PLA2G4E, a candidate gene for resilience in Alzheimer´s disease and a new target for dementia treatment.

Clinical studies revealed that some aged-individuals accumulate a significant number of histopathological Alzheimer´s disease (AD) lesions in their brain, yet without developing any signs of dementia. Animal models of AD represent suitable tools to identify genes that might promote cognitive resilience and hence, this study first set out to identify cognitively resilient individuals in the aged-Tg2576 mouse model. A transcriptomic analysis of these mice identified PLA2G4E as a gene that might confer resistance to dementia. Indeed, a significant decrease in PLA2G4E is evident in the brain of late-stage AD patients, whereas no such changes are observed in early stage patients with AD neuropathological lesions but no signs of dementia. We demonstrated that adeno-associated viral vector-mediated overexpression of PLA2G4E in hippocampal neurons completely restored cognitive deficits in elderly APP/PS1 mice, without affecting the amyloid or tau pathology. These PLA2G4E overexpressing APP/PS1 mice developed significantly more dendritic spines than sham-injected mice, coinciding with the cognitive improvement observed. Hence, these results support the idea that a loss of PLA2G4E might play a key role in the onset of dementia in AD, highlighting the potential of PLA2G4E overexpression as a novel therapeutic strategy to manage AD and other disorders that course with memory deficits.

[Inhibition of Group IVA Phospholipase A2 as a Novel Therapeutic Strategy for Nonalcoholic Steatohepatitis].

Nonalcoholic steatohepatitis (NASH) is a lifestyle-related disease characterized by hepatic fibrosis with the accumulation of fat and inflammation and can progress to cirrhosis or hepatocellular carcinoma. However, effective pharmacotherapeutic strategies for hepatic fibrosis in NASH remain to be established. Among the initiators of inflammation, we have been investigating the possible involvement of group IVA phospholipase A2 (IVA-PLA2), which catalyzes the initial step in the generation of lipid mediators, including eicosanoids and lysophospholipids, in the progression of hepatic fibrosis. We have recently demonstrated that a lack of IVA-PLA2 alleviates hepatic fibrosis in NASH model mice fed a high-fat and high-cholesterol diet and in CCl4-treated mice. CCl4-induced hepatic fibrosis was also prevented by the administration of an orally active, specific IVA-PLA2 inhibitor even after hepatic fibrosis had developed. Based on these findings suggesting that IVA-PLA2 mediates the cellular responses contributing to the progression of hepatic fibrosis, we have been exploring which types of cells in the liver are involved in IVA-PLA2-mediated hepatic fibrosis using cell-specific IVA-PLA2 knockout mice. The preliminary experimental results suggest that IVA-PLA2 in endothelial cells, but not monocyte-derived cells, plays a role, in part, in the hepatic stellate cell-mediated progression of hepatic fibrosis. In this paper, we discuss the possibility that IVA-PLA2 and/or its related molecules are candidate pharmacotherapeutic targets for NASH treatment.

Brain Cytosolic Phospholipase A2α Mediates Angiotensin II-Induced Hypertension and Reactive Oxygen Species Production in Male Mice.

BACKGROUND: Recently, we reported that angiotensin II (Ang II)-induced hypertension is mediated by group IV cytosolic phospholipase A2α (cPLA2α) via production of prohypertensive eicosanoids. Since Ang II increases blood pressure (BP) via its action in the subfornical organ (SFO), it led us to investigate the expression and possible contribution of cPLA2α to oxidative stress and development of hypertension in this brain area. METHODS: Adenovirus (Ad)-green fluorescence protein (GFP) cPLA2α short hairpin (sh) RNA (Ad-cPLA2α shRNA) and its control Ad-scrambled shRNA (Ad-Scr shRNA) or Ad-enhanced cyan fluorescence protein cPLA2α DNA (Ad-cPLA2α DNA) and its control Ad-GFP DNA were transduced into SFO of cPLA2α+/+ and cPLA2α-/- male mice, respectively. Ang II (700 ng/kg/min) was infused for 14 days in these mice, and BP was measured by tail-cuff and radio telemetry. cPLA2 activity, reactive oxygen species production, and endoplasmic reticulum stress were measured in the SFO. RESULTS: Transduction of SFO with Ad-cPLA2α shRNA, but not Ad-Scr shRNA in cPLA2α+/+ mice, minimized expression of cPLA2α, Ang II-induced cPLA2α activity and oxidative stress in the SFO, BP, and cardiac and renal fibrosis. In contrast, Ad-cPLA2α DNA, but not its control Ad-GFP DNA in cPLA2α-/- mice, restored the expression of cPLA2α, and Ang II-induced increase in cPLA2 activity and oxidative stress in the SFO, BP, cardiac, and renal fibrosis. CONCLUSIONS: These data suggest that cPLA2α in the SFO is crucial in mediating Ang II-induced hypertension and associated pathogenesis. Therefore, development of selective cPLA2α inhibitors could be useful in treating hypertension and its pathogenesis.

