Activation Of Cytosolic Phospholipase Research Articles

207 PLATELET COX-1 SUPPORTS THE PRODUCTION OF BOTH PROSTANOIDS AND HETES

<h3>Backround</h3> Prostaglandins (PGs) and hydroxyeicosatetraeinoic acids (HETEs) are synthesized from arachidonic acid (AA), which is released from the membrane phospholipids of cells through the activity of cytosolic phospholipase A_2α (cPLA_2α). It is known that in platelets AA is metabolised through the enzymes cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) leading to the production of a range of eicosanoids. Here we have determined the enzyme sources of the major prostanoid and HETE products released by platelets following activation by selective platelet agonists. To better model platelet activation we used either platelet rich plasma or whole blood within a platelet aggregometer, warmed and stirred at 1200rpm, and assessed whether HETEs originate from platelets or other blood cells. Furthermore, to investigate how the production of these eicosanoids is affected by anti-platelet therapy we examined the effects of the cyclooxygenase inhibitor, aspirin (100 µM), with or without the addition of the P2Y₁₂ receptor blocker, prasugrel (3 µM). <h3>Methods</h3> Blood from healthy volunteers was collected into hirudin and incubated (30 min, 37°C) either intact or following preparation into platelet rich plasma (PRP) in the presence of collagen (30 µg/ml), TRAP-6 (30 µM) or vehicle, in a platelet aggregometer with stirring (1200rpm). Plasma was then separated from the samples and the levels of prostanoids and HETEs were determined by LC/MS/MS analysis. In some experiments, blood and PRP were pre-treated with aspirin (100 µM), prasugrel (3 µM) or aspirin+prasugrel. <h3>Results</h3> LC/MS/MS analysis of collagen- and TRAP-stimulated PRP and blood showed large increases in the levels of thromboxane (TX) A₂, prostaglandin (PG) E₂, and PGD₂, as well as 8-, 11-, 15-and 12-HETE compared to unstimulated samples. Interestingly, as well as inhibiting the production of TXA₂, PGE₂ and PGD₂, aspirin also strongly inhibited the productions of 11-HETE and 15-HETE by both PRP and blood. The production of 12-HETE was reduced by aspirin and prasugrel used in combination. <h3>Conclusions</h3> LC/MS/MS analysis demonstrated that platelets are the main source of both prostanoids (TXA₂, PGE₂ and PGD₂) and HETEs (8-, 11-, 15- and 12) in blood exposed to platelet activators. Interestingly, in addition to abolishing the production of prostanoids, aspirin also blocked the production of 11-HETE and 15-HETE in both PRP and blood, indicating these HETEs are also formed through the activity of platelet COX-1. Aspirin in combination with prasugrel reduced, but did not abolish, the production of 12-HETE, consistent with this drug inhibiting the processes of platelet activation but not directly inhibiting platelet 12-LOX. It is possible these data may well help provide explanations for the beneficial effects of anti-platelet doses of aspirin in a range of cardiovascular diseases and cancers.

Rapid induction of inflammatory lipid mediators by the inflammasome in vivo

Detection of microbial products by host inflammasomes is critical for innate immune surveillance. Inflammasomes activate the CASPASE-1 (CASP1) protease, which processes the cytokines interleukin(IL)-1β and -18, and initiates a lytic host cell death called pyroptosis1. To identify novel CASP1 functions in vivo, we devised a strategy for cytosolic delivery of bacterial flagellin, a specific ligand for the NAIP5 (NLR family, apoptosis inhibitory protein 5)/NLRC4 (NLR family, CARD domain containing 4) inflammasome2–4. Here we show that systemic inflammasome activation by flagellin leads to loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (< 30 minutes) death in mice. This unexpected response depends on the inflammasome components NAIP5, NLRC4, and CASP1, but is independent of IL-1β/-18 production. Instead, inflammasome activation results, within minutes, in an ‘eicosanoid storm’ – a pathological release of signaling lipids that rapidly initiate inflammation and vascular fluid loss. Mice deficient in cyclooxygenase-1 (COX-1), a critical enzyme in prostaglandin biosynthesis, are resistant to these rapid pathological effects of systemic inflammasome activation by either flagellin or anthrax lethal toxin. Inflammasome-dependent biosynthesis of eicosanoids is mediated by activation of cPLA2 (cytosolic phospholipase A2) in resident peritoneal macrophages, which are specifically primed for production of eicosanoids by high expression of eicosanoid biosynthetic enzymes. Thus, our results identify eicosanoids as a novel cell type-specific signaling output of the inflammasome with dramatic physiological consequences in vivo.

