) (PGE(2))-generating functions in various cell types. In several if not all cell types, the N- and C-terminal domains of sPLA(2)-III were proteolytically removed, leading to the production of the form containing only the sPLA(2) domain, which could be further N-glycosylated at two consensus sites. Immunohistochemistry demonstrated that sPLA(2)-III was preferentially expressed in the microvascular endothelium in human tissues with inflammation, ischemic injury, and cancer. In support of this, sPLA(2)-III was induced in cultured microvascular endothelial cells after stimulation with proinflammatory cytokines. Expression of sPLA(2)-III was also associated with various tumor cells, and colorectal cancer cells transfected with sPLA(2)-III exhibited enhanced PGE(2) production and cell proliferation, which required sPLA(2)-III catalytic activity. When implanted into nude mice, the sPLA(2)-III-transfected cells formed larger solid tumors with increased angiogenesis compared with control cells. Moreover, small interfering RNA for sPLA(2)-III significantly reduced PGE(2) production and proliferation of colorectal cancer cells. Taken together, these results reveal unique cell type-specific processing and N-glycosylation of sPLA(2)-III and the potential role of this enzyme in cancer development by stimulating tumor cell growth and angiogenesis.     

Effect of NGF on the Subcellular Localization of Group IIA Secretory Phospholipase A2 (GIIA) in PC12 Cells: Role in Neuritogenesis

Phospholipases A(2) (PLA(2)s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA(2)s (sPLA(2)) as the administration of exogenous sPLA(2)s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA(2) (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA(2) isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.     

Interleukin-1beta-induced substance P release from rat cultured primary afferent neurons driven by two phospholipase A2 enzymes: secretory type IIA and cytosolic type IV

We previously described that recombinant interleukin-1beta (IL-1beta) induced the significant release of substance P (SP) via a cyclooxygenase (COX) pathway in primary cultured rat dorsal root ganglion (DRG) cells. In the present study, we examined the involvement of two types of phospholipase A2 (PLA2) enzymes, which lie upstream of COX in the prostanoid-generating pathway, in the IL-1beta-induced release of SP from DRG cells. The expression of type IIA secretory PLA2 (sPLA2 -IIA) mRNA was undetectable by ribonuclease protection assay in non-treated DRG cells, while in DRG cells incubated with 1 ng/mL of IL-1beta, the expression was induced in a time-dependent manner. On the other hand, type IV cytosolic PLA2 (cPLA2 ) mRNA was constitutively expressed in the non-treated DRG cells, and treatment with 1 ng/mL of IL-1beta for 3 h significantly increased the levels of cPLA2 mRNA. The IL-1beta-induced SP release was significantly inhibited by the sPLA2 inhibitor, thioetheramide phosphorylcholine (TEA-PC), and the cPLA2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3 ). Furthermore AACOCF3 suppressed the induction of sPLA2 -IIA mRNA expression induced by IL-1beta. These observations suggested that two types of PLA2, sPLA2 -IIA and cPLA2, were involved in the IL-1beta-induced release of SP from DRG cells, and that the functional cross-talk between the two enzymes might help to control their activity in the prostanoid-generating system in DRG cells. These events might be key steps in the inflammation-induced hyperactivity in primary afferent neurons of spinal cord.     

Human group III phospholipase A2 suppresses adenovirus infection into host cells. Evidence that group III, V and X phospholipase A2s act on distinct cellular phospholipid molecular species

Of 10 mammalian secreted phospholipase A(2) (sPLA(2)) enzymes identified to date, group V and X sPLA(2)s, which are two potent plasma membrane-acting sPLA(2)s, are capable of preventing host cells from being infected with adenovirus, and this anti-viral action depends on the conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) in the host cell membrane. Here, we show that human group III sPLA(2), which is structurally more similar to bee venom PLA(2) than to other mammalian sPLA(2)s, also has the capacity to inhibit adenovirus infection into host cells. Mass spectrometry (MS) demonstrated that group III sPLA(2) hydrolyzes particular molecular species of PC to generate LPC in human bronchial epithelial cells. Remarkably, in addition to the catalytically active sPLA(2) domain, the N-terminal, but not C-terminal, domain unique to this enzyme was required for the anti-adenovirus effect. To our knowledge, this is the first demonstration that the biological action of group III sPLA(2) depends on its N-terminal domain. Finally, our MS analysis provided additional and novel evidence that group III, V and X sPLA(2)s target distinct phospholipid molecular species in cellular membranes.     

Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.     

Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression

The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.     

A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.     

