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ABSTRACT: Group IVA phospholipase A(2) (IVA-PLA(2)) is an enzyme that intiates the arachidonic acid pathway and plays an important role in inflammation. We demonstrate that IVA-PLA(2) deficiency suppresses lipid deposition in the liver, which was induced by administration of a high-fat and -cholesterol diet (HFCD) for 16 wk in mice. Herein, we performed 2-dimensional gel-based comparative proteomics to further define the suppressive effect of IVA-PLA(2) deficiency on fatty liver formation. In comparisons among 4 groups, wild-type (WT)/normal diet (ND), IVA-PLA(2)-deficient knockout (KO)/ND, WT/HFCD, and KO/HFCD, 4 proteins, 3 of which are associated with hepatic fibrosis, were identified as molecules, of which altered expression by HFCD was suppressed in KO mice compared to WT mice. Therefore, we assessed the effect of IVA-PLA(2) deficiency on hepatic fibrosis induced by HFCD or carbon tetrachloride (CCl(4)) in mouse models. Biochemical and histological analyses revealed that IVA-PLA(2) deficiency markedly reduced overall collagen accumulation in the liver of HFCD- and CCl(4)-derived mouse models. We found that IVA-PLA(2) deficiency prevented activation of hepatic stellate cells and infiltration of F4/80-positive macrophages without affecting other immunocytes such as CD8(+) lymphocytes and natural killer cells. In summary, IVA-PLA(2) deficiency attenuates not only lipid deposition in the liver but also hepatic fibrosis formation.-Ishihara, K., Miyazaki, A., Nabe, T., Fushimi, H., Iriyama, N., Kanai, S., Sato, T., Uozumi, N., Shimizu, T., Akiba, S. Group IVA phospholipase A(2) participates in the progression of hepatic fibrosis.
The FASEB Journal 06/2012; 26(10):4111-21. · 5.71 Impact Factor
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ABSTRACT: In this study, we observed that low glucose or fructose reduces the increase in hypoxia-inducible factor-1alpha (HIF-1alpha) protein under hypoxic conditions. 6-Aminonicotinamide (6-AN), an inhibitor of the pentose phosphate pathway (PPP), also inhibited the increase of HIF-1alpha protein under hypoxic conditions, while the reduced protein levels of HIF-1alpha by low glucose were apparently recovered by the addition of MG-132 or NADPH. Moreover, siRNA for glucose-6-phosphate dehydrogenase, which produces NADPH, reduced the increase in HIF-1alpha protein. On the other hand, cobalt-induced expression of HIF-1alpha protein was not affected by low glucose or 6-AN under normoxic conditions. In conclusion, glucose metabolism through the PPP, but not in glycolysis, plays an important role in the stabilization of HIF-1alpha protein under hypoxic conditions.
FEBS letters 07/2010; 584(14):3073-9. · 3.54 Impact Factor
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ABSTRACT: Hepatic fat deposition with hepatocellular damage, a feature of non-alcoholic fatty liver disease, is mediated by several putative factors including prostaglandins. In the present study, we examined whether group IVA phospholipase A(2) (IVA-PLA(2)), which catalyzes the first step in prostanoid biosynthesis, is involved in the development of fatty liver, using IVA-PLA(2)-knockout mice. Male wild-type mice on high-fat diets (20% fat and 1.25% cholesterol) developed hepatocellular vacuolation and liver hypertrophy with an increase in the serum levels of liver damage marker aminotransferases when compared with wild-type mice fed normal diets. These high-fat diet-induced alterations were markedly decreased in IVA-PLA(2)-knockout mice. Hepatic triacylglycerol content was lower in IVA-PLA(2)-knockout mice than in wild-type mice under normal dietary conditions. Although high-fat diets increased hepatic triacylglycerol content in both genotypes, the degree was lower in IVA-PLA(2)-knockout mice than in wild-type mice. Under the high-fat dietary conditions, IVA-PLA(2)-knockout mice had lower epididymal fat pad weight and smaller adipocytes than wild-type mice. The serum level of prostaglandin E(2), which has a fat storage effect, was lower in IVA-PLA(2)-knockout mice than in wild-type mice, irrespective of the kind of diet. In both genotypes, high-fat diets increased serum leptin levels equally between the two groups, but did not affect the serum levels of adiponectin, resistin, free fatty acid, triacylglycerol, glucose, or insulin. Our findings suggest that a deficiency of IVA-PLA(2) alleviates fatty liver damage caused by high-fat diets, probably because of the lower generation of IVA-PLA(2) metabolites, such as prostaglandin E(2). IVA-PLA(2) could be a promising therapeutic target for obesity-related diseases including non-alcoholic fatty liver disease.