Regulatory role of cytosolic phospholipase A2 alpha in the induction of CD40 in microglia.

BACKGROUND: The aberrant expression of CD40, a co-stimulatory receptor found on the antigen-presenting cells, is involved in the pathogenesis of various degenerative diseases. Our previous study demonstrated that the reduction of cytosolic phospholipase A2 alpha (cPLA2α) protein overexpression and activation in the spinal cord of a mouse model of ALS, hmSOD1 G93A, inhibited CD40 upregulation in microglia. The present study was designed to determine whether cPLA2α has a direct, participatory role in the molecular events leading to CD40 induction. METHODS: Cultures of primary mouse microglia or BV-2 microglia cell line exposed to lipopolysaccharide (LPS) or interferon gamma (IFNγ) for different periods of time, in order to study the role of cPLA2α in the events leading to CD40 protein induction. RESULTS: Addition of LPS or IFNγ caused a significant upregulation of cPLA2α and of CD40, while prevention of cPLA2α upregulation by a specific oligonucleotide antisense (AS) prevented the induction of CD40, suggesting a role of cPLA2α in the induction of CD40. Addition of LPS to microglia caused an immediate activation of cPLA2α detected by its phosphorylated form, while addition of IFNγ induced cPLA2α activation at a later time scale (4 h). The activation of cPLA2α is mediated by ERK activity. Suppression of cPLA2α activity inhibited superoxide production by NOX2-NADPH oxidase and activation of NF-κB detected by the phosphorylation of p65 on serine 536 at 15 min by LPS and at 4 h by IFNγ. Inhibition of NOX2 prevented NF-κB activation and CD40 induction but did not affect cPLA2α activation, suggesting cPLA2α is located upstream to NOX2 and NF-κB. The activation of cPLA2 by LPS was mediated by both adaptor proteins downstream to LPS receptor; TRIF and MyD88, while the activation of cPLA2α by IFNγ was mediated by the secreted TNF-α at 4 h. The early activation of STAT1α (detected by phospho-serine727 and phoshpo-tyrosine701) by IFNγ and the late activation of STAT1α by LPS were not affected in the presence of cPLA2α inhibitors, indicating that STAT1α is not under cPLA2α regulation. CONCLUSIONS: Our results show for the first time that cPLA2 upregulates CD40 protein expression induced by either LPS or IFNγ, and this regulatory effect is mediated via the activation of NOX2-NADPH oxidase and NF-κB. Cumulatively, our results indicate that cPLA2α may serve as a pivotal amplifier of the inflammatory response in the CNS.

A novel read-through transcript JMJD7-PLA2G4B regulates head and neck squamous cell carcinoma cell proliferation and survival.

Recent findings on the existence of oncogenic fusion genes in a wide array of solid tumors, including head and neck squamous cell carcinoma (HNSCC), suggests that fusion genes have become attractive targets for cancer diagnosis and treatment. In this study, we showed for the first time that a read-through fusion gene JMJD7-PLA2G4B is presented in HNSCC, splicing neighboring jumonji domain containing 7 (JMJD7) and phospholipase A2, group IVB (PLA2G4B) genes together. Ablation of JMJD7-PLA2G4B significantly inhibited proliferation of HNSCC cells by promoting G1 cell cycle arrest and increased starvation-induced cell death compared to JMJD7-only knockdown HNSCC cells. Mechanistically, we found that JMJD7-PLA2G4B modulates phosphorylation of Protein Kinase B (AKT) to promote HNSCC cell survival. Moreover, JMJD7-PLA2G4B also regulated an E3 ligase S-phase kinase-associated protein 2 (SKP2) to control the cell cycle progression from G1 phase to S phase by inhibiting Cyclin-dependent kinase inhibitor 1 (p21) and 1B (p27) expression. Our study provides novel insights into the oncogenic control of JMJD7-PLA2G4B in HNSCC cell proliferation and survival, and suggests that JMJD7-PLA2G4B may serve as an important therapeutic target and prognostic marker for HNSCC development and progression.