Poster Sessions CP11: Neurotoxicity and Neuroprotection. Secretory phospholipase A2 signalling partly overlaps glutamate‐mediated events

Here we explored the mechanisms of secretory phospholipase A2 (sPLA2) and glutamate (glu) in neuronal signalling and cell damage. Rats or primary neuronal cultures were treated with MK‐801 and injected with/exposed to sPLA2 or glu. MK‐801 partially inhibited sPLA2‐ and glu‐induced neuronal death as well as [3H]arachidonic acid release. The involvement of cytosolic PLA2 (cPLA2) and plateletactivating factor (PAF) in sPLA2 or glu signalling was explored by treating cells with the selective cPLA2 inhibitor, AACOCF3, PAF‐acetyl hydrolase (PAF‐AH) or the presynaptic PAF‐receptor antagonist, BN52021. AACOCF3 blocked sPLA2‐ and glu‐induced neuronal death by 26 and 77%, respectively. PAF‐AH ameliorated sPLA2 as well as glu neurotoxicity by 31 and 47%, whereas BN52021 inhibited sPLA2 induced neurotoxicity by 11% but did not significantly protect against glu‐induced neurotoxicity. Expression in neurons of early response genes in response to sPLA2 or glu was further examined. An up‐regulation of COX‐2, c‐fos, and c‐jun, but not COX‐1, was observed at earlier time points after rat striatal injection of glu as compared to sPLA2 injection. Moreover we treated neuronal cells with COX‐2 inhibitors and found that neuronal cell death after sPLA2 and glu exposure was inhibited by 35 and 33%, respectively. Thus sPLA2 activates a neuronal signalling cascade that includes activation of cPLA2, AA‐release, production of PAF and induction of COX‐2. Hence sPLA2 and glu signalling are overlapping, but not identical. Cytosolic PLA2 may primarily drive glutamatergic neurotransmission, whereas PAF plays a more crucial role in sPLA2 neuronal signalling. Acknowledgements: Supported by EPSCoR grant NSF/LEQSF(2001‐04)‐RII‐01 from the National Science Foundation.     

Aromatic hydrocarbon 1,2-diacetylbenzene cross-links neurofilament and other proteins

Here we explored the mechanisms of secretory phospholipase A2 (sPLA2) and glutamate (glu) in neuronal signalling and cell damage. Rats or primary neuronal cultures were treated with MK-801 and injected with/exposed to sPLA2 or glu. MK-801 partially inhibited sPLA2- and glu-induced neuronal death as well as [3H]arachidonic acid release. The involvement of cytosolic PLA2 (cPLA2) and plateletactivating factor (PAF) in sPLA2 or glu signalling was explored by treating cells with the selective cPLA2 inhibitor, AACOCF3, PAF-acetyl hydrolase (PAF-AH) or the presynaptic PAF-receptor antagonist, BN52021. AACOCF3 blocked sPLA2- and glu-induced neuronal death by 26 and 77%, respectively. PAF-AH ameliorated sPLA2 as well as glu neurotoxicity by 31 and 47%, whereas BN52021 inhibited sPLA2 induced neurotoxicity by 11% but did not significantly protect against glu-induced neurotoxicity. Expression in neurons of early response genes in response to sPLA2 or glu was further examined. An up-regulation of COX-2, c-fos, and c-jun, but not COX-1, was observed at earlier time points after rat striatal injection of glu as compared to sPLA2 injection. Moreover we treated neuronal cells with COX-2 inhibitors and found that neuronal cell death after sPLA2 and glu exposure was inhibited by 35 and 33%, respectively. Thus sPLA2 activates a neuronal signalling cascade that includes activation of cPLA2, AA-release, production of PAF and induction of COX-2. Hence sPLA2 and glu signalling are overlapping, but not identical. Cytosolic PLA2 may primarily drive glutamatergic neurotransmission, whereas PAF plays a more crucial role in sPLA2 neuronal signalling.
Acknowledgements:   Supported by EPSCoR grant NSF/LEQSF(2001-04)-RII-01 from the National Science Foundation.     

