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Background Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, is recognized as a regulator of lipid metabolism associated with oncogenesis. Though recent studies reveal that high PLA2G3 expression significantly correlates with poor prognosis in several cancers, however, role of PLA2G3 in ovarian cancer (OC) pathogenesis is still undetermined. Methods CRISPR-Cas9 and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal and Transmission electron microscopy analysis, and the cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Regulation of primary ciliation by PLA2G3 downregulation both genetically and by metabolic inhibitor PFK-158 induced autophagy was assessed by immunofluorescence-based confocal analysis and immunoblot. Transient transfection with GFP-RFP-LC3B and confocal analysis was used to assess the autophagic flux in OC cells. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and with platinum drugs was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis. Results Downregulation of PLA2G3 in OVCAR8 and 5 cells inhibited LD biogenesis, decreased growth and sensitized cells to platinum drug mediated cytotoxicity in vitro and in in vivo OVCAR5 xenograft. PLA2G3 knockdown in HeyA8MDR-resistant cells showed sensitivity to carboplatin treatment. We found that both PFK158 inhibitor-mediated and genetic downregulation of PLA2G3 resulted in increased number of percent ciliated cells and inhibited cancer progression. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore primary cilia in OC cells. Of clinical relevance, PFK158 also induces percent ciliated cells in human-derived primary ascites cells and reduces cell viability with sensitization to chemotherapy. Conclusions Taken together, our study for the first time emphasizes the role of PLA2G3 in regulating the OC metastasis. This study further suggests the therapeutic potential of targeting phospholipases and/or restoration of PC for future OC treatment and the critical role of PLA2G3 in regulating ciliary function by coordinating interface between lipogenesis and metastasis.

OBJECTIVES Our aims were to examine the prevalence and genetic predictors of aspirin and clopidogrel high on-treatment platelet reactivity (HoTPR), and associated adverse cardiovascular outcomes in patients with peripheral arterial disease (PAD). BACKGROUND The association of aspirin and clopidogrel HoTPR with outcomes in PAD remains unclear. METHODS This is a prospective cohort study of patients with angiographically documented PAD involving carotid and lower extremity arteries. Aspirin and clopidogrel HoTPR (using the VerifyNow Assay) and associated genetic predictors were compared to clinical outcomes. The primary end-point was a composite of major adverse cardiovascular events: all-cause mortality, myocardial infarction, stroke, target vessel revascularization (TVR) and limb-loss in patients who underwent extremity intervention. RESULTS The study was stopped prematurely due to slow patient enrolment. Of 195 patients enrolled, the primary analysis was performed in 154 patients taking both drugs. Aspirin HoTPR was present in 31 (20%) and clopidogrel HoTPR in 76 (49%) patients. There was a trend toward more primary composite outcome events with PRU ≥ 235 (52% freedom-from-event rate vs. 70% for PRU

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell–deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS–ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3–L-PGDS–DP1 loop that drives mast cell maturation.

Phospholipases A(2) (PLA(2)) are enzymes that cleave the sn-2 bond of membrane phospholipids to yield free fatty acids and lysophospholipids. Secretory PLA2-III (sPLA(2)-III) has been suggested to be important for neuronal differentiation, growth and survival, and is highly expressed in the spinal cord. The aim of this study is to elucidate its expression and distribution in different regions of the adult rat CNS. Quantitative RT-PCR analyses showed high levels of sPLA(2)-III mRNA expression in the brainstem and spinal cord and low expression in the olfactory bulb. Western blot analyses showed high level of expression in the brainstem, spinal cord and cerebral neocortex. A dense band corresponding to the catalytically active, mature/cleaved form, and a faint band corresponding to the full length sPLA(2)-III were detected in post-mitochondrial supernatants, from different parts of the CNS. Subcellular fractionation of spinal cord homogenates showed that sPLA(2)-III protein is present in the 'light membrane/cytosol' fraction, but not the nucleus, synaptosomal membrane or synaptic vesicle-enriched fractions. sPLA(2)-III was immunolocalized to neurons in the cerebral neocortex, Purkinje neurons in the cerebellar cortex, periaqueductal gray, red nucleus, spinal trigeminal nucleus and dorsal horn of the spinal cord. Electron microscopy of the spinal cord and cerebral neocortex showed that sPLA(2)-III was localized in dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled, putatively glutamatergic, axon terminals. The localization of mature/cleaved form of sPLA(2)-III in postsynaptic structures suggest a physiological role of the enzyme in neurotransmission or synaptic plasticity.

Oxidative stress, which plays a critical role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), is intimately linked to aging, the best established risk factor for AD. Studies in neuronal cells subjected to oxidative stress, mimicking such stress in AD brains, are therefore of great interest. PLA2G3 is the most overexpressed gene in a human neuronal model of oxidative stress induced by the free radical-generating xanthine/xanthine oxidase (X-XOD) system, which provokes apoptotic cell death. In this work, we describe that PLA2G3 gene silencing produced a marked inhibition of X-XOD induced cell death, and that PLA2G3 polymorphisms are associated with AD in a Spanish case-control sample. The capacity to respond to oxidative stress may therefore modulate the risk of AD, and PLA2G3 is a potential target to regulate neuronal damage induced by free radicals.