Cytosolic phospholipase A(2)α and eicosanoids regulate expression of genes in macrophages involved in host defense and inflammation.

The role of Group IVA cytosolic phospholipase A2 (cPLA2α) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2α releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFα was lower in C. albicans-stimulated cPLA2α(+/+) than cPLA2α(-/-) macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2α(+/+) than cPLA2α(-/-) macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2α(+/+) and cPLA2α(-/-) macrophages (3 h) was compared by microarray. cPLA2α(+/+) macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2α(-/-) macrophages (≥2-fold, p<0.05). Several pro-inflammatory genes were expressed at lower levels (Tnfα, Cx3cl1, Cd40, Ccl5, Csf1, Edn1, CxCr7, Irf1, Irf4, Akna, Ifnγ, several IFNγ-inducible GTPases). Genes that dampen inflammation (Socs3, Il10, Crem, Stat3, Thbd, Thbs1, Abca1) and genes involved in host defense (Gja1, Csf3, Trem1, Hdc) were expressed at higher levels in cPLA2α(+/+) macrophages. Representative genes expressed lower in cPLA2α(+/+) macrophages (Tnfα, Csf1) were increased by treatment with a prostacyclin receptor antagonist and protein kinase A inhibitor, whereas genes expressed at higher levels (Crem, Nr4a2, Il10, Csf3) were suppressed. The results suggest that C. albicans stimulates an autocrine loop in macrophages involving cPLA2α, cyclooxygenase 1-derived prostaglandins and increased cAMP that globally effects expression of genes involved in host defense and inflammation.

Disruption of group IVA cytosolic phospholipase A(2) attenuates myocardial ischemia-reperfusion injury partly through inhibition of TNF-α-mediated pathway.

Group IVA cytosolic phospholipase A(2) (cPLA(2)α), which preferentially cleaves arachidonic acid from phospholipids, plays a role in apoptosis and tissue injury. Downstream signals in response to tumor necrosis factor (TNF)-α, a mediator of myocardial ischemia-reperfusion (I/R) injury, involve cPLA(2)α activation. This study examined the potential role of cPLA(2)α and its mechanistic link with TNF-α in myocardial I/R injury using cPLA(2)α knockout (cPLA(2)α(-/-)) mice. Myocardial I/R was created with 10-wk-old male mice by 1 h ligation of the left anterior descending coronary artery, followed by 24 h of reperfusion. As a result, compared with wild-type (cPLA(2)α(+/+)) mice, cPLA(2)α(-/-) mice had a 47% decrease in myocardial infarct size, preservation of echocardiographic left ventricle (LV) function (%fractional shortening: 14 vs. 21%, respectively), and lower content of leukotriene B(4) and thromboxane B(2) (62 and 50% lower, respectively) in the ischemic myocardium after I/R. Treatment with the TNF-α inhibitor (soluble TNF receptor II/IgG1 Fc fusion protein, sTNFR:Fc) decreased myocardial I/R injury and LV dysfunction in cPLA(2)α(+/+) mice but not cPLA(2)α(-/-) mice. sTNFR:Fc also suppressed cPLA(2)α phosphorylation in the ischemic myocardium after I/R of cPLA(2)α(+/+) mice. Similarly, sTNFR:Fc exerted protective effects against hypoxia-reoxygenation (H/R)-induced injury in the cultured cardiomyocytes from cPLA(2)α(+/+) mice but not cPLA(2)α(-/-) cardiomyocytes. H/R and TNF-α induced cPLA(2)α phosphorylation in cPLA(2)α(+/+) cardiomyocytes, which was reversible by sTNFR:Fc. In cPLA(2)α(-/-) cardiomyocytes, TNF-α induced apoptosis and release of arachidonic acid to a lesser extent than in cPLA(2)α(+/+) cardiomyocytes. In conclusion, disruption of cPLA(2)α attenuates myocardial I/R injury partly through inhibition of TNF-α-mediated pathways.

