Type 1 interferon-dependent repression of NLRC4 and iPLA2 licenses down-regulation of Salmonella flagellin inside macrophages.

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection with Salmonella and many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication. Salmonella switches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host. However, the host cues that Salmonella perceives to undergo this switch remain unclear. Here, we report an unexpected role of the NLRC4 inflammasome in promoting expression of its microbial ligand, flagellin, and identify a role for type 1 IFN signaling in switching of Salmonella to a flagellin-low phenotype. Early in infection, activation of NLRC4 by flagellin initiates pyroptosis and concomitant release of lysophospholipids which in turn enhance expression of flagellin by Salmonella thereby amplifying its ability to elicit cell death. TRIF-dependent production of type 1 IFN, however, later represses NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids that results in down-regulation of flagellin expression by Salmonella These findings reveal a previously unrecognized immune-modulating regulatory cross-talk between endosomal TLR signaling and cytosolic NLR activation with significant implications for the establishment of infection with Salmonella.

Lowered iPLA2γ activity causes increased mitochondrial lipid peroxidation and mitochondrial dysfunction in a rotenone-induced model of Parkinson's disease.

iPLA2γ, calcium-independent phospholipase A2γ, discerningly hydrolyses glycerophospholipids to liberate free fatty acids. iPLA2γ-deficiency has been associated with abnormal mitochondrial function. More importantly, the iPLA2 family is causative proteins in mitochondrial neurodegenerative disorders such as parkinsonian disorders. However, the mechanisms by which iPLA2γ affects Parkinson's disease (PD) remain unknown. Mitochondrion stress has a key part in rotenone-induced dopaminergic neuronal degeneration. The present evaluation revealed that lowered iPLA2γ function provokes the parkinsonian phenotype and leads to the reduction of dopamine and its metabolites, lowered survival, locomotor deficiencies, and organismal hypersensitivity to rotenone-induced oxidative stress. In addition, lowered iPLA2γ function escalated the amount of mitochondrial irregularities, including mitochondrial reactive oxygen species (ROS) regeneration, reduced ATP synthesis, reduced glutathione levels, and abnormal mitochondrial morphology. Further, lowered iPLA2γ function was tightly linked with strengthened lipid peroxidation and mitochondrial membrane flaws following rotenone treatment, which can cause cytochrome c release and eventually apoptosis. These results confirmed the important role of iPLA2γ, whereby decreasing iPLA2γ activity aggravates mitochondrial degeneration to induce neurodegenerative disorders in a rotenone rat model of Parkinson's disease. These findings may be useful in the design of rational approaches for the prevention and treatment of PD-associated symptoms.

Analyses of Calcium-Independent Phospholipase A2beta (iPLA2ß) in Biological Systems.

The Ca2+-independent phospholipases A2 (iPLA2s) are part of a diverse family of PLA2s, manifest activity in the absence of Ca2+, are ubiquitous, and participate in a variety of biological processes. Among the iPLA2s, the cytosolic iPLA2ß has received considerable attention and ongoing studies from various laboratories suggest that dysregulation of iPLA2ß can have a profound impact on the onset and/or progression of many diseases (e.g., cardiovascular, neurological, metabolic, autoimmune). Therefore, appropriate approaches are warranted to gain a better understanding of the role of iPLA2ß in vivo and its contribution to pathophysiology. Given that iPLA2ß is very labile, its basal expression is low in a number of cell systems, and that crystal structure of iPLA2ß is not yet available, careful and efficient protocols are needed to appropriately assess iPLA2ß biochemistry, dynamics, and membrane association. Here, step-by-step details are provided to (a) measure iPLA2ß-specific activity in cell lines or tissue preparations (using a simple radiolabel-based assay) and assess the impact of stimuli and inhibitors on resting- and disease-state iPLA2ß activity, (b) purify the iPLA2ß to near homogeneity (via sequential chromatography) from cell line or tissue preparations, enabling concentration of the enzyme for subsequent analyses (e.g., proteomics), and (c) employ hydrogen/deuterium exchange mass spectrometry analyses to probe both the structure of iPLA2ß and dynamics of its association with the membranes, substrates, and inhibitors.