PLoS ONE 01/2009; 4(12):e8089. · 4.09 Impact Factor
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ABSTRACT: Prostaglandin (PG) E(2) is considered to participate in the storage of fat in adipocytes and hepatocytes, but roles of group IVA phospholipase A(2) (PLA(2)), a key PLA(2) isozyme in the arachidonic acid cascade, remain unclear. The present study examined the possible involvement of the enzyme using group IVA PLA(2)-deficient mice (C57BL/6 background, 22 weeks of age) fed a normal diet (5.3% fat). The ratio of epididymal fat pad weight to body weight was significantly reduced in group IVA PLA(2)-deficient mice compared to wild-type mice. Histological analysis revealed that in group IVA PLA(2)-deficient mice, the adipocytes were smaller, and hepatocytes bearing cytoplasmic vacuolation were scarce. Hepatic triglyceride content and the serum levels of PGE(2) in the deficient mice were also lower. However, there was no difference in the serum levels of insulin, glucose, non-esterified free fatty acid, or total cholesterol between the deficient and wild-type mice. Our findings suggest that group IVA PLA(2) is involved in the storage of lipids in the adipose tissue and liver and in determining circulating PGE(2) levels.
Prostaglandins & other lipid mediators 07/2008; 86(1-4):12-7. · 2.70 Impact Factor
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ABSTRACT: The migration of vascular smooth muscle cells from the media to intima and their subsequent proliferation are critical causes of arterial wall thickening. In atherosclerotic lesions increases in the thickness of the vascular wall and the impairment of oxygen diffusion capacity result in the development of hypoxic lesions. We investigated the effect of hypoxia on the migration of human coronary artery smooth muscle cells (CASMCs) via HIF-1alpha-dependent expression of thrombospondin-1 (TSP-1). When the cells were cultured under hypoxic conditions, mRNA and protein levels of TSP-1, and mRNA levels of integrin beta(3) were increased with the increase in HIF-1alpha protein. DNA synthesis and migration of the cells were stimulated under the conditions, and a neutralizing anti-TSP-1 antibody apparently suppressed the migration, but not DNA synthesis. The migration was also inhibited by RGD peptide that binds to integrin beta(3). Furthermore, the migration was completely suppressed in HIF-1alpha-knockdown cells exposed to hypoxia, while it was significantly enhanced in HIF-1alpha-overexpressing cells. These results suggest that the hypoxia induces the migration of CASMCs, and that the migration is elicited by TSP-1 of which induction is fully dependent on the stabilization of HIF-1alpha, in autocrine regulation. Thus we suggest that HIF-1alpha plays an important role in the pathogenesis of atherosclerosis.
Journal of Cellular Biochemistry 05/2008; 104(5):1918-26. · 2.87 Impact Factor
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ABSTRACT: Matrix metalloproteinase-9 (MMP-9) is involved in atherogenesis, and the production of MMP-9 in macrophages is considered to be mediated by the arachidonic acid cascade. The present study examined the possible involvement of group IVA phospholipase A2 (IVA-PLA2), a key enzyme in the arachidonic acid cascade, in the production of MMP-9 induced by oxidized low-density lipoprotein (oxLDL) in macrophages and high-fat diet-induced formation of atherosclerotic lesions using IVA-PLA2-deficient mice (C57BL/6 background). In wild-type mouse peritoneal macrophages, oxLDL induced an increase in MMP-9 in the culture medium. The oxLDL-promoted production of MMP-9 was markedly reduced in IVA-PLA2-deficient macrophages compared to wild-type macrophages. Feeding of wild-type mice with a high-fat diet caused the formation of early atherosclerotic lesions in the aortic root with increases in MMP-9 and macrophages in the lesions and with higher serum levels of total cholesterol. Such lesions were apparently less severe in IVA-PLA2-deficient mice fed a high-fat diet, despite higher total cholesterol levels. Under the conditions, a high-fat diet reduced the serum levels of high-density lipoprotein-cholesterol (HDL-C) in wild-type mice. However, IVA-PLA2-deficient mice fed a high-fat diet were protected against the decrease in HDL-C levels. The present results suggest that IVA-PLA2 is involved in the oxLDL-induced production of MMP-9 in macrophages and the high-fat diet-induced formation of early atherosclerotic lesions. The protection against the lesions in IVA-PLA2-deficient mice may be ascribable, in part, to the impaired production of MMP-9 and/or the maintained levels of HDL-C.