Mitochondrial dysfunction and ß-cell failure in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is the most common human endocrine disease and is characterized by peripheral insulin resistance and pancreatic islet ß-cell failure. Accumulating evidence indicates that mitochondrial dysfunction is a central contributor to ß-cell failure in the evolution of T2DM. As reviewed elsewhere, reactive oxygen species (ROS) produced by ß-cell mitochondria as a result of metabolic stress activate several stress-response pathways. This paper focuses on mechanisms whereby ROS affect mitochondrial structure and function and lead to ß-cell failure. ROS activate UCP2, which results in proton leak across the mitochondrial inner membrane, and this leads to reduced ß-cell ATP synthesis and content, which is a critical parameter in regulating glucose-stimulated insulin secretion. In addition, ROS oxidize polyunsaturated fatty acids in mitochondrial cardiolipin and other phospholipids, and this impairs membrane integrity and leads to cytochrome c release into cytosol and apoptosis. Group VIA phospholipase A2 (iPLA2ß) appears to be a component of a mechanism for repairing mitochondrial phospholipids that contain oxidized fatty acid substituents, and genetic or acquired iPLA2ß-deficiency increases ß-cell mitochondrial susceptibility to injury from ROS and predisposes to developing T2DM. Interventions that attenuate ROS effects on ß-cell mitochondrial phospholipids might prevent or retard development of T2DM.

Towards novel radiosensitizing agents: the role of cytosolic PLA2α in combined modality cancer therapy.

The radioresistant nature of some tumors serves as an obstacle to curative therapy for several poor-prognosis malignancies. The radiosensitivity of a cancer is dependent not only on the intrinsic ability of tumor cells to recover from radiation-induced damage, but also the ability of stromal elements (e.g., vasculature) in the tumor microenvironment to survive and continue proliferating in the face of ionizing radiation. In this regard, it is important to understand the initial events activating radiation-induced signal transduction pathways. Among these events is the activation of cytosolic phospholipase A2 α and the subsequent production of the lipid second messengers. These events occur within minutes following exposure to ionizing radiation, and have been shown to enhance cell viability through a number of prosurvival signaling pathways. Furthermore, inhibition of cytosolic phospholipase A2 α has now been shown to reduce the viability of endothelial cells in culture after exposure to ionizing radiation, as well as slowing the growth of tumors in animal models of cancer.

Tumor cell group via phospholipase A2 is involved in prostate cancer development.

BACKGROUND: Prostate cancer (PCa) is one of the most common malignancies among men in the United States. Further understanding of the molecular mechanisms underlying PCa tumorigenic development is critical for advancing treatment strategies for PCa. The role of Group VIA phospholipase A2ß (iPLA2ß) in cancers has recently emerged. However, the biological functions of iPLA2ß in PCa development have been minimally investigated and only in vitro studies have been reported. METHODS: We tested the role of iPLA2ß in host cells using an iPLA2ß deficient mouse model and the role of iPLA2ß in tumor cells by comparing the proliferation, migration, and invasion in vitro and tumorigenesis in vivo. CONCLUSIONS: iPLA2ß deficiency did not affect tumor development in C57BL/6 mice injected with syngeneic PCa cell line TRAMP-C1P3 in any of three models (subcutaneous, orthotopic, or intratibia injection) tested, suggesting that host cell iPLA2ß is not required for PCa tumorigenesis and metastasis. In contrast, when iPLA2ß was down-regulated in TRAMP-C1P3 cells, cell proliferation was reduced in vitro and tumor growth was suppressed in vivo compared to control cells. In particular, iPLA2ß was required for lysophosphatidic acid (LPA)-induced migration and invasion in TRAMP-C1P3 cells. We compared human and mouse PCa cells and showed that they shared high similarities in LPA-stimulated effects and signaling pathways. LPA stimulated cell migration and/or invasion via a PI3K-dependent pathway. Together, our results suggest that the tumor cell iPLA2ß-LPA axis may represent a novel target for PCa.

Urothelial cell platelet-activating factor production mediated by calcium-independent phospholipase A2γ.