Role of prefrontal cortical calcium-independent phospholipase A2 in antinociceptive effect of the norepinephrine reuptake inhibitor antidepresssant maprotiline.

The prefrontal cortex is essential for executive functions such as decision-making and planning. There is also accumulating evidence that it is important for the modulation of pain. In this study, we investigated a possible role of prefrontal cortical calcium-independent phospholipase A2 (iPLA2) in antinociception induced by the norepinephrine reuptake inhibitor (NRI) and tetracyclic (tricyclic) antidepressant, maprotiline. Intraperitoneal injections of maprotiline increased iPLA2 mRNA and protein expression in the prefrontal cortex. This treatment also reduced grooming responses to von-Frey hair stimulation of the face after facial carrageenan injection, indicating decreased sensitivity to pain. The antinociceptive effect of maprotiline was abrogated by iPLA2 antisense oligonucleotide injection to the prefrontal cortex, indicating a role of this enzyme in antinociception. In contrast, injection of iPLA2 antisense oligonucleotide to the somatosensory cortex did not reduce the antinociceptive effect of maprotiline. Lipidomic analysis of the prefrontal cortex showed decrease in phosphatidylcholine species, but increase in lysophosphatidylcholine species, indicating increased PLA2 activity, and release of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) after maprotiline treatment. Differences in sphingomyelin/ceramide were also detected. These changes were not observed in maprotiline-treated mice that received iPLA2 antisense oligonucleotide to the prefrontal cortex. Metabolites of DHA and EPA may help to strengthen a known supraspinal antinociceptive pathway from the prefrontal cortex to the periaqueductal gray. Together, results indicate a role of prefrontal cortical iPLA2 and its enzymatic products in the antinociceptive effect of maprotiline.

Role of cytosolic and calcium independent phospholipases A(2) in insulin secretion impairment of INS-1E cells infected by S. aureus.

Cytosolic PLA2 (cPLA2) and Ca(2+)-independent PLA2 (iPLA2) play a significant role in insulin ß-cells secretion. Bacterial infections may be responsible of the onset of diabetes. The mechanism by which Staphylococcus aureus infection of INS-1 cells alters glucose-induced insulin secretion has been examined. After acute infection, insulin secretion and PLA2 activities significantly increased. Moreover, increased expressions of phospho-cPLA2, phospho-PKCα and phospho-ERK 1/2 were observed. Chronic infection causes a decrease in insulin release and a significant increase of iPLA2 and COX-2 protein expression. Moreover, insulin secretion in infected cells could be restored using specific siRNAs against iPLA2 isoform and specific COX-2 inhibitor.

Discrete roles of intracellular phospholipases A2 in human neutrophil cytotoxicity.

BACKGROUND: The role of calcium-independent phospholipase A2 (iPLA2), a component of the three major PLA2 families, in acute/chronic inflammatory processes remains elusive. Previous investigations have documented iPLA2-mediated respiratory burst of neutrophils (PMNs); however, the causative isoform of iPLA2 is unidentified. We also demonstrated that the iPLA2γ-specific inhibitor attenuates trauma/hemorrhagic shock-induced lung injury. Therefore, iPLA2γ may be implicated in acute inflammation. In addition, arachidonic acid (AA), which is primarily produced by cytosolic PLA2 (cPLA2), is known to display PMN cytotoxicity, although the relationship between AA and the cytotoxic function is still being debated on. We therefore hypothesized that iPLA2γ regulates PMN cytotoxicity via AA-independent signaling pathways. The study aim was to distinguish the role of intracellular phospholipases A2, iPLA2, and cPLA2, in human PMN cytotoxicity and explore the possibility of the presence of signaling molecule(s) other than AA. METHODS: Isolated human PMNs were incubated with the PLA2 inhibitor selective for iPLA2ß, iPLA2γ, or cPLA2 and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). Superoxide production was assayed according to the superoxide dismutase-inhibitable cytochrome c reduction method, and the degree of elastase release was measured using a p-nitroanilide-conjugated elastase-specific substrate. In addition, chemotaxis toward platelet activating factor/fMLP was determined with a modified Boyden chamber system. RESULTS: The iPLA2γ-specific inhibitor reduced the fMLP/PMA-stimulated superoxide generation by 90% and 30%, respectively; in addition, the inhibitor completely blocked the fMLP/PMA-activated elastase release. However, the cPLA2-specific inhibitor did not abrogate these effects to any degree at all concentrations. Likewise, the inhibitor for iPLA2γ, but not iPLA2ß or cPLA2, completely inhibited the platelet activating factor/fMLP-induced chemotaxis. CONCLUSION: iPLA2 is involved in extracellular reactive oxygen species production, elastase release, and chemotaxis in response to well-defined stimuli. In addition, the ineffectiveness of the cPLA2 inhibitor suggests that AA may not be relevant to these cytotoxic functions.

