Satoshi Akiba

Publications of Satoshi Akiba

• Regulatory role of antigen-induced interleukin-10, produced by CD4⁺ T cells, in airway neutrophilia in a murine model for asthma.

  Authors: Takeshi Nabe, Ayumu Ikedo, Fusa Hosokawa, Maki Kishima, Masanori Fujii, Nobuaki Mizutani, Shin Yoshino, Keiichi Ishihara, Satoshi Akiba, David D Chaplin

  European journal of pharmacology. 12/2011; 677(1-3):154-62.

  It has been suggested that interleukin (IL)-10 exerts immunosuppressive effects on allergic inflammation, including asthma, mainly through inhibition of Th2 cell-mediated eosinophilic airwayIt has been suggested that interleukin (IL)-10 exerts immunosuppressive effects on allergic inflammation, including asthma, mainly through inhibition of Th2 cell-mediated eosinophilic airway inflammation. In a model of experimental asthma utilizing multiple intratracheal antigen challenges in sensitized mice, IL-10 production as well as eosinophilia and neutrophilia in the lung were induced by the multiple challenges. In this study, we set out to reveal the cellular source of endogenously produced IL-10, and the roles of IL-10 in airway leukocyte inflammation using an anti-IL-10 receptor monoclonal antibody. Balb/c mice were sensitized i.p. with ovalbumin+Al(OH)(3), and then challenged by intratracheal administration of ovalbumin 4 times. Flow cytometric analyses revealed that the cellular source of IL-10 was CD4(+) T cells lacking the transcription factor, forkhead box P3. Treatment with anti-IL-10 receptor monoclonal antibody prior to the 4th challenge significantly augmented airway neutrophilia as well as the production of IL-1β, and CXC chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2, but not airway eosinophilia, Th2 cytokine (IL-4 and IL-5) production, or a late-phase increase in specific airway resistance. Approximately 40% of IL-10 receptor(+) cells expressed the macrophage marker F4/80, whereas only 3-4% of the IL-10 receptor(+) cells were granulocyte differentiation antigen (Gr)-1(high) cells (neutrophils). In conclusion, multiple airway antigen challenges induced the proliferation of IL-10-expressing CD4(+) T cells in regulating airway neutrophilia. Systemic blockade of IL-10 function coincided with increases in IL-1β and CXC chemokines. Thus, IL-1β and CXC chemokines may be targets for development of novel pharmacotherapy for neutrophilic asthma.

• Regulation of macrophage differentiation and polarization by group IVC phospholipase A₂.

  Authors: Keiichi Ishihara, Asuka Kuroda, Kanako Sugihara, Shiho Kanai, Takeshi Nabe, Satoshi Akiba

  Biochemical and biophysical research communications. 11/2011; 416(3-4):325-30.

  Although the cellular function of group IVC phospholipase A(2) (IVC-PLA(2)) remains to be understood, the expression of IVC-PLA(2) in human monocytic THP-1 cells was increased during phorbolAlthough the cellular function of group IVC phospholipase A(2) (IVC-PLA(2)) remains to be understood, the expression of IVC-PLA(2) in human monocytic THP-1 cells was increased during phorbol ester-induced macrophage differentiation. We therefore examined the role of IVC-PLA(2) in macrophage differentiation using THP-1 cells. Two THP-1 cell lines stably expressing IVC-PLA(2)-specific shRNA were established. Differentiation and maturation into macrophages on treatment with phorbol ester were facilitated by knockdown of IVC-PLA(2) without the compensatory induction of mRNA expression for other group IV and VI PLA(2)s. Furthermore, the enhancement of macrophage differentiation by IVC-PLA(2)-knockdown were abolished by treatment with lysophosphatidylcholine, a metabolite of phospholipids generated by PLA(2)-mediated hydrolysis, indicating that PLA(2) activity is necessary for the inhibition of macrophage differentiation by IVC-PLA(2). Additionally, we found that the differentiation into classically activated M1 macrophage was superior in IVC-PLA(2)-knockdown cells, whereas the differentiation into alternatively activated M2 macrophage was suppressed by IVC-PLA(2)-knockdown. These findings suggest that IVC-PLA(2) is involved in regulations of macrophage differentiation and macrophage polarization.

