cPLA2 Phosphorylation Research Articles

Leukotriene B4/antimicrobial peptide LL‐37 proinflammatory circuits are mediated by BLT1 and FPR2/ALX and are counterregulated by lipoxin A4and resolvin E1

In humans, the antimicrobial peptide LL-37 and leukotriene B(4) (LTB(4)) are important proinflammatory mediators, whereas lipoxin A(4) (LXA(4)) and resolvin E1 (RvE1) possess anti-inflammatory, proresolving properties. Previously, we reported that LTB(4) triggers LL-37 release from human neutrophils (PMNs) and, conversely, that LL-37 promotes LTB(4) production from these cells. Here we show that this effect of LL-37 is mediated via the GPCR FPR2/ALX. LL-37 (5-30 μg/ml) induces intracellular calcium mobilization in a dose-dependent manner, and the signal transduction leading to LTB(4) release involves p38 MAP kinase and phosphorylation of cPLA(2). LXA(4), an endogenous lipid ligand of FPR2/ALX, and a stable LXA(4) analog [benzo-LXA(4)] were ineffective as stimuli at the concentrations of 0.1-10 nM for LTB(4) release from PMNs. Likewise, the BLT1 ligand RvE1, a derivative of eicosapentaenoic acid, inhibited LTB(4)-induced LL-37 production from PMNs at 1-100 nM, whereas chemerin, a peptide ligand of the RvE1 receptor ChemR23, failed to block LTB(4)-induced LL-37 release at the same concentrations. Hence, in human neutrophils, binding of LL-37 to FPR2/ALX promotes LTB(4) production, which can bind to BLT1 and elicit further LL-37 release. This proinflammatory circuit might be inhibited by LXA(4) and RvE(1) acting at FPR2/ALX and BLT1, respectively, leading to dampened mediator release.

Low Energy Laser Light (632.8 nm) Suppresses Amyloid-Beta Peptide-Induced Oxidative and Inflammatory Responses in Astrocytes

Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid β-peptides (Aβ) in mediating these processes. In astrocytes, oligomeric Aβ induces the assembly of NADPH oxidase complexes resulting in its activation to produce anionic superoxide. Aβ also promotes production of pro-inflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by Aβ. Primary rat astrocytes were exposed to Helium-Neon laser (λ= 632.8 nm), followed by the treatment with oligomeric Aβ. Primary rat astrocytes were used to measure Aβ-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47phox subunit and the membrane gp91phox subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A2 (cPLA2), and expressions of pro-inflammatory factors including interleukin-1β (IL-1β) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed Aβ-induced superoxide production, colocalization between NADPH oxidase gp91phox and p47phox subunits, phosphorylation of cPLA2, and the expressions of IL-1β and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD.

Luteolin-7-O-glucoside Suppresses Leukotriene C4 Production and Degranulation by Inhibiting the Phosphorylation of Mitogen Activated Protein Kinases and Phospholipase C.GAMMA.1 in Activated Mouse Bone Marrow-Derived Mast Cells

In this study, luteolin-7-O-glucoside (L7G), an herbal medicine isolated from Ailanthus altissima, inhibited 5-lipoxygenase (5-LOX)-dependent leukotriene C(4) (LTC(4)) production in bone marrow-derived mast cells (BMMCs) in a concentration-dependent manner with an IC(50) of 3.0 µM. To determine the action mechanism of L7G, we performed immunoblotting for cytosolic phospholipase A(2) (cPLA(2)) and mitogen-activated protein kinases (MAPKs) following c-kit ligand (KL)-induced activation of BMMCs with or without L7G. Inhibition of LTC(4) production by L7G was accompanied by a decrease in cPLA(2) phosphorylation, which occurred via the extracellular signal-regulated protein kinase-1/2 (ERK1/2) and p38 and c-Jun N-terminal kinase (JNK) pathways. In addition, L7G also attenuated mast cell degranulation in a dose-dependent manner (IC(50), 22.8 µM) through inhibition of phospholipase Cγ1 (PLCγ1) phosphorylation. Our results suggest that the anti-asthmatic activity of L7G may be mediated in part via the inhibition of LTC(4) generation and mast cell degranulation.