A VE-cadherin–PAR3–α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A2α in endothelial cells

Phospholipase A(2) enzymes hydrolyze phospholipids to liberate arachidonic acid for the biosynthesis of prostaglandins and leukotrienes. In the vascular endothelium, group IV phospholipase A(2)α (cPLA(2)α) enzyme activity is regulated by reversible association with the Golgi apparatus. Here we provide evidence for a plasma membrane cell adhesion complex that regulates endothelial cell confluence and simultaneously controls cPLA(2)α localization and enzymatic activity. Confluent endothelial cells display pronounced accumulation of vascular endothelial cadherin (VE-cadherin) at cell-cell junctions, and mechanical wounding of the monolayer stimulates VE-cadherin complex disassembly and cPLA(2)α release from the Golgi apparatus. VE-cadherin depletion inhibits both recruitment of cPLA(2)α to the Golgi and formation of tubules by endothelial cells. Perturbing VE-cadherin and increasing the soluble cPLA(2)α fraction also stimulated arachidonic acid and prostaglandin production. Of importance, reverse genetics shows that α-catenin and δ-catenin, but not β-catenin, regulates cPLA(2)α Golgi localization linked to cell confluence. Furthermore, cPLA(2)α Golgi localization also required partitioning defective protein 3 (PAR3) and annexin A1. Disruption of F-actin internalizes VE-cadherin and releases cPLA(2)α from the adhesion complex and Golgi apparatus. Finally, depletion of either PAR3 or α-catenin promotes cPLA(2)α-dependent endothelial tubule formation. Thus a VE-cadherin-PAR3-α-catenin adhesion complex regulates cPLA(2)α recruitment to the Golgi apparatus, with functional consequences for vascular physiology.

Attenuation of niacin-induced prostaglandin D2 generation by omega-3 fatty acids in THP-1 macrophages and Langerhans dendritic cells

Niacin, also known as nicotinic acid, is an organic compound that has several cardio-beneficial effects. However, its use is limited due to the induction of a variable flushing response in most individuals. Flushing occurs from a niacin receptor mediated generation of prostaglandins from arachidonic acid metabolism. This study examined the ability of docosahexaenoic acid, eicosapentaenoic acid, and omega-3 polyunsaturated fatty acids (PUFAs), to attenuate niacin-induced prostaglandins in THP-1 macrophages. Niacin induced both PGD2 and PGE2 generation in a dose-dependent manner. Niacin also caused an increase in cytosolic calcium and activation of cytosolic phospholipase A2. The increase in PGD2 and PGE2 was reduced by both docosahexaenoic acid and eicosapentaenoic acid, but not by oleic acid. Omega-3 PUFAs efficiently incorporated into cellular phospholipids at the expense of arachidonic acid, whereas oleic acid incorporated to a higher extent but had no effect on arachidonic acid levels. Omega-3 PUFAs also reduced surface expression of GPR109A, a human niacin receptor. Furthermore, omega-3 PUFAs also inhibited the niacin-induced increase in cytosolic calcium. Niacin and/or omega-3 PUFAs minimally affected cyclooxygenase-1 activity and had no effect on cyclooxygenase -2 activity. The effects of niacin on PGD2 generation were further confirmed using Langerhans dendritic cells. Results of the present study indicate that omega-3 PUFAs reduced niacin-induced prostaglandins formation by diminishing the availability of their substrate, as well as reducing the surface expression of niacin receptors. In conclusion, this study suggests that the regular use of omega-3 PUFAs along with niacin can potentially reduce the niacin-induced flushing response in sensitive patients.