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3–/– mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3–/– mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3–/– mice. Moreover, the gonads of Pla2g3–/– mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction.

PLA2 (phospholipase A2) group III is an atypical sPLA2 (secretory PLA2) that is homologous with bee venom PLA2 rather than with other mammalian sPLA2s. In the present paper, we show that endogenous group III sPLA2 (PLA2G3) is expressed in mouse skin and that Tg (transgenic) mice overexpressing human PLA2G3 spontaneously develop skin inflammation. Pla2g3-Tg mice over 9 months of age frequently developed dermatitis with hyperkeratosis, acanthosis, parakeratosis, erosion, ulcer and sebaceous gland hyperplasia. The dermatitis was accompanied by infiltration of neutrophils and macrophages and by elevated levels of pro-inflammatory cytokines, chemokines and prostaglandin E2. In addition, Pla2g3-Tg mice had increased lymph aggregates and mucus in the airway, lymphocytic sialadenitis, hepatic extramedullary haemopoiesis, splenomegaly with increased populations of granulocytes and monocytes/macrophages, and increased serum IgG1. Collectively, these observations provide the first demonstration of spontaneous development of inflammation in mice with Tg overexpression of mammalian sPLA2.

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

Recent studies suggest that secreted phospholipases A2 (sPLA2s) represent attractive potential tumour biomarkers and therapeutic targets for various cancers. As a first step to address this issue in human colorectal cancer, we examined the expression of the full set of sPLA2s in sporadic adenocarcinomas and normal matched mucosa from 21 patients by quantitative PCR and immunohistochemistry. In normal colon, PLA2G2A and PLA2G12A were expressed at high levels, PLA2G2D, PLA2G5, PLA2G10 and PLA2G12B at moderate levels, and PLA2G1B, PLA2G2F and PLA2G3 at low levels. In adenocarcinomas from left and right colon, the expression of PLA2G3 was increased by up to 40-fold, while that of PLA2G2D and PLA2G5 was decreased by up to 23- and 14-fold. The variations of expression for sPLA2-IID, sPLA2-III and sPLA2-V were confirmed at the protein level. The expression pattern of these sPLA2s appeared to be linked respectively to the overexpression of interleukin-8, defensin alpha6, survivin and matrilysin, and downregulation of SFRP-1 and RLPA-1, all these genes being associated to colon cancer. This original sPLA2 profile observed in adenocarcinomas highlights the potential role of certain sPLA2s in colon cancer and suggests that sPLA2-III might be a good candidate as a novel biomarker for both left and right colon cancers.

Phospholipases A(2) (PLA(2)) are group of enzymes that hydrolyze membrane phospholipids at the sn-2 position. PLA(2) are present in the brain and spinal cord and are implicated in several neurological disorders. Previously, we showed that PLA(2) activity increases following traumatic spinal cord injury and injection of group III secretory PLA(2) (sPLA(2)-III) demyelinates spinal cord axons. Here, we demonstrate that injections of sPLA(2)-III into the cervical dorsolateral funiculus (DLF) resulted in dose-dependent demyelination, loss of oligodendrocytes and astrocytes, as well as axonopathy. Additionally, spared axons within the lesion were remyelinated by Schwann cells between weeks 2 and 3. To assess functional loss and recovery, we employed a modified "Staircase Test" pellet retrieval device and footprint analysis of forelimb function during locomotion. Pellet retrieval assessment sensitively detected the dose dependent lesion and its recovery after sPLA(2)-III injections with greater sensitivity than footprint analysis. We believe that this is the first report of a reaching task being able to discriminate between various grades of cervical white matter damage and varying extents of recovery. Thus, our results indicate that sPLA(2)-III can create white matter pathologies that are remyelinated by Schwann cells 2 to 3 weeks after injury. Additionally, the pellet retrieval test is a sensitive and quantifiable method for assessing the dysfunction and later recovery mediated by sPLA(2)-III injections.

Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa -/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa -/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa -/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD.

Phospholipase A2s (PLA2s) are a group of enzymes that hydrolyze the sn-2 position of phospholipids to release (typically unsaturated) fatty acids and lysophospholipids, which serve as precursors for a variety of bioactive lipid mediators. Among the PLA2 superfamily, secreted PLA2 (sPLA2) enzymes comprise the largest subfamily that includes 11 isoforms with a conserved His–Asp catalytic dyad. Individual sPLA2 enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. Recent studies using transgenic and knockout mice for individual sPLA2 isofoms have revealed their involvement in various pathophysiological events. Here, we overview the current state of knowledge about sPLA2s, specifically their roles in mast cells (MCs) in the context of allergology. In particular, we highlight group III sPLA2 (PLA2G3) as an “anaphylactic sPLA2” that promotes MC maturation and thereby anaphylaxis through a previously unrecognized lipid-orchestrated circuit.