OBJECTIVES: To determine the effect of phospholipase A(2) (PLA(2)) inhibitors on urothelial cell platelet-activating factor (PAF) production in response to tryptase stimulation. METHODS: Urothelial cells isolated from normal human ureters were immortalized with the human papillomavirus type 16E6E7 cell line (TEU-2 cells). PLA(2) activity in TEU-2 cells was measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence and absence of calcium. [(3)H]PAF production was measured in TEU-2 cells prelabeled with [(3)H] acetic acid. PAF-acetylhydrolase activity was measured by determining the amount of [(3)H] acetate hydrolyzed from [(3)H]PAF incubated with TEU-2 cellular protein. Adherence of human polymorphonuclear leukocyte (PMN) to TEU-2 cells was assessed by measuring myeloperoxidase activity in adherent PMNs after incubation with TEU-2 cells. RESULTS: Most PLA(2) activity measured in TEU-2 cells was determined to be membrane-associated, calcium-independent PLA(2) and selective for plasmenylcholine substrate. Stimulation of TEU-2 cells with tryptase results in increased production of PAF and increased PMN adherence that were inhibited completely by pretreatment with the membrane-associated, calcium-independent PLA(2)γ-selective inhibitor (R)-bromoenol lactone. Pretreatment with the cytosolic PLA(2) inhibitor methyl arachidonyl fluorophosphonate resulted in potentiation of tryptase-stimulated PAF production and PMN adherence to TEU-2 cells that is a result of PAF-acetylhydrolase inhibition. CONCLUSIONS: Tryptase stimulation of TEU-2 cells results in activation of membrane-associated, calcium-independent PLA(2)γ, leading to an increase in PAF production and increased PMN adherence. Inhibition of TEU-2 cell PAF-acetylhydrolase activity with methyl arachidonyl fluorophosphonate potentiated tryptase-stimulated PAF production and PMN adherence.

Cytosolic phospholipase A2 group IVA influence on GM-CSF expression in human lung cells: a pilot study.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important mediator in the differentiation, maturation, and survival of inflammatory cells. It might play a significant role in the pathogenesis of asthma. We have shown previously that cytosolic phospholipase A2 group IV A (cPLA2alpha) is able to activate gene expression, through peroxisome proliferator-activated receptor (PPAR)-gamma response elements (PPRE). In the promoter regions of GM-CSF gene (CSF2), we have found potential PPRE. The goal of the current study was to investigate the influence of cPLA2 overexpression and activation on CSF2 gene expression in human lung cells. MATERIAL/METHODS: Subconfluent A549 cells were transfected with GM-CSF reporter gene and cPLA2alpha overexpression vector. Transfected cells were treated with or without calcium ionophore, A23187, or epidermal growth factor (EGF). Cell lysates were collected and assayed for dual luciferase activity. Some cultures were preincubated with a potent cPLA2alpha inhibitor, methyl arachidonyl fluorophosphonate (MAFP); a secretory phospholipase A2 inhibitor, thioetheramide-PC; a calcium-independent phospholipase A2 inhibitor, bromoenol lactone (BEL); or vehicle. After preincubation, cells were treated with A23187. Expression of GM-CSF messenger RNA (mRNA) was measured using real-time polymerase chain reaction mRNA quantification. RESULTS: Overexpression of cPLA2alpha or activation of endogenous cPLA2alpha by calcium ionophore or EGF caused a significant increase in GM-CSF relative luciferase activity. Calcium ionophore significantly increased GM-CSF mRNA in A549 human lung cells. These effects were at least in part inhibited by MAFP treatment, but not by BEL or thioetheramide-PC. CONCLUSIONS: These preliminary data might suggest an influence of cPLA2alpha on GM-CSF gene expression in human lung cells, which could play a role in the pathogenesis of asthma and other inflammatory diseases.

Cytosolic phospholipase A2alpha and peroxisome proliferator-activated receptor gamma signaling pathway counteracts transforming growth factor beta-mediated inhibition of primary and transformed hepatocyte growth.

