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Signalling to Activation of Cytosolic Phospholipase A2 and Arachidonate Release in Macrophages

Hiller, Gösta LU (2000)
Abstract
Eicosanoids include prostaglandins, thromboxanes and leukotrienes, which are arachidonic acid oxygenation products. These lipids make important contributions to the signs and symptoms of inflammatory diseases. The level of free arachidonic acid seems to be a rate-limiting factor in the synthesis of eicosanoids and the enzyme cytosolic phospholipase A2 (cPLA2) has been shown to preferentially liberate arachidonic acid from phospholipids in the cell membranes of macrophages. Stimulation of mouse peritoneal macrophages with Prevotella intermedia, a bacterium involved in periodontitis, led to activation of cPLA2 and release of arachidonate. The use of the inhibitors of mitogen-activated protein kinase (MAP kinase) kinase (PD 98059) and p38 (SB... (More)
Eicosanoids include prostaglandins, thromboxanes and leukotrienes, which are arachidonic acid oxygenation products. These lipids make important contributions to the signs and symptoms of inflammatory diseases. The level of free arachidonic acid seems to be a rate-limiting factor in the synthesis of eicosanoids and the enzyme cytosolic phospholipase A2 (cPLA2) has been shown to preferentially liberate arachidonic acid from phospholipids in the cell membranes of macrophages. Stimulation of mouse peritoneal macrophages with Prevotella intermedia, a bacterium involved in periodontitis, led to activation of cPLA2 and release of arachidonate. The use of the inhibitors of mitogen-activated protein kinase (MAP kinase) kinase (PD 98059) and p38 (SB 203580) revealed that both the MAP kinases ERK-1/2 and p38 were regulating the bacteria- or zymosan-induced activation of cPLA2. Another kinase, phosphatidylinositol 3-kinase (PtdIns 3-kinase) was found to be involved in the activation of ERK-1/2 and p38 and thereby also in the activation of cPLA2 and the subsequent release of arachidonate. However, lipopolysaccharide induced a PtdIns 3-kinase-independent activation of cPLA2. Pretreatment of the cells with an inhibitor of PtdIns 3-kinase (wortmannin) decreased the zymosan-induced translocation of phospholipase C-gamma2 (PLC-gamma2) to the cytoskeleton. Both zymosan and bacteria stimulation induced a tyrosine phosphoprylation of PLC-gamma2. In contrast, stimulation with LPS failed to induce any tyrosine phosphorylation of the enzyme. The synthetic glucocorticoid dexamethasone (dex) was found to reduce the mRNA expression and activation of cPLA2 The effects of dex on cPLA2 activation were not exerted to any major extent at the MAP kinase level. In conclusion, bacteria- and zymosan-induced signalling to activation of cPLA2 and release of arachidonate is mediated via PtdIns 3-kinase, ERK-1/2 and p38. (Less)
Abstract (Swedish)
Popular Abstract in Swedish

Summary in swedish



I en rad olika inflammatoriska sjukdomar såsom astma, reumatoid artrit och parodontit är makrofager involverade. Makrofager är vita blodkroppar som finns i kroppens olika vävnader. De reagerar på främmande partiklar, t ex bakterier, och bekämpar dessa genom att ”dels äta upp” främmande partiklar, sk fagocytos, och dels genom att utsöndra olika substanser såsom prostaglandiner och leukotriener. Dessa bildas från fettsyran arakidonsyra och är viktiga proinflammatoriska ämnen. Prostaglandiner och leukotriener finns normalt i låg koncentration i vävnaden och kan där ha en skyddande mekanism. Vid en inflammation kan koncentrationen av dessa substanser öka... (More)
Popular Abstract in Swedish

Summary in swedish



I en rad olika inflammatoriska sjukdomar såsom astma, reumatoid artrit och parodontit är makrofager involverade. Makrofager är vita blodkroppar som finns i kroppens olika vävnader. De reagerar på främmande partiklar, t ex bakterier, och bekämpar dessa genom att ”dels äta upp” främmande partiklar, sk fagocytos, och dels genom att utsöndra olika substanser såsom prostaglandiner och leukotriener. Dessa bildas från fettsyran arakidonsyra och är viktiga proinflammatoriska ämnen. Prostaglandiner och leukotriener finns normalt i låg koncentration i vävnaden och kan där ha en skyddande mekanism. Vid en inflammation kan koncentrationen av dessa substanser öka okontrollerat och bidraga till kroniska inflammationer. Frisättningen av arakidonsyra från cellmembranen utförs av enzymet fosfolipas A2 (cPLA2). Denna reaktion utgör det initiala och hastighetsreglerande steget i bildandet av prostaglandiner och leukotriener. Det är därför av stort intresse att klargöra vilka mekanismer som reglerar aktiviteten av cPLA2. Syftet med arbetet har varit att klarlägga de signaleringsmekanismer som aktiveras inuti makrofagen och leder till aktivering av cPLA2 och frisättning av arakidonsyra. Bakterier involverade i tandlossning har bl a använts som stimuli.