Biological & Pharmaceutical Bulletin 04/2008; 31(3):363-8. · 1.66 Impact Factor
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ABSTRACT: When the arterial wall thickens and blood-diffusion capacity is low in atherosclerotic lesions, hypoxia is a key factor for the development of atherosclerosis. Under hypoxic conditions, >100 genes, including those encoding many growth factors, are known to be induced by a transcriptional factor, hypoxia-inducible factor-1alpha (HIF-1alpha). In this study, to examine whether HIF-1alpha-dependent induction of growth factors is associated with the proliferation and migration of vascular cells in atherosclerotic lesions, we studied the role of thrombospondin-1 (TSP-1), which is induced by hypoxia, in the pathogenesis and progression of atherosclerosis in human coronary artery smooth muscle cells (CASMCs). Under hypoxic conditions, expression of HIF-1alpha increased time-dependently in human CASMCs with a concomitant increase in the proliferation and migration of cells. Under these conditions, the mRNA and protein levels of TSP-1 and the mRNA level of TSP-1 receptor, integrin beta3, were also enhanced. Neutralizing antibody against TSP-1 reduced hypoxia-induced migration, but not proliferation. Similarly, RGD peptide, which binds to integrin beta3, inhibited cell migration under hypoxia. In HIF-1alpha-knockdown CASMCs, in which expression of HIF-1alpha and TSP-1 mRNA and proteins is suppressed, hypoxia-induced migration was markedly reduced. In conclusion, hypoxia in atherosclerotic lesions induces TSP-1, which plays important roles in acceleration of the migration of human CASMCs and the progression of atherosclerosis.
Yakugaku zasshi journal of the Pharmaceutical Society of Japan 03/2008; 128(3):377-83. · 0.39 Impact Factor
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ABSTRACT: Atherosclerotic lesions are reported to be hypoxic. Since hypoxia is known to induce the production of growth factors, such as vascular endothelial growth factor (VEGF), we examined the implication of hypoxia-induced VEGF in the proliferation of human coronary artery smooth muscle cells (CASMCs).
Cells were cultured under hypoxic conditions (1% O(2), 5% CO(2)) and several responses were measured.
Under hypoxic conditions, the mRNA and protein levels of VEGF, and the mRNA level of VEGF receptor-1 (VEGFR-1) increased with an increase in hypoxia-inducible factor-1alpha (HIF-1alpha) protein, and considerable amounts of VEGF were secreted. Hypoxia enhanced the incorporation of [(3)H]-thymidine by CASMCs, which was completely inhibited by a neutralizing antibody against VEGF. A neutralizing antibody against NADPH-cytochrome P-450 reductase (NPR), which contributes to the stabilization of HIF-1alpha, also attenuated hypoxia-stimulated proliferation. In NPR-knockdown cells, the expression of VEGF, proliferation, and transcriptional activity were attenuated, whereas in NPR-overexpressing cells, they were enhanced.
Hypoxia-induced proliferation of CASMCs is mediated through the expressions of VEGF and VEGFR-1 in an autocrine mechanism. Their expressions are dependent on the stabilization of HIF-1alpha, which is regulated by NPR. We suggest that hypoxia and hypoxia-induced VEGF expression are involved in the pathogenesis of progressive atherosclerosis.
