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Naonori Uozumi

Publications and source records attributed to Naonori Uozumi.

16 recordsLinked to original sources
PubMed2006

Biochemical properties and pathophysiological roles of cytosolic phospholipase A2s.

Phospholipase A(2) (PLA(2)) (EC 3.1.1.4) catalyzes hydrolysis of the sn-2 ester bond of glycerophospholipids. The enzyme is essential for the production of two classes of lipid mediators, fatty acid metabolites and lysophospholipid-related lipids, as well as being involved in the remodeling of membrane phospholipids. Among many mammalian PLA(2)s, cytosolic PLA(2)alpha (cPLA(2)alpha) plays a critical role in various physiological and pathophysiological conditions through generating lipid mediators. Here, we summarize the in vivo significance of cPLA(2)alpha, revealed from the phenotypes of cPLA(2)alpha-null mice, and properties of newly discovered cPLA(2) family enzymes. We also briefly introduce a quantitative lipidomics strategy using liquid chromatography-mass spectrometry, a powerful tool for the comprehensive analysis of lipid mediators.

Animals↗
PubMed2005

Cytosolic phospholipase A2 alpha-deficient mice are resistant to experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2alpha (cPLA2alpha), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2alpha-/- mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2alpha+/- mice, whereas the lesions in cPLA2alpha-/- mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2alpha-/- mice compared with cPLA2alpha+/- mice, which indicates that cPLA2alpha plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2alpha-/- mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2alpha also plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2alpha-/- mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2alpha-/- mice susceptible to EAE. Our data indicate that cPLA2alpha plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype.

Animals↗
PubMed2005

A multiplex quantitation method for eicosanoids and platelet-activating factor using column-switching reversed-phase liquid chromatography-tandem mass spectrometry.

Eicosanoids and platelet-activating factor (PAF) are phospholipid-derived lipid mediators produced by various tissues and cells through a cascade pathway. For a comprehensive analysis of these lipid mediators, a simultaneous quantitation method with sensitivity and reliability is necessary. This article details a development of column-switching reversed-phase liquid chromatography-tandem mass spectrometry for multiplex quantitation of eicosanoids and PAF. The adsorptive nature of lipids caused significant loss of signal in a conventional column-switching configuration. The use of an online-dilution method allowed use of 100% methanol as a sample solvent, which prevented sample adsorption to contacting surfaces. Addition of 0.2% formic acid to the sample solvent was required for the successful introduction of LTC4 to the trapping column and minimizing its carryover. The optimized method provided rapid analysis of 14 lipid mediators with a throughput of 96 samples/24 h, lower limits of quantitation of 5 pg on column, and linear calibration ranges up to 2000-5000 pg. The system was highly compatible with solid-phase-extracted samples, as methanol-eluted fractions were directly injected without reconstitution. The analysis of lipid mediator production of macrophage-like RAW264.7 cells demonstrated that the cell-based assay can be performed in a 96-well format, suitable for metabolomics analyses and/or screening strategies.

Animals↗
PubMed2005

Pathway-oriented profiling of lipid mediators in macrophages.

Macrophages produce various kinds of lipid mediators including eicosanoids and platelet-activating factor. Since they are produced from common precursors, arachidonic acid-containing phospholipids, regulations of metabolic pathways underlie the patterning of lipid mediator production. Here, we report a pathway-oriented profiling strategy of lipid mediators by a newly developed multiplex quantification system. We profiled mouse peritoneal macrophages in different activation states. The analysis of kinetics revealed the differences in the production time course of various lipid mediators, which also differed by the macrophage types. Scatterplot matrix analysis of the inhibitor study revealed correlations of lipid mediator species. The changes of these correlations provided estimates on the effects of lipopolysaccharide priming. We also found a highly linked production of 11-hydroxyeicosatetraenoic acid and prostaglandin E2, implying the in vivo property of cyclooxygenase-mediated 11-hydroxyeicosatetraenoic acid production. The present approach will serve as a strategy for understanding the regulatory mechanism of lipid mediator production.