UNLABELLED: Hepatocellular carcinoma often develops in the setting of abnormal hepatocyte growth associated with chronic hepatitis and liver cirrhosis. Transforming growth factor beta (TGF-beta) is a multifunctional cytokine pivotal in the regulation of hepatic cell growth, differentiation, migration, extracellular matrix production, stem cell homeostasis, and hepatocarcinogenesis. However, the mechanisms by which TGF-beta influences hepatic cell functions remain incompletely defined. We report herein that TGF-beta regulates the growth of primary and transformed hepatocytes through concurrent activation of Smad and phosphorylation of cytosolic phospholipase A(2)alpha (cPLA(2)alpha), a rate-limiting key enzyme that releases arachidonic acid for the production of bioactive eicosanoids. The interplays between TGF-beta and cPLA(2)alpha signaling pathways were examined in rat primary hepatocytes, human hepatocellular carcinoma cells, and hepatocytes isolated from newly developed cPLA(2)alpha transgenic mice. CONCLUSION: Our data show that cPLA(2)alpha activates peroxisome proliferator-activated receptor gamma (PPAR-gamma) and thus counteracts Smad2/3-mediated inhibition of cell growth. Therefore, regulation of TGF-beta signaling by cPLA(2)alpha and PPAR-gamma may represent an important mechanism for control of hepatic cell growth and hepatocarcinogenesis.

Involvement of cytosolic phospholipase A2alpha in MMP-9 up-regulation.

Matrix metalloproteinase-9 (MMP-9) is important in the pathogenesis of periodontitis. Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is involved in MMP-9 up-regulation in human monocytes. We tested the hypothesis that cPLA(2)alpha also regulates MMP-9 induction by Fusobacterium nucleatum and by phorbol 12-myristate-13-acetate (PMA) in gingival epithelial cells. While PMA induced MMP-9 expression considerably, F. nucleatum did so moderately. This time-course study demonstrated that MMP-9 mRNA up-regulation occurred at 3 hours, whereas MMP-9 secretion and activity in cell-free supernatants occurred at 12 hours. cPLA(2)alpha mRNA was constitutively expressed in gingival epithelial cells. Transient activation of cPLA(2) by Ser505 phosphorylation was observed in the nuclei upon stimulation, suggesting its role as a transcription factor, while cPLA(2) protein expression remained unchanged. Induction of MMP-9 expression and activity was significantly inhibited by 1 muM of the specific cPLA(2)alpha inhibitor (P < 0.01). These findings demonstrate the involvement of cPLA(2)alpha in MMP-9 up-regulation.

Interleukin-32 positively regulates radiation-induced vascular inflammation.

PURPOSE: To study the role of interleukin-32 (IL-32), a novel protein only detected in human tissues, in ionizing radiation (IR)-induced vascular inflammation. METHODS AND MATERIALS: Irradiated (0-6 Gy) human umbilical vein endothelial cells treated with or without various agents--a cytosolic phospholipase A2 (cPLA2) inhibitor, a cyclooxygenase-2 (Cox-2) inhibitor, or lysophosphatidylcholines (LPCs)--were used to assess IL-32 expression by Northern blot analysis and quantitative reverse transcriptase-polymerase chain reaction. Expression of cell adhesion molecules and leukocyte adhesion to endothelial cells using human acute monocytic leukemia cell line (THP-1) cells was also analyzed. RESULTS: Ionizing radiation dramatically increased IL-32 expression in vascular endothelial cells through multiple pathways. Ionizing radiation induced IL-32 expression through nuclear factor kappaB activation, through induction of cPLA2 and LPC, as well as induction of Cox-2 and subsequent conversion of arachidonic acid to prostacyclin. Conversely, blocking nuclear factor kappaB, cPLA2, and Cox-2 activity impaired IR-induced IL-32 expression. Importantly, IL-32 significantly enhanced IR-induced expression of vascular cell adhesion molecules and leukocyte adhesion on endothelial cells. CONCLUSION: This study identifies IL-32 as a positive regulator in IR-induced vascular inflammation, and neutralization of IL-32 may be beneficial in protecting from IR-induced inflammation.

Coordinate regulation of TLR-mediated arachidonic acid mobilization in macrophages by group IVA and group V phospholipase A2s.