Eosinophil Cysteinyl Leukotriene Synthesis Mediated by Exogenous Secreted Phospholipase A2 Group X

Secreted phospholipase A(2) group X (sPLA(2)-X) has recently been identified in the airways of patients with asthma and may participate in cysteinyl leukotriene (CysLT; C(4), D(4), and E(4)) synthesis. We examined CysLT synthesis and arachidonic acid (AA) and lysophospholipid release by eosinophils mediated by recombinant human sPLA(2)-X. We found that recombinant sPLA(2)-X caused marked AA release and a rapid onset of CysLT synthesis in human eosinophils that was blocked by a selective sPLA(2)-X inhibitor. Exogenous sPLA(2)-X released lysophospholipid species that arise from phospholipids enriched in AA in eosinophils, including phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine as well as plasmenyl phosphatidylcholine and phosphatidylethanolamine. CysLT synthesis mediated by sPLA(2)-X but not AA release could be suppressed by inhibition of cPLA(2)α. Exogenous sPLA(2)-X initiated Ser(505) phosphorylation of cPLA(2)α, an intracellular Ca(2+) flux, and translocation of cPLA(2)α and 5-lipoxygenase in eosinophils. Synthesis of CysLTs in response to sPLA(2)-X or lysophosphatidylcholine was inhibited by p38 or JNK inhibitors but not by a MEK 1/2 inhibitor. A further increase in CysLT synthesis was induced by the addition of sPLA(2)-X to eosinophils under conditions of N-formyl-methionyl-leucyl-phenylalanine-mediated cPLA(2)α activation. These results indicate that sPLA(2)-X participates in AA and lysophospholipid release, resulting in CysLT synthesis in eosinophils through a mechanism involving p38 and JNK MAPK, cPLA(2)α, and 5-lipoxygenase activation and resulting in the amplification of CysLT synthesis during cPLA(2)α activation. Transactivation of eosinophils by sPLA(2)-X may be an important mechanism leading to CysLT formation in the airways of patients with asthma.

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Thromboxane Generation by Regulating cPLA2 Activation and Calcium Mobilization In Platelets

AbstractAbstract 2020Positive regulatory role of Protein Kinase C (PKC) isoforms in platelets have been extensively studied. However, negative regulatory roles of PKCs in platelets are poorly understood. In this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms involved in this process. Pan PKC inhibition with GF109203X potentiated ADP-induced cPLA2 phosphorylation suggesting that PKCs negatively regulate thromboxane generation by regulating cPLA2 activation. Inhibition of PKCs potentiated ADP-induced ERK activation and intracellular calcium mobilization, two upstream signaling molecules of cPLA2.These data suggest that PKCs negatively regulate thromboxane by regulating ERK activation and calcium mobilization, which inturn regulate cPLA2 activation. Pan-PKC inhibition potentiated ADP-induced, P2Y1 receptor-mediated calcium mobilization in platelets independent of P2Y12-receptor. Pretreatment of platelets with GF109203X, a Pan PKC inhibitor, but not Go-6976, a classical PKC isoform inhibitor, potentiated ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We have previously demonstrated that nPKC η, θ, δ positively regulates agonist-induced thromboxane generation in platelets. Thus, we investigated if the role of nPKC ε in ADP-induced thromboxane generation using PKC ε knockout mice (PKCε KO). ADP-induced thromboxane generation in PKC ε KO murine platelets was ten-fold higher than that of wild type platelets. Furthermore, PKC ε KO mice exhibited shorter times to occlusion in FeCl3-induced arterial injury model and shorter bleeding times in Tail bleeding experiments. We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and thereby offers protection against thrombosis. Disclosures:No relevant conflicts of interest to declare.

Cytosolic and calcium-independent phospholipase A 2 mediate glioma-enhanced proangiogenic activity of brain endothelial cells