Animals↗
PubMed2005

Identification of novel cytosolic phospholipase A(2)s, murine cPLA(2){delta}, {epsilon}, and {zeta}, which form a gene cluster with cPLA(2){beta}.

Phospholipase A(2) hydrolyzes the sn-2 ester bond of glycerophospholipids that produce free fatty acids and lysophospholipids. Cytosolic phospholipase A(2)s (cPLA(2), group IV) are a subgroup of enzymes that act on the intracellular phospholipid membrane. The best investigated cPLA(2)alpha (group IVA) is a key enzyme for lipid mediator production in vivo. Here we report cloning and characterization of novel murine cPLA(2)s: cPLA(2)delta (group IVD), cPLA(2)epsilon (group IVE), and cPLA(2)zeta (group IVF), that form a gene cluster with cPLA(2)beta (group IVB). The deduced amino acid sequences of cPLA(2)delta, epsilon, and zeta demonstrated a conserved domain structure of cPLA(2), i.e. one C2 domain and one lipase domain. The potential catalytic dyad, Ser and Asp, was conserved for these newly cloned cPLA(2)s along with relatively high conservation for the surrounding residues. Transcripts of murine cPLA(2)delta, epsilon, and zeta appeared to be enriched in certain organs rather than ubiquitous distribution. Major Northern signals for cPLA(2)delta were detected in placenta, cPLA(2)epsilon in thyroid, heart, and skeletal muscle, and cPLA(2)zeta in thyroid. Recombinant proteins expressed in human embryonic kidney 293 cells demonstrated molecular sizes of about 100 kDa by Western blotting and exhibited Ca(2+)-dependent PLA(2) activities on 1-palmitoyl-2-[(14)C]arachidonoyl-phosphatidylcholine substrate. In contrast to cPLA(2)alpha, cPLA(2)zeta preferred phosphatidylethanolamine to phosphatidylcholine. Intracellular localization was visualized by green fluorescent-tagged proteins. Each molecule showed specific localization, and cPLA(2)delta translocated from the cytosol to the perinuclear region by calcium-ionophore stimulation. We thus discovered these functional novel cPLA(2) genes, which cluster on murine chromosome 2E5.

Amino Acid Sequence↗
PubMed2005

Group X secretory phospholipase A2 can induce arachidonic acid release and eicosanoid production without activation of cytosolic phospholipase A2 alpha.

Group X secretory phospholipase A2 (sPLA2-X) and cytosolic phospholipase A2 alpha (cPLA2alpha) are involved in the release of arachidonic acid (AA) from membrane phospholipids linked to the eicosanoid production in various pathological states. Recent studies have indicated the presence of various types of cross-talk between sPLA2s and cPLA2alpha resulting in effective AA release. Here we examined the dependence of sPLA2-X-induced potent AA release on the cPLA2alpha activation by using specific cPLA2alpha or sPLA2 inhibitors as well as cPLA2alpha-deficient mice. We found that Pyrrophenone, a cPLA2alpha-specific inhibitor, did not suppress the sPLA2-X-induced potent AA release and prostaglandin E2 formation in mouse spleen cells. Furthermore, the amount of AA released by sPLA2-X from spleen cells was not significantly altered by cPLA2alpha deficiency. These results suggest that sPLA2-X induces potent AA release without activation of cPLA2a, which might be relevant to eicosanoid production in some pathological states where cPLA2a is not activated.

Animals↗
PubMed2004

B-Myb-dependent regulation of c-Myc expression by cytosolic phospholipase A2.