Bioactive Lipoxygenase Metabolites Stimulation of NADPH Oxidases and Reactive Oxygen Species

In mammalian cells, reactive oxygen species (ROS) are produced via a variety of cellular oxidative processes, including the activity of NADPH oxidases (NOX), the activity of xanthine oxidases, the metabolism of arachidonic acid (AA) by lipoxygenases (LOX) and cyclooxygenases (COX), and the mitochondrial respiratory chain. Although NOX-generated ROS are the best characterized examples of ROS in mammalian cells, ROS are also generated by the oxidative metabolism (e.g., via LOX and COX) of AA that is released from the membrane phospholipids via the activity of cytosolic phospholipase A(2) (cPLA(2)). Recently, growing evidence suggests that LOX- and COX-generated AA metabolites can induce ROS generation by stimulating NOX and that a potential signaling connection exits between the LOX/COX metabolites and NOX. In this review, we discuss the results of recent studies that report the generation of ROS by LOX metabolites, especially 5-LOX metabolites, via NOX stimulation. In particular, we have focused on the contribution of leukotriene B(4) (LTB(4)), a potent bioactive eicosanoid that is derived from 5-LOX, and its receptors, BLT1 and BLT2, to NOX stimulation through a signaling mechanism that leads to ROS generation.

Angiotensin II-dependent growth of vascular smooth muscle cells requires transactivation of the epidermal growth factor receptor via a cytosolic phospholipase A 2-mediated release of arachidonic acid

Angiotensin (Ang) II stimulates vascular smooth muscle cell (VSMC) growth via activation of cytosolic phospholipase A 2 (cPLA 2), release of arachidonic acid (ArAc) and activation of mitogen-activated protein kinase (MAPK). The mechanism linking AT 1 receptor stimulation of ArAc release with MAPK activation may involve transactivation of the epidermal growth factor receptor (EGFR). In this study, Ang II increased phosphorylation of the EGFR and MAPK in cultured VSMC and these effects were attenuated by the cPLA 2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF 3), and restored by addition of ArAc. Ang II- or ArAc-induced phosphorylation of the EGFR and MAPK were abolished by the EGFR kinase inhibitor AG1478. Ang II or ArAc also stimulated VSMC growth that was blocked by AG1478 or the MAPK kinase (MEK) inhibitor PD98059. Thus, it appears that the cPLA 2-dependent release of ArAc may provide a mechanism for the transactivation between the AT 1 receptor and the EGFR signaling cascade.

Decrease in expression or activity of cytosolic phospholipase A 2α increases cyclooxygenase-1 action: A cross-talk between key enzymes in arachidonic acid pathway in prostate cancer cells

The eicosanoid pathway is activated in many types of cancers including prostate. Eicosanoids are synthesized from intracellular arachidonic acid (AA), which is released from membrane glycerophospholipids mainly by the action of cytosolic phospholipase A 2α (cPLA 2α). Thus, targeting cPLA 2α has been proposed as a treatment option. The aim of this study was to determine the effect of cPLA 2α inhibition on cyclooxygenase (COX) expression and PGE 2 production. Inhibition of cPLA 2α expression by siRNA or activity by Efipladib in prostate cancer cell lines (PC3 and LNCaP) led to an increase in COX-1 protein and PGE 2 levels in a dose-dependent manner from 24 to 72 h. The COX-2 response was less evident. Efipladib treatment increased COX-1 promoter transcriptional activity without changing the rate of COX-1 protein degradation. Treatment with Efipladib also led to a decrease in most LOX products (HETEs) as measured by LC/MS/MS. Replenishing 5- and 12-HETEs abolished Efipladib-induced COX-1 and PGE 2 levels. Decreasing 5- and 12-HETE production, as a result of treating cells with inhibitors MK886 and Baicalein, respectively, mimicked the effect of Efipladib on COX-1 and PGE 2 levels. Hence, the mechanism underlying the cPLA 2α inhibition-induced COX-1 is likely due to a decrease in LOX products, which may exert a negative feedback on COX-1 gene expression in prostate cancer cells. Considering that PGE 2 is a potent promoter of cancer cell proliferation and survival, understanding the mechanism coupling cPLA 2α with COX-1 is of potential clinical significance.