Negative feedback between secretory and cytosolic phospholipase A2 and their opposing roles in ovalbumin-induced bronchoconstriction in rats

Phospholipase A2 (PLA2) hydrolyzes cell membrane phospholipids (PL) to produce arachidonic acid and lyso-PL. The PLA2 enzymes include the secretory (sPLA2) and cytosolic (cPLA2) isoforms, which are assumed to act synergistically in production of eicosanoids that are involved in inflammatory processes. However, growing evidence raises the possibility that in airways and asthma-related inflammatory cells (eosinophils, basophils), the production of the bronchoconstrictor cysteinyl leukotrienes (CysLT) is linked exclusively to sPLA2, whereas the bronchodilator prostaglandin PGE2 is produced by cPLA2. It has been further reported that the capacity of airway epithelial cells to produce CysLT is inversely proportional to PGE2 production. This seems to suggest that sPLA2 and cPLA2 play opposing roles in asthma pathophysiology and the possibility of a negative feedback between the two isoenzymes. To test this hypothesis, we examined the effect of a cell-impermeable extracellular sPLA2 inhibitor on bronchoconstriction and PLA2 expression in rats with ovalbumin (OVA)-induced asthma. It was found that OVA-induced bronchoconstriction was associated with elevation of lung sPLA2 expression and CysLT production, concomitantly with suppression of cPLA2 expression and PGE2 production. These were reversed by treatment with the sPLA2 inhibitor, resulting in amelioration of bronchoconstriction and reduced CysLT production and sPLA2 expression, concomitantly with enhanced PGE2 production and cPLA2 expression. This study demonstrates, for the first time in vivo, a negative feedback between sPLA2 and cPLA2 and assigns opposing roles for these enzymes in asthma pathophysiology: sPLA2 activation induces production of the bronchoconstrictor CysLT and suppresses cPLA2 expression and the subsequent production of the bronchodilator PGE2.     

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.     

Enhancement of mast cell survival: a novel function of some secretory phospholipase A(2) isotypes

This study tested the hypothesis that certain secretory phospholipase A(2) (sPLA(2)) isotypes act in a cytokine-like fashion through cell surface receptors to influence mast cell survival. Initial experiments revealed that sPLA(2) activity and sPLA(2) receptor expression are increased, and mast cells lost their capacity to maintain membrane asymmetry upon cytokine depletion. Groups IB and III, but not group IIA PLA(2), prevented the loss of membrane asymmetry. Similarly, group IB prevented nucleosomal DNA fragmentation in mast cells. Providing putative products of sPLA(2) hydrolysis to cytokine-depleted mast cells did not influence survival. Furthermore, catalytic inactivation of sPLA(2) did not alter its capacity to prevent apoptosis. Inhibition of protein synthesis using cycloheximide or actinomycin reversed the antiapoptotic effect of sPLA(2). Additionally, both wild-type and catalytically inactive group IB PLA(2) induced IL-3 synthesis in mast cells. However, adding IL-3-neutralizing Ab did not change Annexin V(FITC) binding and only partially inhibited thymidine incorporation in sPLA(2)-supplemented mast cells. In contrast, IL-3-neutralizing Ab inhibited both Annexin V(FITC) binding and thymidine incorporation in mast cells maintained with IL-3. sPLA(2) enhanced phosphoinositide 3'-kinase activity, and a specific inhibitor of phosphoinositide 3'-kinase reversed the antiapoptotic effects of sPLA(2). Likewise, sPLA(2) increased the degradation of I-kappaBalpha, and specific inhibitors of nuclear factor kappa activation (NF-kappaB) reversed the antiapoptotic effects of sPLA(2). Together, these experiments reveal that certain isotypes of sPLA(2) enhance the survival of mast cells in a cytokine-like fashion by activating antiapoptotic signaling pathways independent of IL-3 and probably via sPLA(2) receptors rather than sPLA(2) catalytic products.     

Enhanced tissue expression and elevated circulating level of phospholipase A(2) receptor during murine endotoxic shock

Phospholipase A(2) receptor (PLA(2)R) mediates a variety of biological responses elicited by mammalian secretory phospholipase A(2) (sPLA(2)). In mice, group IB sPLA(2) (sPLA(2)-IB) acts as an endogenous ligand of PLA(2)R, and analysis of PLA(2)R-deficient mice has demonstrated a critical role of the sPLA(2)-IB/PLA(2)R system in the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) in the development of endotoxic shock. Here, we generated specific antibodies against a recombinant soluble form of PLA(2)R and examined its expression in the lung and spleen where a remarkable elevation of TNF-alpha expression has been observed during endotoxemia. Immunohistochemical analysis revealed the expression of PLA(2)R in type II alveolar epithelial cells and a subset of splenic lymphocytes, and its expression levels were markedly enhanced at 1 h after endotoxin challenge. Analysis with a newly developed sandwich enzyme-linked immunosorbent assay system revealed the presence of a soluble form of PLA(2)R in plasma of wild-type mice compared with its absence in plasma of PLA(2)R-deficient mice. After exposure to endotoxin, its circulating level was significantly elevated to the maximum level at 2-3 h after the treatment. These results suggest that tissue expression and the circulating level of PLA(2)R are elevated during murine endotoxemia, which might be relevant to its potential roles in the production of proinflammatory mediators during the development of inflammatory conditions.     