The iPLA(2)γ is identified as the membrane potential sensitive phospholipase in liver mitochondria.

Previous reports from our lab identified a mitochondrial calcium-independent phospholipase A2 activity that is activated when the mitochondrial membrane potential is decreased. This activity was demonstrated to influence occurrence of the permeability transition. Originally, this activity was ascribed to the iPLA2ß protein. Recently, both iPLA2ß and iPLA2γ knock out mice have been generated. It has been shown by others that the iPLA2γ plays a significant role in progression of the permeability transition. In this paper, using the iPLA2ß and iPLA2γ knock out mice we show that the membrane potential sensitive activity is the iPLA2γ.

Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA(2) activity.

Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA2, which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death.

Impaired corticostriatal LTP and depotentiation following iPLA2 inhibition is restored following acute application of DHA.

Docosahexaenoic acid (DHA) is a 22 carbon, six cis-double bonded (22:6, w3) omega-3 polyunsaturated acid (PUFA), found highly enriched with neuronal membranes, and believed to play a critical role in synaptic plasticity and cognitive correlates of learning and memory. DHA is released from the neuronal membrane via the action of the cytostolic calcium-independent phospholipase A2 (iPLA2) enzyme. Previous studies have demonstrated that inhibition of iPLA2 by bromoenol lactone (BEL), results in inhibition of CA1 long-term potentiation (LTP), restored following acute application of DHA. In the present study, we investigated the effect of selective iPLA2 inhibition and acute application of DHA on corticostriatal synaptic plasticity. We demonstrate that acute application of 30µM DHA facilitates cotricostriatal LTP, whilst long-term depression (LTD), basal transmission, and paired-pulse facilitation (PPF) are unaffected. Conversely, selective inhibition of iPLA2, via acute application of 10µM BEL, inhibits the expression of corticostriatal LTP, with no effect on LTD. Furthermore, we show that 10µM BEL inhibition of LTP is reversed following acute application of 30µM DHA. Finally, we demonstrate that 10µM BEL inhibits depotentiation of corticostriatal LTP, which is restored following acute application of 30µM DHA. Our findings indicate that appropriate release of DHA is a critical facet of corticostriatal LTP and depotentiation, and thus provides an exciting cellular target for the positive facilitation of cognitive function observed following DHA dietary supplementation.

Calcium-independent phospholipase A2, group VIA, is critical for RPE cell survival.