Cytosolic phospholipase A 2-α enhances induction of endoplasmic reticulum stress

Induction of endoplasmic reticulum (ER) stress by the complement membrane attack complex is enhanced by activation of cytosolic phospholipase A 2-α (cPLA 2). To address mechanisms by which cPLA 2 may modulate ER stress, we produced a mutant cPLA 2, containing an ER targeting domain (cPLA 2-ERmut). After transfection and fractionation of COS-1 cells, cPLA 2-ERmut was present mainly in the membrane fraction, whereas wild type (wt) cPLA 2 was principally in the cytosol. By fluorescence microscopy, cPLA 2-ERmut was enriched in a perinuclear distribution under basal conditions, colocalizing with the ER protein, calnexin, while cPLA 2-wt was mainly cytosolic. Both forms of cPLA 2 transiently expressed in COS cells showed basal phosphorylation at serine 505, which correlates with catalytic activity. Expression of cPLA 2-wt was ∼ 5-fold greater, compared with cPLA 2-ERmut, but both enzymes produced comparable increases in free arachidonic acid, implying that cPLA 2-ERmut effectively hydrolyzed ER membrane phospholipids. Although transfection of cPLA 2-ERmut or wt did not induce ER stress independently, cPLA 2-ERmut and wt enhanced the induction of ER stress by tunicamycin, dithiothreitol and ionomycin (monitored by induction of grp94 and C/EBP homologous protein-10), and the effect was dependent on the catalytic activity. cPLA 2-ERmut enhanced production of superoxide. Induction of ER stress in tunicamycin-treated cells expressing cPLA 2-ERmut was attenuated in the presence of the antioxidant, N-acetyl cysteine, and reduced glutathione, and was exacerbated by dl-buthionine-(S,R)-sulfoximine (which depletes glutathione). Expression of cPLA 2-ERmut exacerbated tunicamycin-induced apoptosis. Thus, induction of ER stress is facilitated by the activation of cPLA 2 at the ER. The mechanism involves ER membrane phospholipid hydrolysis, and accumulation of reactive oxygen species.

Mechanism of Cytosolic Phospholipase Activation in Ghrelin Protection of Salivary Gland Acinar Cells against Ethanol Cytotoxicity

Ghrelin, a peptide hormone, newly identified in oral mucosal tissues, has emerged recently as an important mediator of the processes of mucosal defense. Here, we report on the mechanism of ghrelin protection against ethanol cytotoxicity in rat sublingual salivary gland cells. The protective effect of ghrelin was associated with the increase in NO and PGE2, and upregulation in cytosolic phospholipase A2 (cPLA2) activity and arachidonic acid (AA) release. The loss in countering effect of ghrelin occurred with cNOS inhibitor, L-NAME, as well as indomethacin and COX-1 inhibitor, SC-560, while COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced cell capacity for NO production, cPLA2 activation and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA2 phosphorylation and S-nitrosylation. Inhibition in ghrelin-induced S-nitrosylation was attained with L-NAME, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA2 protein phosphorylation as well as S-nitrosylation. Thus, ghrelin protection of salivary gland cells against ethanol involves cNOS-derived NO induction of cPLA2 activation through S-nitrosylation for the increase in AA release at the site of COX-1 action for PGE2 synthesis.

Quinacrine protects neuronal cells against heat‐induced injury

The effects of quinacrine (QA) on heat-induced neuronal injury have been explored, with the intention of understanding the mechanisms of QA protection. Primary cultivated striatum neurons from newborn rats were treated with QA 1h before heat treatment at 43 degrees C which lasted for another 1h, and necrosis and apoptosis were detected by Annexin-V-FITC and propidium iodide (PI) double staining. Neuronal apoptosis was determined using terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) techniques. Cell membrane fluidity, activity of cytosolic phospholipase A(2) (cPLA(2)) and the level of arachidonic acid (AA) were also examined. Membrane surface ultrastructure of striatum neurons was investigated by atomic force microscopy (AFM). Results showed that heat treatment induced great striatum neurons death, with many dying neurons undergoing necrosis rather than apoptosis. QA alone had little effect on the survival of striatum neurons, while QA pretreatment before heat treatment decreased necrosis. Heat treatment also resulted in decreased membrane fluidity and increased cPLA(2) activity as well as arachidonic acid level; these effects were reversed by QA pretreatment. QA pretreatment also significantly prevented damage to the membrane surface ultrastructure of heat-treated neurons. These results suggest that QA protects striatum neurons against heat-induced neuronal necrosis, and also demonstrate that inhibition of cPLA(2) activity and stabilization of membranes may contribute to protective effect of quinacrine.