Glioma is characterized by an active production of proangiogenic molecules. We observed that conditioned medium (CM) from C6 glioma significantly enhanced proliferation and migration of immortalized rat brain GP8.3 endothelial cells (ECs) and primary bovine brain microvascular ECs. The glioma CM effect was significantly reduced by cytosolic (cPLA 2) and Ca ++-independent (iPLA 2) phospholipase A 2, cyclooxygenase-2, and protein kinase inhibitors. In GP8.3 ECs, cPLA 2 and iPLA 2 enzyme activities and phosphorylation of cPLA 2, significantly stimulated after 24 h CM co-incubation, were attenuated by PLA 2, PI3-K, MEK-1, and ERK1/2 inhibitors. By confocal microscopy, in glioma CM-stimulated ECs, enhancement of fluorescence signals for phospho-cPLA 2, phospho-ERK1/2, phospho-PKCα, COX-2, and iPLA 2 was in parallel observed. Electroporation of anti-iPLA 2 and cPLA 2 antibodies and siRNAs directed against iPLA 2 and cPLA 2 significantly inhibited cell proliferation and migration. Incubation of CM- or VEGF peptide-stimulated ECs with antibodies against VEGF or VEGFR-1/-2 receptors strongly reduced mitotic rate, cell migration, and phospho-cPLA 2 and iPLA 2 protein levels. The findings suggest that PLA 2 activities are involved in stimulating EC migration and proliferation in the presence of glioma CM and that cPLA 2 is positively regulated upstream by PI3-K, PKCα, and ERK1/2 signal cascades. Our work provides new insights in understanding EC metabolism and signaling during tumor angiogenesis.

Low energy laser light (632.8 nm) suppresses amyloid-β peptide-induced oxidative and inflammatory responses in astrocytes

Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid β-peptides (Aβ) in mediating these processes. In astrocytes, oligomeric Aβ induces the assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complexes resulting in its activation to produce anionic superoxide. Aβ also promotes production of pro-inflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by Aβ. Primary rat astrocytes were exposed to Helium–Neon laser (λ=632.8 nm), followed by the treatment with oligomeric Aβ. Primary rat astrocytes were used to measure Aβ-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47 phox subunit and the membrane gp91 phox subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A 2 cPLA 2 and expressions of pro-inflammatory factors including interleukin-1β (IL-1β) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed Aβ-induced superoxide production, colocalization between NADPH oxidase gp91 phox and p47 phox subunits, phosphorylation of cPLA 2, and the expressions of IL-1β and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD.

Preventive Effects of a Kampo Medicine, Hangeshashinto on Inflammatory Responses in Lipopolysaccharide-Treated Human Gingival Fibroblasts

In the present study, we investigated the effects of a Kampo medicine hangeshashinto (TJ-14) on the production of prostaglandin E2 (PGE2), interleukin (IL)-6 and IL-8 by human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) from Porphyromonas gingivalis. HGFs proliferation was dose-dependently decreased with hangeshashinto at days 3 and 7. However, treatment with LPS (10 ng/ml), hangeshashinto (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Hangeshashinto dose-dependently suppressed LPS-induced PGE2 production but did not alter basal PGE2 level. Hangeshashinto weakly decreased LPS-induced IL-6 and IL-8 productions. Hangeshashinto decreased cyclooxygenase (COX)-1 and COX-2 activities to approximately 60% at 1 mg/ml. Hangeshashinto decreased cytoplasmic phospholipase A2 (cPLA2) expression and LPS-induced COX-2 expression but not affected annexin1 expression. Hangeshashinto weakly suppressed LPS-induced extracellular signal-regulated kinase (ERK) phosphorylation, which is known to lead to ERK activation and cPLA2 phosphorylation. These results suggest that hangeshashinto decreased PGE2 production by (1) suppression of cPLA2 and LPS-induced COX-2 expression, and to a lesser extent, (2) inhibition of COX-2 activity and (3) inhibition of cPLA2 phosphorylation and its activation via inhibition of ERK phosphorylation. Moreover, it is also suggested that hangeshashinto may be useful to improve gingival inflammation in periodontal disease.

Signaling in TRPV1-induced platelet activating factor (PAF) in human esophageal epithelial cells

Transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors were identified in human esophageal squamous epithelial cell line HET-1A by RT-PCR and by Western blot. In fura-2 AM-loaded cells, the TRPV1 agonist capsaicin caused a fourfold cytosolic calcium increase, supporting a role of TRPV1 as a capsaicin-activated cation channel. Capsaicin increased production of platelet activating factor (PAF), an important inflammatory mediator that acts as a chemoattractant and activator of immune cells. The increase was reduced by the p38 MAP kinase (p38) inhibitor SB203580, by the cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF3, and by the lyso-PAF acetyltransferase inhibitor sanguinarin, indicating that capsaicin-induced PAF production may be mediated by activation of cPLA(2), p38, and lyso-PAF acetyltransferase. To establish a sequential signaling pathway, we examined the phosphorylation of p38 and cPLA(2) by Western blot. Capsaicin induced phosphorylation of p38 and cPLA(2). Capsaicin-induced p38 phosphorylation was not affected by AACOCF3. Conversely, capsaicin-induced cPLA(2) phosphorylation was blocked by SB203580, indicating that capsaicin-induced PAF production depends on sequential activation of p38 and cPLA(2). To investigate how p38 phosphorylation may result from TRPV1-mediated calcium influx, we examined a possible role of calmodulin kinase (CaM-K). p38 phosphorylation was stimulated by the calcium ionophore A23187 and by capsaicin, and the response to both agonists was reduced by a CaM inhibitor and by CaM-KII inhibitors, indicating that calcium induced activation of CaM and CaM-KII results in P38 phosphorylation. Acetyl-CoA transferase activity increased in response to capsaicin and was inhibited by SB203580, indicating that p38 phosphorylation in turn causes activation of acetyl-CoA transferase to produce PAF. Thus epithelial cells produce PAF in response to TRPV1-mediated calcium elevation.