PURPOSE: To investigate the significance of calcium-independent phospholipase A2, group VIA (iPLA2-VIA), in RPE cell survival following responses to sodium iodate (SI) in cell cultures. METHODS: The human retinal pigment epithelium (RPE) cell line (ARPE-19) cells and primary mouse-RPE cultures were treated with SI to induce cell death. Cells were transfected with an iPLA2-VIA promoter-luciferase construct to evaluate the regulation of iPLA-VIA after exposure to SI. PCR analysis, western blot analysis, and activity assays were performed to evaluate the mRNA level, protein level, and activity levels of iPLA2-VIA after SI exposure. Inhibitors of iPLA2-VIA were used to explore a potential protective role in cells exposed to SI. Primary RPE cell cultures were grown from iPLA2-VIA knockout mice and wild-type mice. The cultures were exposed to SI to investigate a possible increased protection against SI in iPLA2-VIA knockout mice compared to wild-type mice. RESULTS: The study revealed upregulation of iPLA2-VIA expression (promoter activity, iPLA2-VIA mRNA, iPLA2-VIA protein, and iPLA2-VIA protein activity) in ARPE-19 cells exposed to SI. SI-induced cell death was shown to be inhibited by iPLA2-VIA-specific inhibitors in ARPE-19 cell cultures. RPE cultures from iPLA2-VIA knockout mice were less vulnerable to SI-induced cell death compared to RPE cultures from wild-type mice. CONCLUSIONS: SI -induced RPE cell death involves iPLA2-VIA upregulation and activation, and amelioration of SI-induced RPE cell death can be facilitated by inhibitors of iPLA2-VIA. Thus, we suggest iPLA2-VIA as a possible pharmaceutical target to treat RPE-related retinal diseases.

New potent and selective polyfluoroalkyl ketone inhibitors of GVIA calcium-independent phospholipase A2.

Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as an important pharmaceutical target. Selective and potent GVIA iPLA2 inhibitors can be used to study its role in various neurological disorders. In the current work, we explore the significance of the introduction of a substituent in previously reported potent GVIA iPLA2 inhibitors. 1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)heptan-3-one (GK187) is the most potent and selective GVIA iPLA2 inhibitor ever reported with a XI(50) value of 0.0001, and with no significant inhibition against GIVA cPLA2 or GV sPLA2. We also compare the inhibition of two difluoromethyl ketones on GVIA iPLA2, GIVA cPLA2, and GV sPLA2.

Characterization of FKGK18 as inhibitor of group VIA Ca2+-independent phospholipase A2 (iPLA2ß): candidate drug for preventing beta-cell apoptosis and diabetes.

Ongoing studies suggest an important role for iPLA2ß in a multitude of biological processes and it has been implicated in neurodegenerative, skeletal and vascular smooth muscle disorders, bone formation, and cardiac arrhythmias. Thus, identifying an iPLA2ßinhibitor that can be reliably and safely used in vivo is warranted. Currently, the mechanism-based inhibitor bromoenol lactone (BEL) is the most widely used to discern the role of iPLA2ß in biological processes. While BEL is recognized as a more potent inhibitor of iPLA2 than of cPLA2 or sPLA2, leading to its designation as a "specific" inhibitor of iPLA2, it has been shown to also inhibit non-PLA2 enzymes. A potential complication of its use is that while the S and R enantiomers of BEL exhibit preference for cytosol-associated iPLA2ß and membrane-associated iPLA2γ, respectively, the selectivity is only 10-fold for both. In addition, BEL is unstable in solution, promotes irreversible inhibition, and may be cytotoxic, making BEL not amenable for in vivo use. Recently, a fluoroketone (FK)-based compound (FKGK18) was described as a potent inhibitor of iPLA2ß. Here we characterized its inhibitory profile in beta-cells and find that FKGK18: (a) inhibits iPLA2ß with a greater potency (100-fold) than iPLA2γ, (b) inhibition of iPLA2ß is reversible, (c) is an ineffective inhibitor of α-chymotrypsin, and (d) inhibits previously described outcomes of iPLA2ß activation including (i) glucose-stimulated insulin secretion, (ii) arachidonic acid hydrolysis; as reflected by PGE2 release from human islets, (iii) ER stress-induced neutral sphingomyelinase 2 expression, and (iv) ER stress-induced beta-cell apoptosis. These findings suggest that FKGK18 is similar to BEL in its ability to inhibit iPLA2ß. Because, in contrast to BEL, it is reversible and not a non-specific inhibitor of proteases, it is suggested that FKGK18 is more ideal for ex vivo and in vivo assessments of iPLA2ß role in biological functions.