Activation of phospholipase A 2 is involved in indomethacin-induced damage in Caco-2 cells

Nonsteroidal anti-inflammatory drugs (NSAIDs), widely used in clinical practice, cause adverse effects in the gastrointestinal tract. These effects have been attributed to mechanisms such as drug-induced cyclooxygenase inhibition, oxidative stress, mitochondrial dysfunction and changes in cell membrane lipids. Our previous study showed that indomethacin (an NSAID commonly used in toxicity studies) caused activation of cytosolic phospholipase A 2 (cPLA 2) in the rat small intestine. We hypothesized that activation of cPLA 2 is an important event in the pathogenesis of indomethacin-induced damage in enterocytes. To test this, we incubated enterocyte-like Caco-2 cells with indomethacin, with and without pretreatment with methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of cPLA 2. Cells treated with indomethacin showed decreased viability and evidence of oxidative stress and morphological cell damage. Phospholipids were degraded in these cells, with increases in the levels of lysophospholipids and arachidonic acid. There was no evidence of apoptosis in the cells in response to the drug. Pretreatment of the cells with MAFP attenuated the drug-induced effects seen. This shows that activation of phospholipase A 2 appears to be an important event in the pathogenesis of indomethacin-induced damage in Caco-2 cells. To our knowledge, this is the first report that implicates the involvement of this enzyme in NSAID-induced enteropathy.

Regulatory role of cytosolic phospholipase A2α in NADPH oxidase activity and in inducible nitric oxide synthase induction by aggregated Aβ1–42 in microglia

In Alzheimer's disease, extracellular deposits of amyloid beta(1-42) (Abeta(1-42)) may induce activation of microglial cells by releasing proinflammatory factors that contribute to the neurodegeneration process. Since the activation of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) has been reported in inflammatory conditions, its role in primary rat microglial cell activated by aggregated Abeta(1-42) was elucidated. The results of the present study show that activation of microglia by 5 microM aggregated Abeta(1-42) (as evident by the amoeboid morphology and increased CD68 immunofluorescence reactivity) caused an immediate activation of cPLA(2)alpha, measured by its phosphorylated form and its specific activity, followed by a gradual elevation of its expression and activity during 24 h. Inhibition of cPLA(2)alpha expression and activity by the presence of 1 microM specific antisense resulted in a significant decrease in NADPH oxidase activity that releases superoxides, PGE(2) formation, iNOS expression, and NO production, indicating a major role for cPLA(2)alpha in the regulation of these inflammatory processes. NADPH oxidase activity, which is under cPLA(2)alpha regulation, was found to upregulate cPLA(2)alpha and COX-2 protein expression through the redox-sensitive NFkappaB activation as evident by its phosphorylation on Ser-536, resulting in increased PGE(2) formation. The secreted PGE(2) induced the synthesis of iNOS and the production of NO through the PKA-CREB pathway. Taken together, our results suggest that the response of cPLA(2)alpha to aggregated Abeta(1-42) is probably a key player in the oxidative stress present in AD, regulating potent oxidative agents: the production of superoxides by NADPH oxidase and NO formation by iNOS.