Cytosolic phospholipase A(2) (cPLA(2)) cleaves membrane phospholipids to release arachidonic acid, initiating lipoxygenase and cyclooxygenase pathways. Mice lacking a gene for cPLA(2) suggested important roles of the protein in allergic responses, fertility, and neural cell death. Here we show that cPLA(2) negatively regulates c-Myc expression in a B-Myb-dependent manner. Overexpression of cPLA(2) protein but not a mutant cPLA(2) protein that lacks in vitro binding ability with B-Myb inhibits B-Myb-dependent c-myc gene expression. The inhibition was associated with physical interaction of B-Myb protein with cPLA(2) both in the cytoplasm and the nucleus. Binding site analysis demonstrated that both the N and C termini of cPLA(2) interact with B-Myb. Macrophage colony stimulating factor (MCSF) stimulated cPLA(2) redistribution into the nucleus and also association with B-Myb in human monocytes. Importantly, macrophages from mice with a disrupted cPLA(2) gene demonstrated significantly increased levels of c-Myc protein in the nucleus compared with cells from the wild-type mice, whereas B-Myb levels were similar in the cells from the cPLA(2)(+/+) and cPLA(2)(-/-) mice. Moreover, an introduction of cPLA(2) into cPLA(2)(-/-) mouse macrophages resulted in decreased c-Myc protein levels, and an inhibition of cPLA(2) expression by small interfering RNAs or antisense RNA increased the c-myc transcription in macrophage colony stimulating factor-activated human monocytes. These findings provide new insights into the function of cPLA(2) in B-Myb-dependent gene expression.

Animals↗
PubMed2004

Short report: Lethal malaria in cytosolic phospholipase A2- and phospholipase A2IIA-deficient mice.

Lipid mediators play important roles in the pathogenesis of malaria. Phospholipase A2s are enzymes involved in the production of these mediators, and they function in inflammation. Among them, cytosolic phospholipase A2 (cPLA2) is a key enzyme in the metabolism of arachidonic acid, the first intermediate in the production of lipid mediators. Plasmodium berghei ANKA causes cerebral malaria in CL57B/6 mice, and we recently produced cPLA2-deficient mice with this background. With the expectation of reduced pathogenicity, we performed experimental infection in these mice. Unexpectedly, the infected mice developed cerebral malaria and died at the same time as the control mice, while the parasitemia progressed similarly in both groups. These observations suggest that secretory PLA2s rather than cPLA2 may be involved in the aggravation, although possible compensation by the induction of other enzymes has not been excluded. The present findings are expected to help clarify the involvement of various phospholipase A2s in malaria.

Animals↗
PubMed2003

An essential role of cytosolic phospholipase A2alpha in prostaglandin E2-mediated bone resorption associated with inflammation.

Prostaglandin E (PGE)2 produced by osteoblasts acts as a potent stimulator of bone resorption. Inflammatory bone loss is accompanied by osteoclast formation induced by bone-resorbing cytokines, but the mechanism of PGE2 production and bone resorption in vivo is not fully understood. Using cytosolic phospholipase A2alpha (cPLA2alpha)-null mice, we examined the role of cPLA2alpha in PGE2 synthesis and bone resorption. In bone marrow cultures, interleukin (IL)-1 markedly stimulated PGE2 production and osteoclast formation in wild-type mice, but not in cPLA2alpha-null mice. Osteoblastic bone marrow stromal cells induced the expression of cyclooxygenase (COX)-2 and membrane-bound PGE2 synthase (mPGES) in response to IL-1 and lipopolysaccharide (LPS) to produce PGE2. Osteoblastic stromal cells collected from cPLA2alpha-null mice also induced the expression of COX-2 and mPGES by IL-1 and LPS, but could not produce PGE2 due to the lack of arachidonic acid release. LPS administration to wild-type mice reduced femoral bone mineral density by increased bone resorption. In cPLA2alpha-null mice, however, LPS-induced bone loss could not be observed at all. Here, we show that cPLA2alpha plays a key role in PGE production by osteoblasts and in osteoclastic bone resorption, and suggest a new approach to inflammatory bone disease by inhibiting cPLA2alpha.

Animals↗
PubMed2003

Cytosolic phospholipase A2alpha-deficient mice are resistant to collagen-induced arthritis.