Inhibition of iPLA2 ß and of stretch-activated channels by doxorubicin alters dystrophic muscle function.

BACKGROUND AND PURPOSE: Chronic elevation in intracellular Ca(2+) concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca(2+) overload involve store-operated channels (SOCs) and stretch-activated channels (SACs), which are modulated by the Ca(2+) -independent form of PL A2 (iPLA2 ). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca(2+) handling and muscle function in mdx mice. EXPERIMENTAL APPROACH: Effects of Dox on iPLA2 activity, reactive oxygen species production and on Ca(2+) influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca(2+) concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function. KEY RESULTS: Dox at 1-30 µM inhibited iPLA2 activity in cells and in the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca(2+) influx in myotubes. Stimulated elevations of Ca(2+) concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions. CONCLUSIONS AND IMPLICATIONS: Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment.

A mouse model of in vivo chemical inhibition of retinal calcium-independent phospholipase A2 (iPLA2).

Numerous studies have reported the implication of calcium-independent phospholipase A2 (iPLA2) in various biological mechanisms. Most of these works have used in vitro models and only a few have been carried out in vivo on iPLA2(-/-) mice. The functions of iPLA2 have been investigated in vivo in the heart, brain, pancreatic islets, and liver, but not in the retina despite its very high content in phospholipids. Phospholipids in the retina are known to be involved in several various key mechanisms such as visual transduction, inflammation or apoptosis. In order to investigate the implication of iPLA2 in these processes, this work was aimed to build an in vivo model of iPLA2 activity inhibition. After testing the efficacy of different chemical inhibitors of iPLA2, we have validated the use of bromoenol lactone (BEL) in vitro and in vivo for inhibiting the activity of iPLA2. Under in vivo conditions, a dose of 6µg/g of body weight of BEL in mice displayed a 50%-inhibition of retinal iPLA2 activity 8-16h after intraperitoneal administration. Delivering the same dose twice a day to animals was successful in producing a similar inhibition that was stable over one week. In summary, this novel mouse model exhibits a significant inhibition of retinal iPLA2 activity. This model of chemical inhibition of iPLA2 will be useful in future studies focusing on iPLA2 functions in the retina.

Fluoroketone inhibition of Ca(2+)-independent phospholipase A2 through binding pocket association defined by hydrogen/deuterium exchange and molecular dynamics.

The mechanism of inhibition of group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)) by fluoroketone (FK) ligands is examined by a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models for iPLA(2) were built by homology with the known structure of patatin and equilibrated by extensive MD simulations. Empty pockets were identified during the simulations and studied for their ability to accommodate FK inhibitors. Ligand docking techniques showed that the potent inhibitor 1,1,1,3-tetrafluoro-7-phenylheptan-2-one (PHFK) forms favorable interactions inside an active-site pocket, where it blocks the entrance of phospholipid substrates. The polar fluoroketone headgroup is stabilized by hydrogen bonds with residues Gly486, Gly487, and Ser519. The nonpolar aliphatic chain and aromatic group are stabilized by hydrophobic contacts with Met544, Val548, Phe549, Leu560, and Ala640. The binding mode is supported by DXMS experiments showing an important decrease of deuteration in the contact regions in the presence of the inhibitor. The discovery of the precise binding mode of FK ligands to the iPLA(2) should greatly improve our ability to design new inhibitors with higher potency and selectivity.

Genetic ablation of calcium-independent phospholipase A(2)γ (iPLA(2)γ) attenuates calcium-induced opening of the mitochondrial permeability transition pore and resultant cytochrome c release.