Journal of atherosclerosis and thrombosis 03/2008; 15(1):26-33. · 2.69 Impact Factor
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ABSTRACT: Macrophage-derived foam cells are formed as a result of the accumulation of cholesteryl ester (CE) not only in cytoplasm where CE is produced by the reesterification of free cholesterol derived from oxidized low density lipoprotein (OxLDL) undergoing hydrolysis, but also in lysosomes where the remaining CE of OxLDL is deposited. We examined the possible involvement of cytosolic phospholipase A(2)s (cPLA(2)s) in the production of CE through the reesterification and in the formation of foam cells. In [(3)H]oleic acid-labeled human acute monocytic leukemia (THP-1) cell-derived macrophages (THP-M) and mouse peritoneal macrophages (MPM), which possessed at least cPLA(2)alpha and cPLA(2)gamma, stimulation with OxLDL induced the production of [(3)H]cholesteryl oleate ([(3)H]CE).The production was suppressed by an inhibitor of cPLA(2)s. However, the inhibitor tended to slightly decrease total intracellular levels of CE, and did not affect the formation of foam cells, as estimated by staining with Oil Red O. In cPLA(2)alpha-knockout MPM, OxLDL-induced increases in [(3)H]CE and total CE did not differ from those in wild-type MPM. Our results suggest that cPLA(2)s other than cPLA(2)alpha contribute to the supply of fatty acids, which are utilized for the production of CE through the reesterification, in OxLDL-stimulated macrophages. However, the formation of foam cells could not be inhibited only by the suppression of cPLA(2)-mediated CE production.
Biological & Pharmaceutical Bulletin 02/2008; 31(1):6-12. · 1.66 Impact Factor
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ABSTRACT: An extract of Ginkgo Biloba L. was shown to have preventive effects on cardiovascular disorders, but the molecular mechanisms of its actions remain to be elucidated. Since matrix metalloproteinases (MMPs) are implicated in the rupture of atherosclerotic plaques and the subsequent occurrence of acute coronary syndrome, we examined the effects of a leaf extract (Ginkgolon-24) on the production of MMP-1 in human coronary smooth muscle cells stimulated with oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal, which are factors proposed to play a pivotal role in atherogenesis.
The production of MMP-1 and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 were estimated by immunoblotting. The tyrosine-phosphorylated form of platelet-derived growth factor receptor beta (PDGFR-beta) was analyzed by immunoprecipitation of the receptor followed by immunoblotting.
oxLDL and 4-hydroxynonenal accelerated the production of MMP-1 with the preceding phosphorylation of ERK1/2 and PDGFR-beta;. Pretreatment with Ginkgolon-24 inhibited the production of MMP-1 and phosphorylation of ERK1/2 induced by oxLDL and 4-hydroxynonenal, but did not affect the production and phosphorylation induced by phorbol ester. Furthermore, Ginkgolon-24 prevented tyrosine phosphorylation of the receptor induced by oxLDL and 4-hydroxynonenal.
These results suggest that Ginkgo Biloba extract suppresses the oxLDL- and 4-hydroxynonenal-induced production of MMP-1, probably through the inhibition of PDGFR-beta activation in human coronary smooth muscle cells.
Journal of atherosclerosis and thrombosis 11/2007; 14(5):219-25. · 2.69 Impact Factor
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ABSTRACT: We investigated the role of Ca2+-independent phospholipase A2 (iPLA2) as well as cytosolic phospholipase A2 (cPLA2) in hypoxia-inducible factor-1 (HIF-1)-dependent gene expression. An inhibitor of both iPLA2 and cPLA2, methyl arachidonyl fluorophosphonate (MAFP), prevented hypoxia-induced erythropoietin mRNA expression without affecting HIF-1alpha accumulation in Hep3B cells. The DNA-binding of HIF-1alpha was suppressed by MAFP as confirmed by luciferase reporter gene assays with the hypoxia response element. Translocation of HIF-1alpha to the nucleus assessed by its presence in the nuclear extracts of cells exposed to hypoxia, was diminished by MAFP. However, hypoxia-dependent gene expression was not affected in mesangial cells obtained from cPLA2alpha null mice. Furthermore, a specific iPLA2 inhibitor, bromoenol lactone, suppressed erythropoietin mRNA expression and HIF-1alpha translocation to the nucleus under hypoxic conditions. Thus, iPLA2, but not cPLA2alpha, may play an important role in regulating the transport of HIF-1alpha to the nucleus.