Atorvastatin Reduces Collagen-Induced Platelet Function in Aspirin-Treated Patients with Acute Myocardial Infarction: Mechanisms Involved.

Abstract 3126Poster Board III-63Treatment with statin at the onset of acute myocardial infarction (AMI) has demonstrated a clinical benefit. The effects of statins on platelet TXA2 synthesis or platelet function could be involved in the clinical benefit, but has not been previously evaluated in patients with AMI. Collagen-induced (1μg/mL)-platelet aggregation, activation (14C-5HT release, TXA2 synthesis) and platelet recruitment (proaggregatory activity of activated platelets cell-free releasate) (1, 2) were evaluated in 184 patients with AMI within 48h of the onset of the acute event. Patients were divided into 2 groups: treated with aspirin (ASA) alone (n=139) and treated with both ASA and atorvastatin (AT; n=45). Insufficient inhibition of TXA2 (<95% vs. ASA-free normal subjects) was detected in 25% of patients treated with ASA vs. 9% in the group treated with ASA+AT (p<0.05), and persistent TXA2 synthesis was reduced by 87.5% in patients treated with ASA+AT vs. ASA alone. Reduction of TXA2 by AT was associated with significant inhibitions of collagen-induced: aggregation (30%), 14C-5HT release (39%) and recruitment (33%). To evaluate the biochemical mechanisms involved in the AT effect on TXA2 synthesis, experiments in vitro with washed platelets from normal subjects were performed. Incubation of AT alone (1-20μM) or AT plus a suboptimal ASA concentration (1μM) resulted in a significant inhibition of collagen-induced (1μg/mL) TXA2 synthesis and aggregation, confirming the results in the patients. In contrast, AT did not influence arachidonic acid-induced aggregation, suggesting an effect of the statin on the cytosolic phospholipase A2 (cPLA2) activity rather than on cyclooxygenase-1. cPLA2 activity is known to be regulated by its phosphorylation by MAPK (p38 or erk 1/2) and the increase in cytosolic Ca2+. In kinetic studies we found that AT reduced collagen-induced cPLA2 and erk 1/2 phosphorylation, but did not modify p38 phosphorylation. In addition, AT almost blocked collagen-induced increase in cytosolic Ca2+. In conclusion, AT associated with ASA significantly reduced collagen-induced persistent TXA2 synthesis and platelet reactivity early at the onset of AMI, reducing aspirin resistance in these patients. This effect is mediated by the AT control of cPLA2 activity via both erk1/2-mediated cPLA2 phosphorylation and blockade of collagen-induced elevation of cytosolic calcium. 1. Santos MT et.al. J Clin Invest 1991;87:571-80; 2. Vallés J et.al. Blood 2002;99:3978-84.FIS07/0463/MMA2006/RD06/0026 DisclosuresNo relevant conflicts of interest to declare.