Herein, we demonstrate that calcium-independent phospholipase A(2)γ (iPLA(2)γ) is a critical mechanistic participant in the calcium-induced opening of the mitochondrial permeability transition pore (mPTP). Liver mitochondria from iPLA(2)γ(-/-) mice were markedly resistant to calcium-induced swelling in the presence or absence of phosphate in comparison with wild-type littermates. Furthermore, the iPLA(2)γ enantioselective inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one ((R)-BEL) was markedly more potent than (S)-BEL in inhibiting mPTP opening in mitochondria from wild-type liver in comparison with hepatic mitochondria from iPLA(2)γ(-/-) mice. Intriguingly, low micromolar concentrations of long chain fatty acyl-CoAs and the non-hydrolyzable thioether analog of palmitoyl-CoA markedly accelerated Ca(2+)-induced mPTP opening in liver mitochondria from wild-type mice. The addition of l-carnitine enabled the metabolic channeling of acyl-CoA through carnitine palmitoyltransferases (CPT-1/2) and attenuated the palmitoyl-CoA-mediated amplification of calcium-induced mPTP opening. In contrast, mitochondria from iPLA(2)γ(-/-) mice were insensitive to fatty acyl-CoA-mediated augmentation of calcium-induced mPTP opening. Moreover, mitochondria from iPLA(2)γ(-/-) mouse liver were resistant to Ca(2+)/t-butyl hydroperoxide-induced mPTP opening in comparison with wild-type littermates. In support of these findings, cytochrome c release from iPLA(2)γ(-/-) mitochondria was dramatically decreased in response to calcium in the presence or absence of either t-butyl hydroperoxide or phenylarsine oxide in comparison with wild-type littermates. Collectively, these results identify iPLA(2)γ as an important mechanistic component of the mPTP, define its downstream products as potent regulators of mPTP opening, and demonstrate the integrated roles of mitochondrial bioenergetics and lipidomic flux in modulating mPTP opening promoting the activation of necrotic and necroapoptotic pathways of cell death.

Interaction between VEGF and calcium-independent phospholipase A2 in proliferation and migration of retinal pigment epithelium.

PURPOSE: Inhibition of VEGF in the eye is an important treatment modality for reducing proliferation and migration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). Additionally, previous studies suggest calcium-independent phospholipase A(2) group VIA (iPLA(2)-VIA) to be a potential regulator of cell proliferation and migration, and evidence show abundant expression of iPLA(2)-VIA in RPE cells. The aim of the present study was to evaluate the potential role of iPLA(2)-VIA in VEGF-induced proliferation and migration of RPE cells. MATERIALS AND METHODS: The human RPE cell line, ARPE-19, was used in all assays. To explore the role of iPLA(2)-VIA in VEGF-induced RPE proliferation and migration, iPLA(2)-VIA inhibition by the iPLA(2)-VIA specific inhibitor, bromoenol lactone, was done. RPE cell proliferation and migration were evaluated by measurements of incorporated radioactive thymidine in DNA and by a Boyden chamber technique, respectively. A luciferase assay monitored the VEGF-induced iPLA(2)-VIA transcriptional activity. Western blot analysis and an activity assay were used to detect the protein levels and activity of iPLA(2)-VIA respectively after treatment with VEGF. RESULTS: RPE cells treated with VEGF showed significant increased proliferation and migration. Furthermore, inhibition of iPLA(2)-VIA significantly reduced the spontaneous proliferation and migration as well as the VEGF-induced proliferation and migration. Finally, inhibition of iPLA(2)-VIA reduced the VEGF-induced iPLA(2)-VIA-activity, -protein level, and -promoter activity. CONCLUSIONS: A significant interaction between VEGF and iPLA(2)-VIA in the regulation of RPE cells appears to be relevant in elucidating the exact mechanisms of action in the proliferative and migratory phenotype of RPE cells in AMD.

Activation of mitochondrial calcium-independent phospholipase A2γ (iPLA2γ) by divalent cations mediating arachidonate release and production of downstream eicosanoids.