European Journal of Pharmacology 12/2006; 549(1-3):58-62. · 2.52 Impact Factor
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ABSTRACT: Increases in matrix metalloproteinases (MMPs) at atherosclerotic lesions are involved in the migration of smooth muscle cells (SMCs) into the intima and to the rupture of plaques, being implicated in the progression of atherosclerosis. The present study examined the mechanisms underlying the production of MMP-1, interstitial collagenase-1, induced by oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal (4-HNE), factors proposed to play a pivotal role in atherogenesis, in human coronary SMCs. oxLDL promoted the production of MMP-1 with the preceding phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Immunoprecipitation of platelet-derived growth factor receptor beta (PDGFR-beta) revealed that oxLDL induced tyrosine phosphorylation of the receptor. Inhibition of the activation of PDGFR-beta and ERK1/2 resulted in a suppression of the production of MMP-1. Consistently, 4-HNE also elicited the production of MMP-1 with the preceding phosphorylation of PDGFR-beta and ERK1/2. The 4-HNE-induced production of MMP-1 was prevented when the activation of PDGFR-beta and ERK1/2 was inhibited. The present results suggest that the activation of PDGFR-beta and ERK1/2 is involved in the production of MMP-1 in oxLDL- and 4-HNE-stimulated human coronary SMCs.
Biochimica et Biophysica Acta 09/2006; 1763(8):797-804. · 4.66 Impact Factor
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Baoliang Guo,
Ken Inoki,
Motohide Isono,
Hiroyuki Mori,
Keizo Kanasaki,
Toshiro Sugimoto,
Satoshi Akiba, Takashi Sato,
Baofeng Yang,
Ryuichi Kikkawa,
Atsunori Kashiwagi,
Masakazu Haneda,
Daisuke Koya
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ABSTRACT: Receptor-regulated Smads and/or mitogen-activated protein kinases (MAPKs) are involved in transforming growth factor-beta (TGF-beta)-induced expression of various genes, including plasminogen activator inhibitor-1 (PAI-1). Because the sequence of the promoter region in rat PAI-1 gene differs from that in the human gene, we examined the mechanisms of TGF-beta-induced rat PAI-1 expression in rat mesangial cells.
TGF-beta1-induced PAI-1 and c-fos mRNA expressions were determined by Northern blot analysis. Activation of MAPKs and Smad proteins was evaluated by an immunoblot analysis. DNA binding activities of nuclear protein were examined by using an electrophoretic mobility shift assay (EMSA). The activities of PAI-1 promoter were measured by a luciferase reporter assay.
Extracellular-regulated kinase (ERK) and c-Jun NH-terminal kinase (JNK) phosphorylation, c-fos mRNA expression, and activator protein-1 (AP-1) DNA binding activity stimulated by TGF-beta1 were completely suppressed by the ERK kinase (MEK) inhibitors. EMSA and reporter analysis revealed that an AP-1-like sequence located in the proximal region of the rat PAI-1 promoter was the target for TGF-beta1, and the disruption of this AP-1-like sequence suppressed basal and TGF-beta1-induced promoter activation. TGF-beta1 also stimulated nuclear translocation of Smads and binding to palindromic Smad binding element (SBE) located in the rat PAI-1 promoter, without being affected by MEK inhibitor. Point mutation and deletion of palindromic SBE did not affect TGF-beta1-induced rat PAI-1 promoter activity. Moreover, interferon-gamma (IFN-gamma) inhibited TGF-beta1-induced PAI-1 expression through selectively suppressing the ERK-AP-1 pathway.
These results suggest that the essential requirement of MAPK/AP-1 activation for TGF-beta1-induced PAI-1 expression is unique to rat mesangial cells.