PKCα-MAPK/ERK-phospholipase A 2 signaling is required for human melanoma-enhanced brain endothelial cell proliferation and motility

The largely undefined signal transduction mechanisms and cross-talk between human melanoma cell (HMC) lines and brain endothelial cells (ECs) involved in tumor cell interaction and adhesion were investigated. In immortalized rat brain GP8.3 EC cultures, conditioned media (CM) prepared from SK-MEL28 and OCM-1 melanoma cells significantly enhanced arachidonic acid release, cytosolic phospholipase A 2 (cPLA 2) and Ca +-independent phospholipase A 2 (iPLA 2) specific activities, and cell growth by 24 h. Inhibitors such as wortmannin and LY294002 (vs. PI3 kinase activity), AACOCF 3, (vs. cPLA 2 and iPLA 2), PD98059 (vs. ERK1/2 activity) and NS-398 (vs. cyclooxygenase-2 activity, COX-2) were all able to block cell proliferation and motility determined using a scratch wound healing assay in melanoma CMs-stimulated EC monolayers. These media also support the enhanced cell proliferation of primary ECs derived from rat brain (BBEC). Electroporation of anti-cPLA 2 antibody into ECs markedly inhibited the EC proliferation in response to CMs. With both CMs, phosphorylation of cPLA 2, PKCα, ERK1/2, protein and mRNA expression of cPLA 2 and iPLA 2, and COX-2 protein expression were significantly stimulated after 24 h coincubation, and attenuated by specific inhibitors. By confocal microscopy, activation of cPLA 2, ERK1/2, PKCα and COX-2 in perinuclear and membrane regions of ECs grown in CM-stimulated cultures were clearly observed. Thus MEK-PKCα-ERK1/2 and PI3-K/Akt survival pathways are activated in EC cultures during the interaction with CM from both melanoma cell lines, providing new insight in understanding EC metabolism and signaling. These pathways represent potential therapeutic targets to inhibit or enhance tumor angiogenesis.

LRP1 Controls cPLA2 Phosphorylation, ABCA1 Expression and Cellular Cholesterol Export

BackgroundATP-binding cassette transporter A1 mediates apolipoprotein AI-dependent efflux of cholesterol and thereby removes cholesterol from peripheral tissues. ABCA1 expression is tightly regulated and deficiency of this cholesterol transporter results in cholesterol accumulation within cells. Low-density lipoprotein receptor-related protein 1 (LRP1) participates in lipid metabolism and energy homeostasis by endocytosis of apolipoprotein E-containing lipoproteins and modulation of cellular proliferation signals.Methods and Principal FindingsIn the present study, we demonstrate a new role for LRP1 in reverse cholesterol transport. Absence of LRP1 expression results in increased PDGFRβ signaling and sequential activation of the mitogen-activated protein kinase signaling pathway, which increases phosphorylation of cytosolic phospholipase A2 (cPLA2). Phosphorylated and activated cPLA2 releases arachidonic acid from the phospholipid pool. Overproduction of arachidonic acid suppresses the activation of LXR/RXR heterodimers bound to the promoter of LXR regulated genes such as ABCA1, resulting in greatly reduced ABCA1 expression.Conclusions and SignificanceLRP1 regulates LXR-mediated gene transcription and participates in reverse cholesterol transport by controlling cPLA2 activation and ABCA1 expression. LRP1 thus functions as a physiological integrator of cellular lipid homeostasis with signals that regulate cellular proliferation and vascular wall integrity.

Involvement of the ERK1/2 Pathway During Oxytocin-Induced PGF2alpha Production in the Bovine Endometrial Epithelial Cell Line bEEL.

In ruminants, oxytocin (OT) initiates the pulsatile release of progstaglandin F2α (PGF2α) from the endometrial epithelial cells through the G protein coupled oxytocin receptor (OTR) that is up-regulated during the luteolytic phase. It has been proposed that the embryonic signal interferon tau exerts its antiluteolytic effect by disrupting the OT signaling axis. In this respect, we have studied the signaling pathway of OT-induced PGF2α production in bovine endometrial epithelial cell line (bEEL) using kinase inhibitors. Uncoupling OTR with suramin (100μM) inhibited OT (500nM)-induced PGF2α release confirming mediation through G protein subunits. OT induces intracellular Ca2+ mobilization suggesting Gαq/11 mediated signaling as was shown for myometrial OTRs. Surprisingly, however, neither the Ca2+ chelator BAPTA nor a PKC inhibitor had any effect on OT-induced PGF2α release. In contrast, the MEK inhibitor PD 98059 (10μM) and Raf1 inhibitor GW5074 (1μM) practically abolished OT-induced PGF2α production in bEEL cells, thus pointing at the involvement of the of ERK pathway. Inhibition of the small GTPase Ras by munanmyin (1μM) inhibited PGF2α by 50%; whereas LY 294002 (25μM) and AG1879 (10μM) inhibited OT-induced PGF2α production by 80-90% indicating the involvement of PI3K and Src, respectively. In addition, pertussis toxin (PTX 200 ngml-1) inhibited PGF2α production by 50% suggesting a role for Gαi/o; however, increasing intracellular cAMP by 8 bromo cAMP, forskolin or IBMX did not inhibit OT-induced PGF2α production suggesting involvement of Gαo rather than Gαi as the PTX sensitive subtype. Surprisingly, the inhibition of OT-induced PGF2α production was not accompanied by down regulation of COX2 expression. Therefore, phosphorylation of cPLA2, inhibition of COX2 activity, or uncoupling of PGF synthase from COX2 might be considered as alternate mechanisms behind the reduced PGF2α production. Our results suggest that OT-induced PGF2α production in bEEL cells is mediated principally through Gβ γ subunit initiating phosphorylation of downstream molecules such as PI3K, Src, Ras, Raf1, MEK and ERK kinases and co-regulated by PTX sensitive Gα. (poster)