Arachidonic acid: A key molecule for astrocyte survival to peroxynitrite

Nontoxic concentrations of peroxynitrite (ONOO(-)) nevertheless commit rat astrocytes to mitochondrial permeability transition-dependent toxicity, however prevented by a signaling response driven by arachidonic acid (ARA). The lipid messenger was released upon ONOO(-)-dependent activation of cytosolic phospholipase A(2) and its pharmacological inhibition, or knock-down, was invariably associated with a prompt apoptotic response sensitive to exogenous ARA, but insensitive to other polyunsaturated fatty acids, as eicosapentaenoic or linoleic acid. Interestingly, while microglia also used ARA to cope with ONOO(-), cerebellar granule cells were killed by the same concentrations of ONOO(-) employed in astrocyte/microglia experiments via a mechanism sensitive to inhibition of ARA release. These results collectively support the notion that resistance of glial cells to ONOO(-), a species extensively produced under neuroinflammatory conditions, is largely based on a critical survival signaling triggered by the inflammatory product ARA. In remarkable contrast with these results, the lipid messenger appears to mediate toxicity in neuronal cells.

Leptin-induced cytosolic phospholipase A2 activation in gastric mucosal protection against ethanol cytotoxicity involves epidermal growth factor receptor transactivation

A pluripotent cytokine, leptin, released locally within the mucosal tissue is an important mediator of the processes of gastric mucosal defense and repair. Here, we report that leptin protection of gastric mucosal cells against ethanol cytotoxicity requires epidermal growth factor receptor (EGFR) participation. We show that the protective effect of leptin against ethanol cytotoxicity was associated with the increased EGFR and cPLA(2) phosphorylation, and characterized by a marked increase in arachidonic acid (AA) release and prostaglandin (PGE(2)) generation. The loss in countering capacity of leptin on the ethanol-induced cytotoxicity was attained with Src kinase inhibitor, PP2, and EGFR kinase inhibitor, AG1478, as well as ERK inhibitor, PD98059. Moreover, all three agents evoked also the inhibition in leptin-induced upregulation in cPLA(2) activity, AA release, and PGE(2) generation. Furthermore, changes caused by leptin in EGFR phosphorylation and cPLA(2) activation were susceptible to suppression by GM6001, a metalloprotease inhibitor of membrane-anchored EGFR ligand cleavage. These findings disclose an important link between leptin-induced and Src kinase-mediated EGFR transactivation and the activation of cytosolic phospholipase A(2) that leads to up-regulation in PGE2 production, thus providing new insights into the mechanism of gastric mucosal protection by leptin.

Cytosolic phospholipase A2, lipoxygenase metabolites, and reactive oxygen species

Reactive oxygen species (ROS) are generated in mammalian cells via both enzymatic and non-enzymatic mechanisms. Although certain ROS production pathways are required for the performance of specific physiological functions, excessive ROS generation is harmful, and has been implicated in the pathogenesis of a number of diseases. Among the ROS-producing enzymes, NADPH oxidase is widely distributed among mammalian cells, and is a crucial source of ROS for physiological and pathological processes. Reactive oxygen species are also generated by arachidonic acid (AA) metabolites, which are released from membrane phospholipids via the activity of cytosolic phospholipase A(2) (cPLA(2)). In this study, we describe recent studies concerning the generation of ROS by AA metabolites. In particular, we have focused on the manner in which AA metabolism via lipoxygenase (LOX) and LOX metabolites contributes to ROS generation. By elucidating the signaling mechanisms that link LOX and LOX metabolites to ROS, we hope to shed light on the variety of physiological and pathological mechanisms associated with LOX metabolism.

Tumor Necrosis Factor-α Develops Late Anaphylactic Reaction through Cytosolic Phospholipase A2 Activation