Macrophages can be activated through TLRs for a variety of innate immune responses. In contrast with the wealth of data existing on TLR-dependent gene expression and resultant cytokine production, very little is known on the mechanisms governing TLR-mediated arachidonic acid (AA) mobilization and subsequent eicosanoid production. We have previously reported the involvement of both cytosolic group IVA phospholipase A(2) (cPLA(2)) and secreted group V phospholipase A(2) (sPLA(2)-V) in regulating the AA mobilization response of macrophages exposed to bacterial LPS, a TLR4 agonist. In the present study, we have used multiple TLR agonists to define the role of various PLA(2)s in macrophage AA release via TLRs. Activation of P388D(1) and RAW2647.1 macrophage-like cells via TLR1/2, TLR2, TLR3, TLR4, TLR6/2, and TLR7, but not TLR5 or TLR9, resulted in AA mobilization that appears to involve the activation of both cPLA(2) and sPLA(2) but not of calcium-independent phospholipase A(2). Furthermore, inhibition of sPLA(2)-V by RNA interference or by two cell-permeable compounds, namely scalaradial and manoalide, resulted in a marked reduction of the phosphorylation of ERK1/2 and cPLA(2) via TLR1/2, TLR2, TLR3, and TLR4, leading to attenuated AA mobilization. Collectively, the results suggest a model whereby sPLA(2)-V contributes to the macrophage AA mobilization response via various TLRs by amplifying cPLA(2) activation through the ERK1/2 phosphorylation cascade.

Phospholipase A2 regulation of bovine endometrial (BEND) cell prostaglandin production.

BACKGROUND: Prostaglandins (PG), produced by the uterine endometrium, are key regulators of several reproductive events, including estrous cyclicity, implantation, pregnancy maintenance and parturition. Phospholipase A2 (PLA2) catalyzes the release of arachidonic acid from membrane phospholipids, the rate-limiting step in PG biosynthesis. The bovine endometrial (BEND) cell line has served as a model system for investigating regulation of signaling mechanisms involved in uterine PG production but information concerning the specific PLA2 enzymes involved and their role in regulation of this process is limited. The objectives of this investigation were to evaluate the expression and activities of calcium-dependent group IVA (PLA2G4A) and calcium-independent group VI (PLA2G6) enzymes in the regulation of BEND cell PG production. METHODS: Cells were grown to near-confluence and treated with phorbol 12, 13 dibutyrate (PDBu), interferon-tau (IFNT), the PLA2G4A inhibitor pyrrolidine-1 (PYR-1), the PLA2G6 inhibitor bromoenol lactone (BEL) and combinations of each. Concentrations of PGF2alpha and PGE2 released into the medium were determined. Western blot analysis was performed on cellular protein to determine effects of treatment on expression of PLA2G4A, PLA2G6 and PLA2G4C. PLA2 assays were performed on intact cells by measuring arachidonic acid and linoleic acid release and group-specific PLA2 activity assays were performed on cell lysates. RESULTS: BEND cells produced about 10-fold more PGE2 than PGF2alpha under resting conditions. Production of both PGs increased significantly in response to PDBu-stimulation. PYR-1 significantly diminished production of both PGs by resting cells and abolished the stimulatory effect of PDBu. BEL stimulated production of both PGs. IFNT reduced both PGE2 and PGF2alpha production by resting cells and diminished PDBu stimulation of PG production. Conversely, IFNT did not significantly reduce BEL stimulation of PG production. Cellular expression of PLA2G4A was enhanced by PDBu and this response was diminished by IFNT. Expression of PLA2G6 was not observed to be affected by treatments and no PLA2G4C expression was observed. Arachidonic acid release from intact cells was significantly increased by PDBu and this effect was attenuated by PYR-1 but not by BEL. Release of linoleic acid from intact cells was stimulated by PDBu and inhibited by BEL but not PYR-1. Group specific PLA2-activity assays demonstrated both PLA2G4A and PLA2G6 activity. CONCLUSION: Results from this study demonstrate that PGE2 and PGF2-alpha production by BEND cells is mediated by the activity and expression of PLA2G4A. Interferon-tau treatment diminished expression of PLA2G4A and PG production. BEND cells were shown to express PLA2G6 but, unlike primary or early passage luminal bovine endometrial cells, stimulation of PLA2G6 activity was not associated with increased PG production.