Calcium-independent phospholipase A(2)γ (iPLA(2)γ) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)γ gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)γ in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)γ resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)γ mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)γ selective), but not its enantiomer, (S)-BEL (iPLA(2)ß selective) or pyrrolidine (cytosolic PLA(2)α selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)γ activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)γ. Intriguingly, Ca(2+)-induced iPLA(2)γ activation was completely inhibited by long-chain acyl-CoA (IC(50) ∼20 µm) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)γ is activated by divalent cations and inhibited by acyl-CoA modulating the generation of biologically active metabolites that regulate mitochondrial bioenergetic and signaling functions.

Group VIA PLA2 (iPLA2ß) is activated upstream of p38 mitogen-activated protein kinase (MAPK) in pancreatic islet ß-cell signaling.

Group VIA phospholipase A(2) (iPLA(2)ß) in pancreatic islet ß-cells participates in glucose-stimulated insulin secretion and sarco(endo)plasmic reticulum ATPase (SERCA) inhibitor-induced apoptosis, and both are attenuated by pharmacologic or genetic reductions in iPLA(2)ß activity and amplified by iPLA(2)ß overexpression. While exploring signaling events that occur downstream of iPLA(2)ß activation, we found that p38 MAPK is activated by phosphorylation in INS-1 insulinoma cells and mouse pancreatic islets, that this increases with iPLA(2)ß expression level, and that it is stimulated by the iPLA(2)ß reaction product arachidonic acid. The insulin secretagogue D-glucose also stimulates ß-cell p38 MAPK phosphorylation, and this is prevented by the iPLA(2)ß inhibitor bromoenol lactone. Insulin secretion induced by d-glucose and forskolin is amplified by overexpressing iPLA(2)ß in INS-1 cells and in mouse islets, and the p38 MAPK inhibitor PD169316 prevents both responses. The SERCA inhibitor thapsigargin also stimulates phosphorylation of both ß-cell MAPK kinase isoforms and p38 MAPK, and bromoenol lactone prevents both events. Others have reported that iPLA(2)ß products activate Rho family G-proteins that promote MAPK kinase activation via a mechanism inhibited by Clostridium difficile toxin B, which we find to inhibit thapsigargin-induced ß-cell p38 MAPK phosphorylation. Thapsigargin-induced ß-cell apoptosis and ceramide generation are also prevented by the p38 MAPK inhibitor PD169316. These observations indicate that p38 MAPK is activated downstream of iPLA(2)ß in ß-cells incubated with insulin secretagogues or thapsigargin, that this requires prior iPLA(2)ß activation, and that p38 MAPK is involved in the ß-cell functional responses of insulin secretion and apoptosis in which iPLA(2)ß participates.

Phospholipase A2 superfamily members play divergent roles after spinal cord injury.

Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be preventable as it occurs, secondary to the trauma. We show that the phospholipase A(2) (PLA(2)) superfamily plays important roles in SCI. PLA(2) enzymes hydrolyze membrane glycerophospholipids to yield a free fatty acid and lysophospholipid. Some free fatty acids (arachidonic acid) give rise to eicosanoids that promote inflammation, while some lysophospholipids (lysophosphatidylcholine) cause demyelination. We show in a mouse model of SCI that two cytosolic forms [calcium-dependent PLA(2) group IVA (cPLA(2) GIVA) and calcium-independent PLA(2) group VIA (iPLA(2) GVIA)], and a secreted form [secreted PLA(2) group IIA (sPLA(2) GIIA)] are up-regulated. Using selective inhibitors and null mice, we show that these PLA(2)s play differing roles. cPLA(2) GIVA mediates protection, whereas sPLA(2) GIIA and, to a lesser extent, iPLA(2) GVIA are detrimental. Furthermore, completely blocking all three PLA(2)s worsens outcome, while the most beneficial effects are seen by partial inhibition of all three. The partial inhibitor enhances expression of cPLA(2) and mediates its beneficial effects via the prostaglandin EP1 receptor. These findings indicate that drugs that inhibit detrimental forms of PLA(2) (sPLA(2) and iPLA2) and up-regulate the protective form (cPLA2) may be useful for the treatment of SCI.