Kidney International 10/2005; 68(3):972-84. · 6.61 Impact Factor
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ABSTRACT: We examined the mechanisms underlying the activation of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) contributing to the supply of fatty acids required for the formation of cholesteryl ester in oxidized low-density lipoprotein (oxLDL)-stimulated macrophages. The possible involvement of oxidized lipids was also examined. In [(3)H]arachidonic acid-labeled mouse peritoneal macrophages, oxLDL stimulated the release of arachidonic acid, which was suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cPLA(2)alpha inhibitor. oxLDL induced an increase in PLA(2)alpha levels in the membrane fraction without affecting those in whole cells or the activity in the lysate. Among 13-hydroxyoctadecadienoic acid (13-HODE), 7-ketocholesterol, and 25-hydroxycholesterol, oxidized lipids present in oxLDL particles, only 13-HODE induced the release of arachidonic acid, which was also sensitive to MAFP. Under conditions where addition of Ca(2+) to the cell lysate induced an increase in cPLA(2)alpha protein in the membrane fraction, preincubation with 13-HODE facilitated the Ca(2+)-dependent translocation of cPLA(2)alpha. Furthermore, 13-HODE increased cholesteryl ester formation in the presence of [(3)H]cholesterol. These results suggest that 13-HODE mediates the oxLDL-induced activation of cPLA(2)alpha through an increase in cPLA(2)alpha protein in the membranes, thus contributing, in part, to the supply of fatty acids required for the esterification of cholesterol in macrophages.
Biochimica et Biophysica Acta 12/2004; 1686(1-2):77-84. · 4.66 Impact Factor
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ABSTRACT: Since prostanoids such as prostaglandin E2 play a pivotal role in modulating renal function, we investigated the involvement of ceramide in expression of secretory phospholipase A2 (sPLA2) and cyclooxygenase-2 (COX-2) in tumor necrosis factor-alpha (TNF-alpha)-stimulated mesangial cells. TNF-alpha stimulation increased ceramide generation in parallel with a decrease in sphingomyelin. Pretreatment with exogenous sphingomyelinase (SMase) dose-dependently enhanced TNF-alpha-stimulated increases in COX-2 protein and sPLA) activity. SMase also augmented TNF-alpha-mediated nuclear factor kappaB (NF-kappaB) activation. N-acetylcysteine (NAC), an antioxidant, completely inhibited the SMase-induced increase in sPLA2 activity, whereas NAC inhibited partially the activity stimulated with TNF-alpha alone. Under the conditions, NAC completely inhibited reactive oxygen species (ROS) production induced by SMase followed by TNF-alpha. These results suggest that ceramide elicits up-regulation of NF-kappaB through ROS production, which, in turn, leads to stimulation of COX-2 and sPLA2 expression. Therefore, ceramide may be implicated in the pathogenesis of renal abnormalities.
Cellular Signalling 09/2004; 16(8):967-74. · 4.06 Impact Factor
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ABSTRACT: The catalytic activity of calcium-independent phospholipase A2 (iPLA2), which is classified as a group VI PLA2, is regulated by protein kinase C, calmodulin, and others such as reactive oxygen species. Numerous findings have shown that iPLA2 is involved in stimulus-induced arachidonic acid release and lysophospholipid generation, although the participation is dependent upon the cell type and stimulus. The catalytic action of iPLA2 is known to be responsible for phospholipid remodeling as a housekeeping function. However, it has been widely accepted that arachidonic acid and lysophospholipid generated by iPLA2 act as a signaling molecule in cellular functions. Those include eicosanoid production, glucose-induced insulin secretion, Fas-induced apoptosis, cellular proliferation, membrane traffic in fusion, contribution to myocardial ischemia, and others. In this review, the functional role of iPLA2 in cellular responses upon stimulation is the focus.
Biological & Pharmaceutical Bulletin 09/2004; 27(8):1174-8. · 1.66 Impact Factor
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ABSTRACT: 1 The leaf extract of Ginkgo Biloba L. exhibits a variety of pharmacological effects through an antioxidant action. We examined the effects of the leaf extract (Ginkgolon-24) on the production of fibronectin induced by oxidized low-density lipoprotein (oxLDL) in rat mesangial cells. 2 Stimulation with oxLDL accelerated the production of fibronectin with the preceding generation of reactive oxygen species (ROS). Pretreatment with Ginkgolon-24 inhibited the oxLDL-induced fibronectin production as well as ROS generation. 3 oxLDL also elicited the activation of SP-1, nuclear factor-kappaB, and cAMP response element-binding protein, which are transcription factors involved in the fibronectin production. Among these activated transcription factors, Ginkgolon-24 inhibited the activation of SP-1 only. 4 Furthermore, 7-ketocholesterol, an oxidized lipid in oxLDL particles, induced the production of fibronectin and the activation of SP-1, which were also suppressed by Ginkgolon-24. 5 These results suggest that the leaf extract of Ginkgo Biloba L. inhibits the oxLDL-induced production of fibronectin probably through inhibitory effects on ROS generation and SP-1 activation in rat mesangial cells.