• Synthesis of N-(trifluoromethyl-2-pyridinyl)arenesulfonamides as an inhibitor of secretory phospholipase A₂.

  Authors: Hitoshi Nakayama, Yuka Morita, Hirohiko Kimura, Keiichi Ishihara, Satoshi Akiba, Jun'ichi Uenishi

  Chemical & pharmaceutical bulletin. 01/2011; 59(6):783-6.

  A series of N-(trifluoromethyl-2-pyridinyl)alkane- and arenesulfonamides 2-5 have been synthesized by the substitution reaction of 2-chloro(trifluoromethyl)pyridines 6 with alkane- andA series of N-(trifluoromethyl-2-pyridinyl)alkane- and arenesulfonamides 2-5 have been synthesized by the substitution reaction of 2-chloro(trifluoromethyl)pyridines 6 with alkane- and arenesulfonamides 7. Their inhibitory activities against secretory phospholipase A₂ of porcine pancreas were examined and the analog N-[4,5-bis(trifluoromethyl)-2-pyridinyl]-4-trifluoromethylbenzenesulfonamide 4i was shown to have the highest inhibitory activity, with an IC(50) value of 0.58 mM.

• Synthesis of N-[2-(2,4-Difluorophenoxy)trifluoromethyl-3-pyridyl]sulfonamides and their inhibitory activities against secretory phospholipase A₂.

  Authors: Hitoshi Nakayama, Keiichi Ishihara, Satoshi Akiba, Jun'ichi Uenishi

  Chemical & pharmaceutical bulletin. 01/2011; 59(8):1069-72.

  N-[2-(2,4-Difluorophenoxy)trifluoromethyl-3-pyridyl]sulfonamide derivatives 3-6 were prepared by the reaction of 3-pyridylamines and sulfonyl chlorides. Inhibitory activities of these compoundsN-[2-(2,4-Difluorophenoxy)trifluoromethyl-3-pyridyl]sulfonamide derivatives 3-6 were prepared by the reaction of 3-pyridylamines and sulfonyl chlorides. Inhibitory activities of these compounds toward secretory phospholipase A₂ (sPLA₂) were examined and N-[2-(2,4-difluorophenoxy)-5-trifluoromethyl-3-pyridyl]-2-naphthalenesulfonamide (5c) was found to be the most potent against sPLA₂ with an IC₅₀ value of 90 µM.

• Triacylglycerol deposition with group IVC phospholipase A2 expression in oleate- and linoleate-stimulated Huh-7 hepatocytes.

  Authors: Keiichi Ishihara, Kengo Tachibana, Asuka Kuroda, Ayano Terakawa, Shinsuke Baba, Shiho Kanai, Satoshi Akiba

  Biological & pharmaceutical bulletin. 01/2011; 34(2):191-6.

  The accumulation of hepatocellular triacylglycerol (TG), a major symptom of fatty liver, is associated with the excessive incorporation of exogenous free fatty acids into hepatocytes, the free fattyThe accumulation of hepatocellular triacylglycerol (TG), a major symptom of fatty liver, is associated with the excessive incorporation of exogenous free fatty acids into hepatocytes, the free fatty acids inducing an increase in TG bearing acyl chains derived from not only themselves but also endogenous fatty acids. However, the mechanisms responsible for the supply of endogenous fatty acids, which are mainly esterified into phospholipids, remain unclear. In the present study, we examined the possible involvement of intracellular phospholipase A(2) (PLA(2))s including group IVA, IVC, VIA, and VIB PLA(2)s, which catalyze the release of endogenous fatty acids, in the deposition of TG in hepatocytes. Stimulation of human hepatoma Huh-7 cells with oleate or linoleate for 48 h increased TG contents time-dependently. Under the conditions, increased expression of group IVC PLA(2) mRNA and protein was observed at 6-12 h and 24-48 h after the stimulation, respectively. However, mRNA levels of group IVA, VIA, or VIB PLA(2) did not change. When cells were treated with methyl arachidonyl fluorophosphonate used as an inhibitor of group IVC PLA(2), the fatty acid-induced deposition of TG was partially but significantly suppressed at 48 h, although no significant inhibition was observed at 24 h. Overexpression of wild-type group IVC PLA(2) but not a catalytically inactive mutant of group IVC PLA(2) tended to increase cellular TG levels. The present findings suggest that stimulation of Huh-7 hepatocytes with free fatty acids induces the expression of group IVC PLA(2), which is involved in the fatty acid-induced deposition of TG.