NAD(P)H oxidase-mediated reactive oxygen species production alters astrocyte membrane molecular order via phospholipase A2

ROS (reactive oxygen species) overproduction is an important underlying factor for the activation of astrocytes in various neuropathological conditions. In the present study, we examined ROS production in astrocytes and downstream effects leading to changes in the signalling cascade, morphology and membrane dynamics using menadione, a redox-active compound capable of inducing intracellular ROS. NAD(P)H oxidase-mediated menadione-induced ROS production, which then stimulated phosphorylation of p38 MAPK (mitogen-activated protein kinase) and ERK1/2 (extracellular-signal-regulated kinase 1/2), and increased actin polymerization and cytoskeletal protrusions. We also showed that astrocyte plasma membranes became more molecularly ordered under oxidative stress, which was abrogated by down-regulating cPLA2 (cytosolic phospholipase A2) either with a pharmacological inhibitor or by RNA interference. In addition, mild disruption of F-actin with cytochalasin D suppressed menadione-enhanced phosphorylation of cPLA2 and membrane alterations. Taken together, these results suggest an important role for ROS derived from NAD(P)H oxidase in activation of astrocytes to elicit biochemical, morphological and biophysical changes reminiscent of reactive astrocytes in pathological conditions.

Cytosolic Phospholipase A2 Activation by Candida albicans in Alveolar Macrophages

Candida albicans is an increasingly important pulmonary fungal pathogen. Resident alveolar macrophages are important in host defense against opportunistic fungal infections. Activation of Group IVA cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in macrophages initiates arachidonic acid (AA) release for production of eicosanoids, which regulate inflammation and immune responses. We investigated the ability of C. albicans to activate cPLA(2)alpha in unprimed alveolar macrophages and after priming with granulocyte macrophage colony-stimulating factor (GM-CSF), which regulates alveolar macrophage maturation. AA was released within minutes by GM-CSF-primed but not unprimed alveolar macrophages in response to C. albicans, and was blocked by soluble glucan phosphate (S-GP). The expression of the beta-glucan receptor dectin-1 was increased in GM-CSF-primed macrophages, and AA release from GM-CSF-primed dectin-1(-/-) alveolar macrophages was reduced to basal levels. The enhanced activation of extracellular signal-regulated kinases and phosphorylation of cPLA(2)alpha on Ser-505 that occurred in GM-CSF-primed macrophages were reduced by MEK1 and Syk inhibitors, which also suppressed AA release. At later times after C. albicans infection (6 h), unprimed and GM-CSF-primed macrophages released similar levels of AA. The expression of cyclooxygenase 2 and prostanoid production at 6 hours was higher in GM-CSF-primed macrophages, but the responses were not dependent on dectin-1. However, dectin-1 contributed to the C. albicans-stimulated increase in TNF-alpha production that occurred in GM-CSF-primed macrophages. The results demonstrate that dectin-1 mediates the acute activation of cPLA(2)alpha in GM-CSF-primed alveolar macrophages, but not in the more delayed phase of AA release and GM-CSF-dependent prostanoid production.

A novel hypothesis regarding the possible involvement of cytosolic phospholipase 2 in insulin‐stimulated proliferation of vascular smooth muscle cells

Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA2) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA2 are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA2 and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA2 by 1.5-fold ( p < 0.01), which was blocked by the cPLA2 inhibitor MAFP (10 microM; 15 min). Similarly, the PI3K inhibitor LY294002 (10 microM; 15 min) and ERK1/2 inhibitor PD98059 (20 microM; 15 min) abolished the INS-mediated increase in cPLA2 phosphorylation by 59% (p < 0.001), and by 75% ( p < 0.001), respectively. Further, inhibition of cPLA2 with cPLA2 inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% ( p < 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% ( p < 0.001). The effect of INS on VSMCs proliferation was significantly ( p < 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA2 and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA2 activity.