Platelet-activating factor production in the spinal cord of experimental allergic encephalomyelitis mice via the group IVA cytosolic phospholipase A2-lyso-PAFAT axis.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) plays a critical role in inflammatory disorders including experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Although PAF accumulation in the spinal cord (SC) of EAE mice and cerebrospinal fluid of MS patients has been reported, little is known about the metabolic processing of PAF in these diseases. In this study, we demonstrate that the activities of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase (LysoPAFAT) are elevated in the SC of EAE mice on a C57BL/6 genetic background compared with those of naive mice and correlate with disease severity. Correspondingly, levels of groups IVA, IVB, and IVF cytosolic PLA(2)s, group V secretory PLA(2), and LysoPAFAT transcripts are up-regulated in the SC of EAE mice. PAF acetylhydrolase activity is unchanged during the disease course. In addition, we show that LysoPAFAT mRNA and protein are predominantly expressed in microglia. Considering the substrate specificity and involvement of PAF production, group IVA cytosolic PLA(2) is likely to be responsible for the increased PLA(2) activity. These data suggest that PAF accumulation in the SC of EAE mice is profoundly dependent on the group IVA cytosolic PLA(2)/LysoPAFAT axis present in the infiltrating macrophages and activated microglia.

Release of arachidonic acid by 2-arachidonoyl glycerol and HU210 in PC12 cells; roles of Src, phospholipase C and cytosolic phospholipase A(2)alpha.

The phospholipase A(2) (PLA(2))-prostanoid cascade is involved in cannabinoid receptor-mediated neuronal functions. We investigated the signaling mechanism for the release of arachidonic acid by cannabinoids, 2-arachidonoyl glycerol (2-AG) and HU210, in rat PC12 cells and in primary cultured cells from the mouse cerebellum. The effect of selective inhibitors for signaling pathways and/or enzymes (alpha type cytosolic PLA(2) (cPLA(2)alpha), G protein, Src kinases, phospholipase C, protein kinase C) was assessed. Methods included translocation of the chimeric protein GFP-cPLA(2)alpha, the activities of Src family kinases, Ca(2+)-dependent fluorescence and cyclic AMP accumulation. Treatment with 2-AG and HU210 at greater concentrations than 3 muM caused the release of arachidonic acid, and the response was inhibited by AM251 (an antagonist of cannabinoid CB(1) receptor) and by pyrrophenone (a selective inhibitor of cPLA(2)alpha) in PC12 cells. The cannabinoid treatment caused the intracellular translocation of cPLA(2)alpha and an increase in the intracellular Ca(2+) level. Treatment with HU210 caused tyrosine phosphorylation of Src and Fyn, and increased their kinase activities. Pretreatment with inhibitors of tyrosine kinases or phospholipase C abolished the cannabinoids-induced release of arachidonic acid and Ca(2+) response, and protein kinase C inhibitor reduced the release of arachidonic acid. 2-AG caused the release of arachidonic acid from cultured cells of the mouse cerebellum via similar mechanisms. These data reveal that cannabinoids activated cPLA(2)alpha in a Src-phospholipase C-protein kinase C-dependent manner probably via cannabinoid CB(1) receptor and/or CB(1)-like receptor in neuronal cells.

Inherited human cPLA(2alpha) deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction.

Cytosolic phospholipase A2alpha (cPLA2alpha) hydrolyzes arachidonic acid from cellular membrane phospholipids, thereby providing enzymatic substrates for the synthesis of eicosanoids, such as prostaglandins and leukotrienes. Considerable understanding of cPLA2alpha function has been derived from investigations of the enzyme and from cPLA2alpha-null mice, but knowledge of discrete roles for this enzyme in humans is limited. We investigated a patient hypothesized to have an inherited prostanoid biosynthesis deficiency due to his multiple, complicated small intestinal ulcers despite no use of cyclooxygenase inhibitors. Levels of thromboxane B2 and 12-hydroxyeicosatetraenoic acid produced by platelets and leukotriene B4 released from calcium ionophore-activated blood were markedly reduced, indicating defective enzymatic release of the arachidonic acid substrate for the corresponding cyclooxygenase and lipoxygenases. Platelet aggregation and degranulation induced by adenosine diphosphate or collagen were diminished but were normal in response to arachidonic acid. Two heterozygous single base pair mutations and a known SNP were found in the coding regions of the patient's cPLA2alpha genes (p.[Ser111Pro]+[Arg485His; Lys651Arg]). The total PLA2 activity in sonicated platelets was diminished, and the urinary metabolites of prostacyclin, prostaglandin E2, prostaglandin D2, and thromboxane A2 were also reduced. These findings characterize what we believe is a novel inherited deficiency of cPLA2.