• Glucose is necessary for stabilization of hypoxia-inducible factor-1alpha under hypoxia: contribution of the pentose phosphate pathway to this stabilization.

  Authors: Mayuko Osada-Oka, Yasushi Hashiba, Satoshi Akiba, Susumu Imaoka, Takashi Sato

  FEBS letters. 07/2010; 584(14):3073-9.

  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 inhibitorIn 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.

• Alleviation of high-fat diet-induced fatty liver damage in group IVA phospholipase A2-knockout mice.

  Authors: Hiromi Ii, Naoki Yokoyama, Shintaro Yoshida, Kae Tsutsumi, Shinji Hatakeyama, Takashi Sato, Keiichi Ishihara, Satoshi Akiba

  PloS one. 01/2009; 4(12):e8089.

  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 examinedHepatic 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.

• Group IVA phospholipase A2 is associated with the storage of lipids in adipose tissue and liver.

  Authors: Hiromi Ii, Shinji Hatakeyama, Kae Tsutsumi, Takashi Sato, Satoshi Akiba

  Prostaglandins & other lipid mediators. 07/2008; 86(1-4):12-7.

  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 arachidonicProstaglandin (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.

• Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1alpha-dependent expression of thrombospondin-1.

  Authors: Mayuko Osada-Oka, Takako Ikeda, Satoshi Akiba, Takashi Sato

  Journal of cellular biochemistry. 05/2008; 104(5):1918-1926.

  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 theThe 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. J. Cell. Biochem. 104: 1918-1926, 2008. (c) 2008 Wiley-Liss, Inc.

• Group IVA phospholipase A2-associated production of MMP-9 in macrophages and formation of atherosclerotic lesions.

  Authors: Hiromi Ii, Naoya Hontani, Issei Toshida, Mayuko Oka, Takashi Sato, Satoshi Akiba

  Biological & pharmaceutical bulletin. 04/2008; 31(3):363-8.

  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 examinedMatrix 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.

• [Role of thrombospondin-1 in hypoxia-induced migration of human vascular smooth muscle cells]

  Authors: Minoru Takahashi, Mayuko Oka, Takako Ikeda, Satoshi Akiba, Takashi Sato

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan. 03/2008; 128(3):377-83.

  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,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.

• VEGF-enhanced proliferation under hypoxia by an autocrine mechanism in human vascular smooth muscle cells.

  Authors: Mayuko Osada-Oka, Takako Ikeda, Susumu Imaoka, Satoshi Akiba, Takashi Sato

  Journal of atherosclerosis and thrombosis. 03/2008; 15(1):26-33.

  AIM: 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 theAIM: 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). METHODS: Cells were cultured under hypoxic conditions (1% O(2), 5% CO(2)) and several responses were measured. RESULTS: 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. CONCLUSION: 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.

• Inhibition of cytosolic phospholipase A(2) suppresses production of cholesteryl ester through the reesterification of free cholesterol but not formation of foam cells in oxidized LDL-stimulated macrophages.

  Authors: Hiromi Ii, Mayuko Oka, Atsushi Yamashita, Keizo Waku, Naonori Uozumi, Takao Shimizu, Takashi Sato, Satoshi Akiba

  Biological & pharmaceutical bulletin. 02/2008; 31(1):6-12.

  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 fromMacrophage-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.

• Suppression of oxidized LDL-induced PDGF receptor beta activation by ginkgo biloba extract reduces MMP-1 production in coronary smooth muscle cells.

  Authors: Satoshi Akiba, Hidenori Yamaguchi, Satomi Kumazawa, Mayuko Oka, Takashi Sato

  Journal of atherosclerosis and thrombosis. 11/2007; 14(5):219-25.