British Journal of Pharmacology 07/2004; 142(3):419-24. · 4.41 Impact Factor
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ABSTRACT: We examined the mechanisms responsible for the production of fibronectin induced by oxidized low-density lipoprotein (oxLDL) in rat mesangial cells. oxLDL accelerated the production of fibronectin with the preceding generation of reactive oxygen species (ROS). Pretreatment with N-acetylcysteine suppressed the oxLDL-induced fibronectin production as well as ROS generation. oxLDL also elicited the activation of SP-1, nuclear factor-kappaB, and cAMP response element-binding protein, but not activator protein-1. Among these activated transcription factors, N-acetylcysteine inhibited the activation of SP-1 only. 7-Ketocholesterol, an oxidized lipid in oxLDL particles, induced the production of fibronectin and the activation of SP-1, those which were suppressed by N-acetylcysteine. Furthermore, mithramycin A, an inhibitor of SP-1, also suppressed the oxLDL- and 7-ketocholesterol-stimulated production of fibronectin. These results suggest that oxLDL stimulates fibronectin production, at least in part, through the ROS-dependent activation of SP-1 in rat mesangial cells, and further that the ROS-dependent cellular responses may be elicited by 7-ketocholesterol.
Biochemical and Biophysical Research Communications 11/2003; 310(2):491-7. · 2.48 Impact Factor
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ABSTRACT: We examined the roles of phospholipase A2 (PLA2) in oxidized LDL (oxLDL)-induced cholesteryl ester formation in macrophages. In [3H]oleic acid-labeled RAW264.7 cells and mouse peritoneal macrophages, oxLDL induced [3H]cholesteryl oleate formation with an increase in free [3H]oleic acid and a decrease in [3H]phosphatidylcholine. The changes in these lipids were suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cytosolic PLA2 (cPLA2) inhibitor. However, MAFP had no effect on the ACAT activity or the binding and/or uptake of oxLDL. Stimulation with oxLDL in the presence of [3H]cholesterol increased [3H]cholesteryl ester bearing fatty acyl chains derived from cellular and/or exogenous (oxLDL) lipids. The formation of cholesteryl ester under this condition was also inhibited by MAFP, and the inhibitory effect was reversed by adding oleic acid. While oxLDL did not affect the activity or amounts of cPLA2, preincubation with oxLDL enhanced the release of oleic acid and arachidonic acid induced by ionomycin in RAW264.7 cells. 13(S)-hydroxyoctadecadienoic acid, but not 7-ketocholesterol, also enhanced ionomycin-induced oleic acid release. These results suggest that oxLDL induces cPLA2 activation, which contributes, at least in part, to the supply of fatty acids required for the cholesteryl esterification, probably through the acceleration by oxidized lipids of the catalytic action of cPLA2 in macrophages.
The Journal of Lipid Research 10/2003; 44(9):1676-85. · 5.56 Impact Factor
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ABSTRACT: The involvement of cytosolic phospholipase A(2) (cPLA(2)) and ceramide in the accumulation of cholesteryl ester induced by the uptake of oxidized low-density lipoproteins (oxLDL) in macrophages was investigated. Uptake of oxLDL by [(3)H]oleic acid-labeled macrophages stimulated the formation of cholesteryl oleate, and this process was completely inhibited by a cPLA(2) inhibitor. Under the conditions, a time-dependent increase in ceramide was observed, while sphingomyelin levels were unaffected. The production of ceramide was completely inhibited by fumonisin B1, an inhibitor of the de novo synthesis of ceramide, and oxLDL-induced cholesteryl oleate formation was inhibited partially. Treatment of the cells with sphingomyelinase accelerated the formation of cholesteryl ester. Furthermore, sphingomyelinase or cell-permeable ceramide induced the release of oleic acid, and this was inhibited by a cPLA(2) inhibitor. These results suggest that activation of cPLA(2) is responsible for the formation of cholesteryl ester induced by the uptake of oxLDL in macrophages, and that de novo-synthesized ceramide is implicated, at least in part, in this process.
Cellular Signalling 09/2002; 14(8):695-701. · 4.06 Impact Factor