Background: We have recently reported that tumor necrosis factor (TNF)-α plays an important role in the development of a late anaphylactic reaction, but the downstream pathway beyond TNF-α remains unclear. Objective: It was the aim of this study to examine whether TNF-α induces late-phase anaphylaxis via the activation of cytosolic phospholipase A₂ (cPLA₂). Methods: Using a murine model of active systemic anaphylaxis to penicillin V, the induction of the late phase of anaphylaxis was quantified by measuring the increase in hematocrit value as well as the plasma level of platelet-activating factor in TNF-α knockout mice. Phosphorylation of mitogen-activated protein kinases (MAPKs) and cPLA₂ was measured by immunoprecipitation. cPLA₂ activity was assessed by using 1-stearoyl-2-[1-¹⁴C] arachidonyl-sn-glycero-3-phosphocholine as the substrate. Results: Phosphorylation and enzymatic activity of cPLA₂, and phosphorylation of the 3 known MAPKs, i.e. p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase, were markedly increased in a TNF-α-dependent way in the lungs of mice undergoing anaphylaxis. A specific cPLA₂ inhibitor significantly attenuated the late anaphylactic symptoms. Either p38 or an ERK inhibitor significantly attenuated not only cPLA₂ phosphorylation and activity, but also the late-phase anaphylaxis. Conclusion: TNF-α-induces cPLA₂ activation through the pathway involving p38 MAPK and ERK activation and appears to be the key mechanism leading to the development of late-phase anaphylaxis.

Toxoplasma gondii‐derived heat shock protein 70 induces lethal anaphylactic reaction through activation of cytosolic phospholipase A2 and platelet‐activating factor via Toll‐like receptor 4/myeloid differentiation factor 88

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) was proven to induce IFN-gamma-dependent lethal anaphylactic reaction in T. gondii-infected mice through an alternative PAF-mediated pathway, but not the classical immunoglobulin (Ig)E-dependent pathway. Although marked IFN-gamma production was observed by CD11b(+), CD11c(+), CD4(+) and CD8(+) splenocytes, CD11b(+) and CD11c(+) cells were shown to be the key effecter cells which generated pro-inflammatory lipid such as PAF and caused T.g.HSP70-induced anaphylactic reaction. In the present study, we found that the T.g.HSP70-induced anaphylactic reaction was not observed in TLR 4-deficient ((-/-)) mice, whereas it was observed in WT and TLR2(-/-) mice. The mRNA expression of PAF-AH, the main enzyme for PAF degradation, increased in T. gondii-infected WT and TLR2(-/-) but not in TLR4(-/-) mice after T.g.HSP70 injection. Furthermore, phosphorylation of cPLA(2), which is the key enzyme for pro-inflammatory lipid generation, was detected in CD11b(+) splenocytes of WT and TLR2(-/-) mice but not in TLR4(-/-) mice. Subsequently, cPLA(2) activation was suppressed by inhibiting the TLR4-directed p38 and p44/42 MAPK pathways. However, T.g.HSP70-induced anaphylactic reaction was observed in TRIF(-/-) mice, but not in MyD88(-/-) mice. These findings indicate the cPLA(2) activated-PAF production via TLR4/MyD88-dependent, but not TRIF-dependent, signaling pathway in T.g.HSP70-induced anaphylactic reaction in T. gondii-infected mice.

Activation of cytosolic phospholipase A2 and fatty acid transacylase is essential but not sufficient for thrombin-induced smooth muscle cell proliferation

Thrombin is a potent stimulant of smooth muscle cell (SMC) proliferation in inflammatory conditions, leading to pathological thickening of vascular walls in atherosclerosis and airway remodeling in asthma. Cell proliferation requires the formation and remodeling of cell membrane phospholipids (PLs), involving the activation of PL-metabolizing enzymes. Yet, the role of specific PL-metabolizing enzymes in SMC proliferation has hardly been studied. To bridge this gap, in the present study, we investigated the role of key enzymes involved in PL metabolism, the PL-hydrolyzing enzyme phospholipase A2 (PLA2) and the PL-synthesizing enzyme lysophosphatidic acid-fatty acid transacylase (LPAAT), in thrombin-induced proliferation of bovine aortic SMCs (BASMCs). Concomitantly with the induction of BASMC proliferation, thrombin activated cytosolic PLA2 (cPLA2-alpha), expressed by selective release of arachidonic acid and mRNA expression, as well as LPAAT, expressed by nonselective incorporation of fatty acid and mRNA expression. Specific inhibitors of these enzymes, arachidonyl-trifluoromethyl-ketone for cPLA2 and thimerosal for LPAAT, suppressed their activities, concomitantly with suppression of BASMC proliferation, suggesting a mandatory requirement for cPLA2 and LPAAT activation in thrombin-induced SMC proliferation. Thrombin acts through the protease-activated receptor (PAR-1), and, accordingly, we found that thrombin-induced BASMC proliferation was suppressed by the PAR-1 inhibitor SCH-79797. However, the PAR-1 inhibitor did not prevent thrombin-induced mRNA expression of cPLA2 and LPAAT, implying that the activation of cPLA2 and LPAAT is essential but not sufficient for thrombin-induced proliferation of BASMCs.

