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Molecular targets for glucocorticoids in macrophages: cytosolic phospholipase A2 and cytokine formation.

Gewert, Karin LU (2000)
Abstract
The aim of this investigation has been to study mechanisms by which glucocorticoids inhibit inflammatory processes in vitro. Type IV cytosolic 85 kDa phospholipase A2 (cPLA2) is an important modulator of inflammatory responses that is present in macrophages as well as in other cell types. In macrophages, the synthetic glucocorticoid dexamethasone (dex) was found to reduce the expression of cPLA2 and further to inhibit the activation of cPLA2. Stimulation with phorbol 12-myristate 13-acetate (PMA), zymosan or the protein phosphatase inhibitor okadaic acid increased the cPLA2 activity. After treatment with dex, okadaic acid, but not PMA or zymosan, was able to up-regulate cPLA2 activity. These results indicate that the effect of okadaic acid... (More)
The aim of this investigation has been to study mechanisms by which glucocorticoids inhibit inflammatory processes in vitro. Type IV cytosolic 85 kDa phospholipase A2 (cPLA2) is an important modulator of inflammatory responses that is present in macrophages as well as in other cell types. In macrophages, the synthetic glucocorticoid dexamethasone (dex) was found to reduce the expression of cPLA2 and further to inhibit the activation of cPLA2. Stimulation with phorbol 12-myristate 13-acetate (PMA), zymosan or the protein phosphatase inhibitor okadaic acid increased the cPLA2 activity. After treatment with dex, okadaic acid, but not PMA or zymosan, was able to up-regulate cPLA2 activity. These results indicate that the effect of okadaic acid was exerted at, or downstream of, the dex-sensitive step(s). Phosphorylation of cPLA2 occurred on multiple sites. Upon stimulation with PMA or bacteria, the most C-terminal fragment (residues 698-749) of cPLA2 was the most heavily phosphorylated. We did not find any evidence for down-regulation of protein kinase C isoforms after dex treatment. Neither was the expression, nor the activation of, the mitogen-activated protein kinases (MAP kinases), extracellular signal- regulated kinase-2 (ERK-2) or p38, affected to the same extent as the cPLA2 activity. Although a minor inhibition of the zymosan-induced activation of the MAP kinases was observed, our results suggest that dex-mediated inhibition of cPLA2 activation is exerted down-stream of the MAP kinases. The effect of dex on the formation of two other inflammatory mediators in macrophages, the cytokines interleukin-1beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha) was also investigated. Treatment with dex inhibited bacteria- induced IL-1beta expression primarily at the transcriptional level whereas TNF-alpha expression was only partially inhibited at the level of transcription but further inhibited at the translational level. The translational inhibition after dex treatment was overcome by okadaic acid. Also the post-translational processing of TNF-alpha was modified by okadaic acid. TNF-alpha was found both in the cleaved and uncleaved form after okadaic acid treatment. The different TNF-alpha forms exhibited different sensitivity to dex treatment. Based on these data, we suggest that the effect of dex on TNF-alpha translation is mediated by an okadaic acid-sensitive protein phosphatase. In conclusion, dex inhibits the synthesis of cPLA2. Studies with okadaic acid revealed protein phosphatases to be likely targets for dex both in the inhibition of cPLA2 activation and in inhibition of TNF-alpha translation. (Less)
Abstract (Swedish)
Popular Abstract in Swedish

En studie av hur glokokortikoider hämmar inflammation



Glukokortikosteroider, varav kortison är den mest kända, är hormoner som reglerar många funktioner i kroppen och även används som läkemedel för att dämpa inflammatoriska tillstånd som t.ex. astma och reumatiska sjukdomar. Glukokortikosteroider är mycket potenta läkemedel och används flitigt med mycket goda effekter. Vid långvarigt systemiskt bruk av höga doser förekommer tyvärr biverkningar. Det är därför av stort intresse att klargöra glukokortikosteroidernas verkningsmekanismer för att utifrån dessa fakta kunna framställa mer specifika läkemedel med färre biverkningar. I denna studie har vi använt dexametason som är en... (More)
Popular Abstract in Swedish

En studie av hur glokokortikoider hämmar inflammation



Glukokortikosteroider, varav kortison är den mest kända, är hormoner som reglerar många funktioner i kroppen och även används som läkemedel för att dämpa inflammatoriska tillstånd som t.ex. astma och reumatiska sjukdomar. Glukokortikosteroider är mycket potenta läkemedel och används flitigt med mycket goda effekter. Vid långvarigt systemiskt bruk av höga doser förekommer tyvärr biverkningar. Det är därför av stort intresse att klargöra glukokortikosteroidernas verkningsmekanismer för att utifrån dessa fakta kunna framställa mer specifika läkemedel med färre biverkningar. I denna studie har vi använt dexametason som är en syntetisk glukokortikosteroid.



Effekterna av dexametason har studerats på celler som kallas makrofager. Makrofager är en sorts vita blodkroppar som deltar i kroppens försvar mot främmande organismer. Vid aktivering av makrofager är fosfolipas A2 det enzym som reglerar hastigheten med vilken olika inflammationssubstanser såsom prostaglandiner och leukotriener bildas. Cytokiner är ett annat exempel på substanser som bildas och frisätts av makrofager och medverkar vid inflammationer.