Vi fann att bakterier aktiverar två sk proteinkinaser, ERK och p38, vilka i sin tur reglerar aktiviteten av cPLA2 och frisättningen av arakidonsyra. Vidare fann vi att den inflammationsdämpande substansen dexametason inte hämmar aktiveringen av ERK och p38 men däremot hämmar bildningen av proteinet cPLA2. Förutom att aktiveras (fosforyleras) av proteinkinaser behöver cPLA2 en ökning av den intracellulära kalciumkoncentrationen för att kunna frisätta arakidonsyra från cellmembranet. Enzymet fosfolipas C (PLC) reglerar calciumkoncentrationen i cellen. En viss forma av detta enzym, PLC-gamma2, aktiveras av lipidkinaset fosfatidylinositol 3-kinase (PtdIns 3-kinas) vilket leder till att PLC-gamma2 byter lokalisation inuti makrofagen. Vi fann vidare att PtdIns 3-kinas på ett stimuliberoende sätt reglerar aktiveringen av ERK och p38. Detta medför att PtdIns 3-kinas indirekt reglerar aktiveringen av cPLA2 och frisättningen av arakidonsyra i makrofager. Sammanfattningsvis så leder denna kunskap till en bättre förståelse för hur bakterier orsakar inflammation och kan därmed på sikt få betydelse för utvecklingen av nya behandlingsmetoder. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Prof Huwiler, Andrea, Zentrum der Pharmacologie, Klinikum der Johann Wolfgang Goete-Universität, Frankfurt am Main, Germany
organization
publishing date
type
Thesis
publication status
published
subject
keywords
signal transduction, dexamethasone, macrophages, phospholipase C, phosphatidylinositol 3-kinase, mitogen-activated protein kinases, cytosolic phospholipase A2, arachidonic acid, Histology, cytochemistry, histokemi, cytokemi, tissue culture, Histologi, vävnadskultur, histochemistry
pages
93 pages
publisher
Department of Cell and Molecular Biology, Lund University
defense location
Hörsal B, Kemicentrum, Sölvegatan 39, Lund
defense date
2000-11-02 09:30:00
external identifiers
  • other:ISRN: LUMEDW/MECM- -00/1051- -SE
ISBN
91-628-4381-8
language
English
LU publication?
yes
id
f3f4202f-6e35-403a-a375-bec6e2d87314 (old id 40877)
date added to LUP
2016-04-04 11:40:01
date last changed
2018-11-21 21:06:22
@phdthesis{f3f4202f-6e35-403a-a375-bec6e2d87314,
  abstract     = {{Eicosanoids include prostaglandins, thromboxanes and leukotrienes, which are arachidonic acid oxygenation products. These lipids make important contributions to the signs and symptoms of inflammatory diseases. The level of free arachidonic acid seems to be a rate-limiting factor in the synthesis of eicosanoids and the enzyme cytosolic phospholipase A2 (cPLA2) has been shown to preferentially liberate arachidonic acid from phospholipids in the cell membranes of macrophages. Stimulation of mouse peritoneal macrophages with Prevotella intermedia, a bacterium involved in periodontitis, led to activation of cPLA2 and release of arachidonate. The use of the inhibitors of mitogen-activated protein kinase (MAP kinase) kinase (PD 98059) and p38 (SB 203580) revealed that both the MAP kinases ERK-1/2 and p38 were regulating the bacteria- or zymosan-induced activation of cPLA2. Another kinase, phosphatidylinositol 3-kinase (PtdIns 3-kinase) was found to be involved in the activation of ERK-1/2 and p38 and thereby also in the activation of cPLA2 and the subsequent release of arachidonate. However, lipopolysaccharide induced a PtdIns 3-kinase-independent activation of cPLA2. Pretreatment of the cells with an inhibitor of PtdIns 3-kinase (wortmannin) decreased the zymosan-induced translocation of phospholipase C-gamma2 (PLC-gamma2) to the cytoskeleton. Both zymosan and bacteria stimulation induced a tyrosine phosphoprylation of PLC-gamma2. In contrast, stimulation with LPS failed to induce any tyrosine phosphorylation of the enzyme. The synthetic glucocorticoid dexamethasone (dex) was found to reduce the mRNA expression and activation of cPLA2 The effects of dex on cPLA2 activation were not exerted to any major extent at the MAP kinase level. In conclusion, bacteria- and zymosan-induced signalling to activation of cPLA2 and release of arachidonate is mediated via PtdIns 3-kinase, ERK-1/2 and p38.}},
  author       = {{Hiller, Gösta}},
  isbn         = {{91-628-4381-8}},
  keywords     = {{signal transduction; dexamethasone; macrophages; phospholipase C; phosphatidylinositol 3-kinase; mitogen-activated protein kinases; cytosolic phospholipase A2; arachidonic acid; Histology; cytochemistry; histokemi; cytokemi; tissue culture; Histologi; vävnadskultur; histochemistry}},
  language     = {{eng}},
  publisher    = {{Department of Cell and Molecular Biology, Lund University}},
  school       = {{Lund University}},
  title        = {{Signalling to Activation of Cytosolic Phospholipase A2 and Arachidonate Release in Macrophages}},
  year         = {{2000}},
}