Activation of Cytosolic Phospholipase A2 in Dorsal Root Ganglion Neurons by Ca2+/Calmodulin-Dependent Protein Kinase II after Peripheral Nerve Injury

BackgroundPeripheral nerve injury leads to a persistent neuropathic pain state in which innocuous stimulation elicits pain behavior (tactile allodynia), but the underlying mechanisms have remained largely unknown. We have previously shown that spinal nerve injury induces the activation of cytosolic phospholipase A2 (cPLA2) in injured dorsal root ganglion (DRG) neurons that contribute to tactile allodynia. However, little is known about the signaling pathway that activates cPLA2 after nerve injury. In the present study, we sought to determine the mechanisms underlying cPLA2 activation in injured DRG neurons in an animal model of neuropathic pain, focusing on mitogen-activated protein kinases (MAPKs) and Ca2+/calmodulin-dependent protein kinase II (CaMKII).ResultsPharmacological inhibition of either p38 or extracellular signal-regulated kinase (ERK) in the injured DRG, which led to suppression of the development of tactile allodynia, did not affect cPLA2 phosphorylation and translocation after nerve injury. By contrast, a CaMKII inhibitor prevented the development and expression of nerve injury-induced tactile allodynia and reduced both the level of cPLA2 phosphorylation and the number of DRG neurons showing translocated cPLA2 in response to nerve injury. Applying ATP to cultured DRG neurons increased the level of both phosphorylated cPLA2 and CaMKII in the vicinity of the plasma membrane and caused physical association of these two proteins. In addition, ATP-stimulated cPLA2 and CaMKII phosphorylation were inhibited by both a selective P2X3R/P2X2+3R antagonist and a nonselective voltage-dependent Ca2+ channel (VDCC) blocker.ConclusionThese results suggest that CaMKII, but not MAPKs, has an important role in cPLA2 activation following peripheral nerve injury, probably through P2X3R/P2X2+3R and VDCCs in primary afferent neurons.

Role of cytosolic phospholipase A2 in Pseudomonas aeruginosa-induced inflammation

Phospholipase A2 (PLA2) belongs to a superfamily of enzymes that hydrolyse phospholipids. This family can be classified into secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2) and Ca 2+ -independent PLA2 (iPLA2). cPLA2 selectively release arachidonic acid (AA) from membrane phospholipids. AA metabolites play a role in inflammation and have cytotoxic effects. Pseudomonas aeruginosa , a Gram-negative bacteria, is an opportunistic pathogen that infects immuno-compromised and cystic fibroblast patients. This results in high mortality due to the notorious resistance to antibiotics of this bacterium. But, the down-stream enzymes of the host involved in the pathogenesis of lung inflammation induced by P aeruginosa have not been identified. We postulated that cPLA2 plays an important role in P aeruginosa -induced inflammation. Alveolar epithelial cells A459 were pre-incubated with the indicated drugs 1 h before infection with laboratory P aeruginosa strains PAK or PAO1 or stimulation with bacterial toxins, PMA or TNFα A release was measured using [ 3 H]-AA labeled cells. IL-8 and PGE2 levels were determined using ELISA methods. Activation of MAPKs and expression of COX enzymes were analyzed by western blot. results showed that incubation of A549 epithelial cells with a wild-type strain of P aeruginosa , PAK, led to an increased cPLA2 phosphorylation which reflects its activation. This suggests that cPLA2 may be involved in P aeruginosa -induced inflammation. We found that either PAK or another P aeruginosa strain, PAO1, induced a time-dependent AA release from A549 cells. The production of prostaglandin E2 (PGE2), a downstream metabolite of AA, was increased significantly after stimulation with PAK PAK also induced IL-8 synthesis. In addition, our results showed that PAK induced p38MAPK phosphorylation in a time- and concentration- dependent manner. SB2035803, a specific inhibitor of p38 MAPK, abolished the effect of PAK on both PGE2 and IL-8 synthesis. Neither Erk nor Junk MAPK was involved in this process. The expression of cyclooxygenase (COX), which converts AA into PGE2, was increased after PAK stimulation. Finally, the effects of different virulence factors isolated from P aeruginosa , including LPS, flagellin and pili were investigated. LPS had no effect on PGE2 release and IL-8 synthesis. Flagellin stimulation increased IL- 8 expression but had no effect on PGE2 release. The δflic strain, in which the flagellin gene was deleted, had less effect on IL-8 synthesis but had similar effect on PGE2, as compared to the PAK strain. Collectively, our results show that cPLA2 is involved in P aeruginosa -induced inflammation through a process probably involving p38MAPK. However, the virulence factors involved in cPLA2 activation remains to be identified.