Pathogenic mechanisms relevant to rheumatoid arthritis occur in the mouse model of collagen-induced arthritis (CIA). Cytosolic phospholipase A2alpha (cPLA2alpha) releases arachidonic acid from cell membranes to initiate the production of prostaglandins and leukotrienes. These inflammatory mediators have been implicated in the development of CIA. To test the hypothesis that cPLA2alpha plays a key role in the development of CIA, we backcrossed cPLA2alpha-deficient mice on the DBA/1LacJ background that is susceptible to CIA. The disease severity scores and the incidence of disease were markedly reduced in cPLA2alpha-deficient mice compared with wild-type littermates. At completion of the study, >90% of the wild-type mice had developed disease whereas none of the cPLA2alpha-deficient mice had more than one digit inflamed. Furthermore, visual disease scores correlated with severity of disease determined histologically. Pannus formation, articular fibrillation, and ankylosis were all dramatically reduced in the cPLA2alpha-deficient mice. Although the disease scores differed significantly between cPLA2alpha mutant and wild-type mice, anti-collagen antibody levels were similar in the wild-type mice and mutant littermates. These data demonstrate the critical role of cPLA2alpha in the pathogenesis of CIA.

5-Lipoxygenase-Activating Proteins↗
PubMed2002

A potent inhibitor of cytosolic phospholipase A2, arachidonyl trifluoromethyl ketone, attenuates LPS-induced lung injury in mice.

Acute respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and sepsis syndrome is one of the most frequent causes of ARDS. Metabolites of arachidonic acid, including thromboxanes and leukotrienes, are proinflammatory mediators and potentially involved in the development of ARDS. A key enzyme for the production of these inflammatory mediators is cytosolic phospholipase A(2) (cPLA(2)). Recently, it has been reported that arachidonyl trifluoromethyl ketone (ATK) is a potent inhibitor of cPLA(2). In the present study, we hypothesized that pharmacological intervention of cPLA(2) could affect acute lung injury. To test this hypothesis, we examined the effects of ATK in a murine model of acute lung injury induced by septic syndrome. The treatment with ATK significantly attenuated lung injury, polymorphonuclear neutrophil sequestration, and deterioration of gas exchange caused by lipopolysaccharide and zymosan administration. The current observations suggest that pharmacological intervention of cPLA(2) could be a novel therapeutic approach to acute lung injury caused by sepsis syndrome.

Animals↗
PubMed2002

Human group IVC phospholipase A2 (cPLA2gamma). Roles in the membrane remodeling and activation induced by oxidative stress.

To create the unique properties of a certain cellular membrane, both the composition and the metabolism of membrane phospholipids are key factors. Phospholipase A(2) (PLA(2)), with hydrolytic enzyme activities at the sn-2 position in glycerophospholipids, plays critical roles in maintaining the phospholipid composition as well as producing bioactive lipid mediators. In this study we examined the contribution of a Ca(2+)-independent group IVC PLA(2) isozyme (cPLA(2)gamma), a paralogue of cytosolic PLA(2)alpha (cPLA(2)alpha), to phospholipid remodeling. The enzyme was localized in the endoplasmic reticulum and Golgi apparatus, as seen using green fluorescence fusion proteins. Electrospray ionization mass spectrometric analysis of membrane extracts revealed that overexpression of cPLA(2)gamma increased the proportion of polyunsaturated fatty acids in phosphatidylethanolamine, suggesting that the enzyme modulates the phospholipid composition. We also found that H(2)O(2) and other hydroperoxides induced arachidonic acid release in cPLA(2)gamma-transfected human embryonic kidney 293 cells, possibly through the tyrosine phosphorylation pathway. Thus, we propose that cPLA(2)gamma is constitutively expressed in the endoplasmic reticulum and plays important roles in remodeling and maintaining membrane phospholipids under various conditions, including oxidative stress.

Animals↗
PubMed2002

Discrete role for cytosolic phospholipase A(2)alpha in platelets: studies using single and double mutant mice of cytosolic and group IIA secretory phospholipase A(2).