The arachidonic acid metabolism (AAM) pathway promotes tumour progression. Chemical inhibitors of AAM pathway prolong post-treatment survival of cancer patients. Here we test whether non-synonymous somatic mutations in genes of this pathway, acting as natural inhibitors, increase post-treatment survival. We identify loss-of-function somatic mutations in 15 (18%) of 84 treatment-naive oral cancer patients by whole-exome sequencing, which we map to genes of AAM pathway. Patients (n = 53) who survived ≥ 12 months after surgery without recurrence have significantly (P = 0.007) higher proportion (26% versus 3%) of mutations than those who did not (n = 31). Patients with mutations have a significantly (P = 0.003) longer median disease-free survival (24 months) than those without (13 months). Compared with the presence of a mutation, absence of any mutation increases the hazard ratio for death (11.3) significantly (P = 0.018). The inferences are strengthened when we pool our data with The Cancer Genome Atlas (TCGA) data. In patients with AAM pathway mutations, some downstream pathways, such as the PI3K-Akt pathway, are downregulated.

Colorectal cancer (CRC) is the third most common malignant tumor and the fourth leading cause of cancer death worldwide. The crucial role of fatty acids for a number of important biological processes suggests a more in-depth analysis of inter-individual differences in fatty acid metabolizing genes as contributing factor to colon carcinogenesis. We examined the association between genetic variability in 43 fatty acid metabolism-related genes and colorectal risk in 1225 CRC cases and 2032 controls participating in the European Prospective Investigation into Cancer and Nutrition study. Three hundred and ninety two single-nucleotide polymorphisms were selected using pairwise tagging with an r2 cutoff of 0.8 and a minor allele frequency of >5%. Conditional logistic regression models were used to estimate odds ratios and corresponding 95% confidence intervals. Haplotype analysis was performed using a generalized linear model framework. On the genotype level, hydroxyprostaglandin dehydrogenase 15-(NAD) (HPGD), phospholipase A2 group VI (PLA2G6) and transient receptor potential vanilloid 3 were associated with higher risk for CRC, whereas prostaglandin E receptor 2 (PTGER2) was associated with lower CRC risk. A significant inverse association (P < 0.006) was found for PTGER2 GGG haplotype, whereas HPGD AGGAG and PLA2G3 CT haplotypes were significantly (P < 0.001 and P = 0.003, respectively) associated with higher risk of CRC. Based on these data, we present for the first time the association of HPGD variants with CRC risk. Our results support the key role of prostanoid signaling in colon carcinogenesis and suggest a relevance of genetic variation in fatty acid metabolism-related genes and CRC risk. © The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org.

In the non-amyloidogenic pathway, amyloid precursor protein (APP) is cleaved by alpha-secretases to produce alpha-secretase-cleaved soluble APP (sAPP(alpha)) with neuroprotective and neurotrophic properties; therefore, enhancing the non-amyloidogenic pathway has been suggested as a potential pharmacological approach for the treatment of Alzheimer's disease. Here, we demonstrate the effects of type III secretory phospholipase A(2) (sPLA(2)-III) on sAPP(alpha) secretion. Exposing differentiated neuronal cells (SH-SY5Y cells and primary rat neurons) to sPLA(2)-III for 24 h increased sAPP(alpha) secretion and decreased levels of Abeta(1-42) in SH-SY5Y cells, and these changes were accompanied by increased membrane fluidity. We further tested whether sPLA(2)-III-enhanced sAPP(alpha) release is due in part to the production of its hydrolyzed products, including arachidonic acid (AA), palmitic acid (PA), and lysophosphatidylcholine (LPC). Addition of AA but neither PA nor LPC mimicked sPLA(2)-III-induced increases in sAPP(alpha) secretion and membrane fluidity. Treatment with sPLA(2)-III and AA increased accumulation of APP at the cell surface but did not alter total expressions of APP, alpha-secretases, and beta-site APP cleaving enzyme. Taken together, these results support the hypothesis that sPLA(2)-III enhances sAPP(alpha) secretion through its action to increase membrane fluidity and recruitment of APP at the cell surface.

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

Secreted phospholipase A(2) (sPLA(2)) has been shown to cause both necrotic and apoptotic neuronal injury in the central nervous system. The acute effects of group III sPLA(2) (sPLA(2)-III) include an ability to potentiate glutamate-induced necrosis and calcium influx in primary rat neuronal cultures. Thus, a brief exposure to glutamate and sPLA(2)-III results in an immediate potentiated calcium influx and a potentiated necrosis the following day after treatment, compared to addition of either agonist alone. In contrast to these acute effects, sPLA(2) from both groups IB and IIA have been shown to induce neuronal cell death via apoptosis. Here, we offer the first evidence that group III sPLA(2) potently causes extensive neuronal apoptosis in rat cortical cultures, as evidenced by DNA fragmentation. Distinct from this effect, even high concentrations of glutamate did not cause neuronal damage under the same conditions, and there was no evidence of sPLA(2) damage to cortical astrocytes.