Upregulation and antiapoptotic role of endogenous Alzheimer amyloid precursor protein in dorsal root ganglion neurons

The amyloid precursor protein (APP) is a transmembrane protein whose abnormal processing is associated with the pathogenesis of Alzheimer's disease. In this study, we examined the expression and role of cell-associated APP in primary dorsal root ganglion (DRG) neurons. When dissociated DRG cells prepared from mouse embryos were treated with nerve growth factor (NGF), neuronal APP levels were transiently elevated. DRG neurons treated with an antibody against cell surface APP failed to mature and underwent apoptosis. When NGF was withdrawn from the cultures after a 36-h NGF treatment, virtually all neurons underwent apoptosis by 48 h. During the course of apoptosis, some neurons with intact morphology contained increased levels of APP immunoreactivity, whereas the APP levels were greatly reduced in apoptotic neurons. Furthermore, affected neurons contained immunoreactivities for activated caspase-3, a caspase-cleaved APP fragment (APPDeltaC31), and Abeta. Downregulation of endogenous APP expression by treatment with an APP antisense oligodeoxynucleotide significantly increased the number of apoptotic neurons in NGF-deprived DRG cultures. Furthermore, overexpression of APP by adenovirus vector-mediated gene transfer reduced the number of apoptotic neurons deprived of NGF. These results suggest that endogenous APP is upregulated to exert an antiapoptotic effect on neurotrophin-deprived DRG neurons and subsequently undergoes caspase-dependent proteolysis.     

Secretory phospholipase A2-potentiated inducible nitric oxide synthase expression by macrophages requires NF-kappa B activation

The effect of secretory group II phospholipase A2 (sPLA2) on the expression of the inducible NO synthase (iNOS) and the production of NO by macrophages was investigated. sPLA2 by itself barely stimulated nitrite production and iNOS expression in Raw264.7 cells. However, in combination with LPS, the effects were synergistic. This potentiation was shown for sPLA2 enzymes from sPLA2-transfected stable cells or for purified sPLA2 from human synovial fluid. The effect of PLA2 on iNOS induction appears to be specific for the secretory type of PLA2. LPS-stimulated activation of iNOS was inhibited by the well-known selective inhibitors of sPLA2 such as 12-epi-scalaradial and p-bromophenacyl bromide. In contrast, the cytosolic PLA2-specific inhibitors methyl arachidonyl fluorophosphate and arachidonyltrifluoromethyl ketone did not affect LPS-induced nitrite production and iNOS expression. Moreover, when we transfected cDNA-encoding type II sPLA2, we observed that the sPLA2-transfected cells produced two times more nitrites than the empty vector or cytosolic PLA2-transfected cells. The sPLA2-potentiated iNOS expression was associated with the activation of NF-kappa B. We found that the NF-kappa B inhibitor pyrrolidinedithiocarbamate prevented nitrite production, iNOS induction, and mRNA accumulation by sPLA2 plus LPS in Raw264.7 cells. Furthermore, EMSA analysis of the activation of the NF-kappa B involved in iNOS induction demonstrated that pyrrolidinedithiocarbamate prevented the NF-kappa B binding by sPLA2 plus LPS. Our findings indicated that sPLA2, in the presence of LPS, is a potent activator of macrophages. It stimulates iNOS expression and nitrite production by a mechanism that requires the activation of NF-kappa B.     

Inhibition of Rho modulates cytokine-induced prostaglandin E2 formation in renal mesangial cells