Among several different types of phospholipase A(2) (PLA(2)), cytosolic PLA(2) (cPLA(2))alpha and group IIA (IIA) secretory PLA(2) (sPLA(2)) have been studied intensively. To determine the discrete roles of cPLA(2)alpha in platelets, we generated two sets of genetically engineered mice (cPLA(2)alpha(-/-)/sPLA(2)-IIA(-/-) and cPLA(2)alpha(-/-)/sPLA(2)-IIA(+/+)) and compared their platelet function with their respective wild-type C57BL/6J mice (cPLA(2)alpha(+/+)/sPLA(2)-IIA(-/-)) and C3H/HeN (cPLA(2)alpha(+/+)/sPLA(2)-IIA(+/+)). We found that cPLA(2)alpha is needed for the production of the vast majority of thromboxane (TX)A(2) with collagen stimulation of platelets. In cPLA(2)alpha-deficient mice, however, platelet aggregation in vitro is only fractionally decreased because small amounts of TX produced by redundant phospholipase enzymes sufficiently preserve aggregation. In comparison, adenosine triphosphate activation of platelets appears wholly independent of cPLA(2)alpha and sPLA(2)-IIA for aggregation or the production of TX, indicating that these phospholipases are specifically linked to collagen receptors. However, the lack of high levels of TX limiting vasoconstriction explains the in vivo effects seen: increased bleeding times and protection from thromboembolism. Thus, cPLA(2)alpha plays a discrete role in the collagen-stimulated production of TX and its inhibition has a therapeutic potential against thromboembolism, with potentially limited bleeding expected.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2002

Roles for cytosolic phospholipase A2alpha as revealed by gene-targeted mice.

Cytosolic phospholipase A2alpha (cPLA2alpha) has unique characteristics among phospholipase A2 (PLA2) family members. Under regulation by intracellular signaling system, cytosolic phospholipase A2alpha liberates arachidonic acid that can be metabolized by downstream enzymes to generate prostaglandins (PGs) and leukotrienes (LTs). Mice deficient in this enzyme have been generated by gene-targeting techniques. Cytosolic phospholipase A2alpha-deficient mice have a normal appearance and grow normally. Close examinations have revealed a renal concentration defect and intestinal ulcerative lesions. There may also be other disadvantages that are not manifested in well-regulated housing conditions. Although female mice are fertile, they become pregnant less frequently and have small litter sizes; moreover, impaired parturition results in few surviving pups. Primary cultured cells prepared from cytosolic phospholipase A2alpha-deficient mice produce significantly smaller amounts of prostaglandins and leukotrienes. Various disease models such as anaphylaxis, acute lung injury, brain injury induced by ischemia/reperfusion and neurotoxin, and polyposis have been investigated. In all these settings, cytosolic phospholipase A2alpha-deficient mice show significantly milder phenotypes. The mechanisms by which deficiencies of this enzyme exert protective effects may differ, but, a cytosolic phospholipase A2alpha inhibitor could have a wide spectrum of clinical targets. Specific functions of cytosolic phospholipase A2alpha have been clearly demonstrated using the gene-targeted mice. Also, comparisons with mice in which related enzymes and receptors have been manipulated using genetic technologies provide further insights into roles of lipid mediators in physiology and pathology.

Anaphylaxis↗
PubMed2002

A pivotal role of cytosolic phospholipase A(2) in bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is an interstitial disorder of the lung parenchyma whose mechanism is poorly understood. Potential mechanisms include the infiltration of inflammatory cells to the lungs and the generation of pro-inflammatory mediators. In particular, idiopathic pulmonary fibrosis is a progressive and fatal form of the disorder characterized by alveolar inflammation, fibroblast proliferation and collagen deposition. Here, we investigated the role of cytosolic phospholipase A(2) (cPLA(2)) in pulmonary fibrosis using cPLA(2)-null mutant mice, as cPLA(2) is a key enzyme in the generation of pro-inflammatory eicosanoids. Disruption of the gene encoding cPLA(2) (Pla2g4a) attenuated IPF and inflammation induced by bleomycin administration. Bleomycin-induced overproduction of thromboxanes and leukotrienes in lung was significantly reduced in cPLA(2)-null mice. Our data suggest that cPLA(2) has an important role in the pathogenesis of pulmonary fibrosis. The inhibition of cPLA(2)-initiated pathways might provide a novel therapeutic approach to pulmonary fibrosis, for which no pharmaceutical agents are currently available.

Animals↗