Altered Endothelin‐1 Signaling in the Activation of Cytosolic Phospholipase A2 (cPLA2) and Thromboxane A2 (TXA2) Productions in Kupffer Cells (KC) of the Prefibrotic Rat Liver: A Possible Mechanism in the Early Development of Portal Hypertension in Cirrhosis

We sought to investigate altered ET‐1 signaling in cPLA2 activation in liver fibrosis. KC isolated from rats of 1 week bile duct ligation (BDL) or sham operation were treated with ET‐1 following pretreatment with inhibitors of Gi or Gq/G11 and intermediates in the signaling pathways. The activation of cPLA2 measured by phosphorylation and TXA2 productions in KC were determined. Inhibitions of cPLA2 with AACOCF3 or [Ca2+]i chelation with BAPTA‐AM, respectively, completely abolished ET‐1 induced releases of TXA2 from KC of both Sham and BDL, indicating that KC productions of TXA2 in response to ET‐1 are mediated primarily by Ca2+ dependent cPLA2. Inhibitions of MEK1/2‐ERK1/2 pathway with PD98059 or Gq signaling with GP2A, respectively, completely in Sham but only partially in BDL abrogated releases of TXA2 in ET‐1 stimulated KC. The inhibition of p38 MAPK with SB203580 or the blockade of Gi using pertussis toxin had no effect in Sham while markedly abolished ET‐1 induced releases of TXA2 in BDL. In addition, BDL resulted in a significant increase in Gαi but decrease in Gqα and G11α compared to Sham. While BDL caused no significant change in the phosphorylation of ERK1/2 in KC in response to ET‐1, it greatly enhanced the phosphorylation of p38 MAPK compared to Sham. All data suggest that ET‐1 signaling in cPLA2 activation may shift from Gq/G11 mediated activations of MEK12‐ERK1/2 to Gi mediated activations of p38 MAPK in KC in response to fibrosis.

Activation of cytosolic phospholipase A 2 and 15-lipoxygenase by oxidized low-density lipoproteins in cultured human lung fibroblasts

In cell cultures of human lung fibroblasts, we found that oxidized LDL (oxLDL), after 24-h treatment, stimulated arachidonic acid release. A putative role for phospholipases A 2 and MAPK activities in this process was postulated. Consequently, we studied the contribution of either Ca 2+-dependent, cytosolic phospholipase A 2 (cPLA 2) or Ca 2+-independent phospholipase A 2 (iPLA 2), and the role of the MAP kinase family in oxLDL toxicity to fibroblastic cells in vitro. Activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH 2-terminal kinase (JNK) was also assessed with Western blotting. Compared with cellular samples untreated or treated with native LDL, treatment with oxLDL (50-100 μM hydroperoxides) for 24 h significantly increased the levels of either cPLA 2 protein expression or constitutively phosphorylated cPLA 2 protein; in addition we observed enzyme translocation to membranes. iPLA 2 activity was not stimulated by oxLDL. Arachidonic acid release appeared to be associated with phosphorylation of ERK1/2 which was significantly enhanced in a dose-dependent manner whereas no activation of p38 and JNKs was found, indicating that these MAPKs are not involved in mediating the maximal oxLDL response. Western blotting on subcellular fractions and confocal microscopy analyses confirmed an increase in 15-lipoxygenase (15-LO) protein expression and translocation upon activation. A significant increase of cyclooxygenase-2 expression into membrane fraction was also found. Collectively, the data presented link the stimulation of ERK–cPLA 2–15-LO pathway by oxLDL to the prooxidant mechanism of the lipoprotein complex. It may initially stimulate the fibroblast reaction against the oxidation challenge as well as metabolic repair, such as during lung inflammation and pulmonary fibrosis.