  AIM: 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 matrixAIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

• Involvement of Ca2+-independent phospholipase A2 in the translocation of hypoxia-inducible factor-1alpha to the nucleus under hypoxic conditions.

  Authors: Mayuko Osada-Oka, Minoru Takahashi, Satoshi Akiba, Takashi Sato

  European journal of pharmacology. 12/2006; 549(1-3):58-62.

  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 ofWe 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.

• Acceleration of matrix metalloproteinase-1 production and activation of platelet-derived growth factor receptor beta in human coronary smooth muscle cells by oxidized LDL and 4-hydroxynonenal.

  Authors: Satoshi Akiba, Satomi Kumazawa, Hidenori Yamaguchi, Naoya Hontani, Takeyoshi Matsumoto, Takako Ikeda, Mayuko Oka, Takashi Sato

  Biochimica et biophysica acta. 09/2006; 1763(8):797-804.

  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 inIncreases 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.

• Mitochondrial dysfunction is related to necrosis-like programmed cell death induced by A23187 in CEM cells.

  Authors: Keigo Hamahata, Souichi Adachi, Hiroshi Matsubara, Masayuki Okada, Tsuyoshi Imai, Ken-Ichiro Watanabe, Shin-ya Toyokuni, Masaki Ueno, Shigeo Wakabayashi, Yuki Katanosaka, Satoshi Akiba, Masaru Kubota, Tatsutoshi Nakahata

  European journal of pharmacology. 07/2005; 516(3):187-96.

  We have previously reported that calcium ionophore A23187 differentially induces necrosis in CEM cells, a T-lymphoblastic leukemia cell line, and apoptosis in HL60 cells, a promyelocytic leukemiaWe have previously reported that calcium ionophore A23187 differentially induces necrosis in CEM cells, a T-lymphoblastic leukemia cell line, and apoptosis in HL60 cells, a promyelocytic leukemia cell line. Stimulation with VP16, however, induces typical apoptosis in both cell lines. Necrosis in CEM cells, characterized by cell shrinkage and clustering, began within 5 min of treatment. Swelling of the mitochondria, lumpy chromatin condensation and intact plasma membranes were evident by electron microscopy. These A23187-mediated changes in CEM cells were suppressed by clonazepam or CGP37157, inhibitors of the mitochondrial Na(+)/Ca(2+) exchanger. The changes, however, were not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. In both CEM and HL60 cells, intra-cellular calcium increased with similar amplitude within 1 min of treatment with 2 microM A23187. Intra-mitochondrial calcium increased with clonazepam pre-treatment alone in both CEM and HL60 cells. However, intra-mitochondrial calcium did not change drastically in response to A23187 in CEM or HL60 cells, either untreated or pre-treated with clonazepam. A23187 induces necrosis in CEM cells concurrent with mitochondrial dysfunction, which is independent of the mitochondrial permeability transition, but affected by intra-mitochondrial calcium, while HL60 cells lack these early changes. Differences in the responses to A23187 between these two cell lines might derive from differences in the susceptibility of the mitochondrial membrane to rapid increases in intra-cellular calcium.

• Translocation of phospholipase A2 to membranes by oxidized LDL and hydroxyoctadecadienoic acid to contribute to cholesteryl ester formation.

  Authors: Satoshi Akiba, Hiromi Ii, Yukimasa Yoneda, Takashi Sato

  Biochimica et biophysica acta. 12/2004; 1686(1-2):77-84.

  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 esterWe 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.

• Cellular function of calcium-independent phospholipase A2.

  Authors: Satoshi Akiba, Takashi Sato

  Biological & pharmaceutical bulletin. 09/2004; 27(8):1174-8.

  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 oxygenThe 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.

• Ceramide-induced enhancement of secretory phospholipase A2 expression via generation of reactive oxygen species in tumor necrosis factor-alpha-stimulated mesangial cells.

  Authors: Kazuyuki Kitatani, Satoshi Akiba, Takashi Sato

  Cellular signalling. 09/2004; 16(8):967-74.

  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) andSince 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.