Stimulation of rat mesangial cells for 24 h with interleukin-1beta (IL- 1beta) plus forskolin (Fk) leads to a marked increase in prostaglandin E2 (PGE2) synthesis. This effect is further enhanced by the small G-protein Rho inhibitor toxin A. A similar increase in PGE2 formation is obtained with Y27632, a Rho-dependent kinase inhibitor, and with lovastatin, a hydroxymethylglutaryl-coenzyme A inhibitor which depletes cells from geranylgeranyl moieties and thus blocks Rho activation. In parallel to the increased PGE2 synthesis, a potentiation of IL-1beta-induced secretory group IIA phospholipases A2 (sPLA2-IIA) protein expression also occurs by Rho inhibition. However, only toxin A triggers an increased sPLA2-IIA activity consistent with the elevated levels of protein expression, whereas Y27632 and lovastatin rather reduced IL-1beta-induced sPLA2-IIA activity. In vitro activity studies reveal that Y27632 and lovastatin can directly block sPLA2-IIA enzyme activity in a concentration-dependent manner. Interestingly, in the absence of IL-1beta/Fk stimulation and the lack of sPLA2-IIA protein expression, all Rho inhibitors exert a small but significant increase in PGE2 formation suggesting that additional PLA2s or downstream enzymes like cyclooxygenases or prostaglandin synthases may be activated by Rho inhibitors. Western blot analyses of toxin A-, Y27632- and lovastatin-stimulated cells reveal that the cytosolic group IV PLA2 (cPLA2) and the cytosolic PGE2 synthase (cPGES), but not the sPLA2-IIA, cyclooxygenase-2 or the microsomal PGE2 synthase (mPGES), are upregulated compared to unstimulated cells. Furthermore, the Rho inhibitors induced arachidonic acid release from intact cells which is blocked by the cPLA2 inhibitor methyl arachidonyl fluorophosphonate (MAFP). In summary, these data show that inhibition of the small G-protein Rho, either by toxin A, lovastatin, or Y27632, exert a dual effect on mesangial cells: (i) in the absence of an inflammatory stimulus it activates the constitutive cPLA2 and cPGE2 synthase and generates low amount of PGE2. (ii) In the presence of inflammatory cytokines it potentiates sPLA2-IIA expression and subsequent PGE2 formation. In addition, we identified lovastatin and Y27632 as direct inhibitors of sPLA2-IIA in a cell-free system.     

PLA2R1 kills cancer cells by inducing mitochondrial stress

Little is known about the biological functions of the phospholipase A2 receptor (PLA2R1) except that it has the ability to bind a few secreted phospholipases A2 (sPLA2′s). We have previously shown that PLA2R1 regulates senescence in normal human cells. In this study, we investigated the ability of PLA2R1 to control cancer cell growth. Analysis of expression in cancer cells indicates a marked PLA2R1 decrease in breast cancer cell lines compared to normal or nontransformed human mammary epithelial cells. Accordingly, PLA2R1 ectopic expression in PLA2R1-negative breast cancer cell lines led to apoptosis, whereas a prosenescence response was predominantly triggered in normal cells. PLA2R1 structure–function studies and the use of chemical inhibitors of sPLA2-related signaling pathways suggest that the effect of PLA2R1 is sPLA2-independent. Functional experiments demonstrate that PLA2R1 regulation of cell death is driven by a reactive oxygen species (ROS)-dependent mechanism. While screening for ROS-producing complexes involved in PLA2R1 biological responses, we identified a critical role for the mitochondrial electron transport chain in PLA2R1-induced ROS production and cell death. Taken together, this set of data provides evidence for an important role of PLA2R1 in controlling cancer cell death by influencing mitochondrial biology.     

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.     

Hair follicular expression and function of group X secreted phospholipase A2 in mouse skin

Although perturbed lipid metabolism can often lead to skin abnormality, the role of phospholipase A(2) (PLA(2)) in skin homeostasis is poorly understood. In the present study we found that group X-secreted PLA(2) (sPLA(2)-X) was expressed in the outermost epithelium of hair follicles in synchrony with the anagen phase of hair cycling. Transgenic mice overexpressing sPLA(2)-X (PLA2G10-Tg) displayed alopecia, which was accompanied by hair follicle distortion with reduced expression of genes related to hair development, during a postnatal hair cycle. Additionally, the epidermis and sebaceous glands of PLA2G10-Tg skin were hyperplasic. Proteolytic activation of sPLA(2)-X in PLA2G10-Tg skin was accompanied by preferential hydrolysis of phosphatidylethanolamine species with polyunsaturated fatty acids as well as elevated production of some if not all eicosanoids. Importantly, the skin of Pla2g10-deficient mice had abnormal hair follicles with noticeable reduction in a subset of hair genes, a hypoplasic outer root sheath, a reduced number of melanin granules, and unexpected up-regulation of prostanoid synthesis. Collectively, our study highlights the spatiotemporal expression of sPLA(2)-X in hair follicles, the presence of skin-specific machinery leading to sPLA(2)-X activation, a functional link of sPLA(2)-X with hair follicle homeostasis, and compartmentalization of the prostanoid pathway in hair follicles and epidermis.