Linoleic acid stimulates gluconeogenesis via Ca2+/PLC, cPLA2, and PPAR pathways through GPR40 in primary cultured chicken hepatocytes

Fatty acids serve vital functions as sources of energy, building materials for cellular structures, and modulators of physiological responses. Therefore, this study examined the effect of linoleic acid on glucose production and its related signal pathways in primary cultured chicken hepatocytes. Linoleic acid (double-unsaturated, long chain) increased glucose production in a dose (> or =10(-4) M)- and time (> or =8 h)-dependent manner. Both oleic acid (monounsaturated, long chain) and palmitic acid (saturated, long chain) also increased glucose production, whereas caproic acid (saturated, short chain) failed to increase glucose production. Linoleic acid increased G protein-coupled receptor 40 (GPR40; also known as free fatty acid receptor-1) protein expression and glucose production that was blocked by GPR40-specific small interfering RNA. Linoleic acid increased intracellular calcium concentration, which was blocked by EGTA (extracellular calcium chelator)/BAPTA-AM (intracellular calcium chelator), U-73122 (phospholipase C inhibitor), nifedipine, or methoxyverapamil (L-type calcium channel blockers). Linoleic acid increased cytosolic phospholipase A(2) (cPLA(2)) phosphorylation and the release of [(3)H]-labeled arachidonic acid. Moreover, linoleic acid increased the level of cyclooxygenase-2 (COX-2) protein expression, which stimulated the synthesis of prostaglandin E(2) (PGE(2)). The increase in PGE(2) production subsequently stimulated peroxisome proliferator-activated receptor (PPAR) expression, and MK-886 (PPAR-alpha antagonist) and GW-9662 (PPAR-delta antagonist) inhibited glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. In addition, linoleic acid-induced glucose production was blocked by inhibition of extracellular and intracellular calcium, cPLA(2), COX-2, or PPAR pathways. In conclusion, linoleic acid promoted glucose production via Ca(2+)/PLC, cPLA(2)/COX-2, and PPAR pathways through GPR40 in primary cultured chicken hepatocytes.

Amyloid-β peptide induces temporal membrane biphasic changes in astrocytes through cytosolic phospholipase A 2

Oligomeric amyloid-β peptide (Aβ) is known to induce cytotoxic effects and to damage cell functions in Alzheimer's disease. However, mechanisms underlying the effects of Aβ on cell membranes have yet to be fully elucidated. In this study, Aβ 1–42 (Aβ 42) was shown to cause a temporal biphasic change in membranes of astrocytic DITNC cells using fluorescence microscopy of Laurdan. Aβ 42 made astrocyte membranes became more molecularly-disordered within the first 30 min to 1 h, but gradually changed to more molecularly-ordered after 3 h. However, Aβ 42 caused artificial membranes of vesicles made of rat whole brain lipid extract to become more disordered only. The trend for more molecularly-ordered membranes in astrocytes induced by Aβ 42 was abrogated by either an NADPH oxidase inhibitor, apocynin, or an inhibitor of cytosolic phospholipase A 2 (cPLA 2), but not by an inhibitor of calcium-independent PLA 2 (iPLA 2). Apocynin also suppressed the increased production of superoxide anions (O 2 −) and phosphorylation of cPLA 2 induced by Aβ 42. In addition, hydrolyzed products of cPLA 2, arachidonic acid (AA), but not lysophosphatidylcholine (LPC) caused astrocyte membranes to become more molecularly-ordered. These results suggest (1) a direct interaction of Aβ 42 with cell membranes making them more molecularly-disordered, and (2) Aβ 42 also indirectly makes membranes become more molecularly-ordered by triggering the signaling pathway involving NADPH oxidase and cPLA 2 in astrocytes.