I den första studien fann vi att dexametason hämmar både bildningen av proteinet fosfolipas A2 (cPLA2) och dess aktivitet. Många proteiners aktivitet regleras genom fosforylering (ett s.k. kinas kopplar på en fosfatgrupp på en aminosyra) respektive defosforylering (borttagning av fosfatgrupp som utförs av fosfatas). Vi fann att vi genom att tillsätta en substans som hämmar fosfataser (okadainsyra) kan upphäva effekten av dexametason. Detta indikerar att hämningen av cPLA2 aktiviteten eventuellt sker genom induktion av ett fosfatas. Vi har också arbetat med att kartlägga vilka aminosyror det är som fosforyleras vid aktivering av cPLA2. Tre olika kinaser har tidigare föreslagits fosforylera cPLA2. Våra data ger inte något stöd för att dexametason skulle hämma något av dessa i samma utsträckning som cPLA2 hämmas. Dex påverkar också cytokiner. Vi har valt att studera främst interleukin-1 och tumour necrosis factor vilka är två cytokiner med stor betydelse vid inflammatoriska tillstånd. Regleringen av cytokiner är viktig eftersom de behövs för att kroppen ska kunna bekämpa infektioner och även cancer, men överaktivering av cytokiner kan ha dödlig utgång. Vi fann att dexametason hämmar bildningen av dessa cytokiner. De båda cytokinerna hämmas under olika betingelser och i olika steg i processen som leder fram till det färdiga proteinet. Även för regleringen av cytokiner med dexametason fann vi att fosfataser är av stor betydelse.



Sammanfattningsvis hämmas långt ifrån alla proteiner som är involverade vid inflammationer av dexametason. Dock utövade glukokortikosteroiderna en kraftfullt hämmande effekt på flera olika viktiga proteiner varav ett är cPLA2. En sådan komplex verkan är med all sannolikhet av stor betydelse för finjusteringen av balansen i kroppens immunsvar. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Assoc. Prof. Johansen, Berit, Trondheim, Norway
organization
publishing date
type
Thesis
publication status
published
subject
keywords
pharmacognosy, Pharmacological sciences, signal transduction, dexamethasone, cytosolic phospholipase A2, glucocorticoids, macrophages, protein kinase C, mitogen-activated protein kinases, TNF-alpha, IL-1beta, protein phosphatases, okadaic acid, pharmacy, toxicology, Farmakologi, farmakognosi, farmaci, toxikologi
pages
108 pages
publisher
Center for Molecular Biomedicine, Lund University
defense location
Hörsal B, Kemicentrum, Lund
defense date
2000-03-01 10:15:00
external identifiers
  • other:ISRN: LUMEDW/MECM--00/1039--SE
language
English
LU publication?
yes
id
bb109f82-9658-4178-b2a2-aa33fdee4e67 (old id 40246)
date added to LUP
2016-04-04 12:04:53
date last changed
2018-11-21 21:08:53
@phdthesis{bb109f82-9658-4178-b2a2-aa33fdee4e67,
  abstract     = {{The aim of this investigation has been to study mechanisms by which glucocorticoids inhibit inflammatory processes in vitro. Type IV cytosolic 85 kDa phospholipase A2 (cPLA2) is an important modulator of inflammatory responses that is present in macrophages as well as in other cell types. In macrophages, the synthetic glucocorticoid dexamethasone (dex) was found to reduce the expression of cPLA2 and further to inhibit the activation of cPLA2. Stimulation with phorbol 12-myristate 13-acetate (PMA), zymosan or the protein phosphatase inhibitor okadaic acid increased the cPLA2 activity. After treatment with dex, okadaic acid, but not PMA or zymosan, was able to up-regulate cPLA2 activity. These results indicate that the effect of okadaic acid was exerted at, or downstream of, the dex-sensitive step(s). Phosphorylation of cPLA2 occurred on multiple sites. Upon stimulation with PMA or bacteria, the most C-terminal fragment (residues 698-749) of cPLA2 was the most heavily phosphorylated. We did not find any evidence for down-regulation of protein kinase C isoforms after dex treatment. Neither was the expression, nor the activation of, the mitogen-activated protein kinases (MAP kinases), extracellular signal- regulated kinase-2 (ERK-2) or p38, affected to the same extent as the cPLA2 activity. Although a minor inhibition of the zymosan-induced activation of the MAP kinases was observed, our results suggest that dex-mediated inhibition of cPLA2 activation is exerted down-stream of the MAP kinases. The effect of dex on the formation of two other inflammatory mediators in macrophages, the cytokines interleukin-1beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha) was also investigated. Treatment with dex inhibited bacteria- induced IL-1beta expression primarily at the transcriptional level whereas TNF-alpha expression was only partially inhibited at the level of transcription but further inhibited at the translational level. The translational inhibition after dex treatment was overcome by okadaic acid. Also the post-translational processing of TNF-alpha was modified by okadaic acid. TNF-alpha was found both in the cleaved and uncleaved form after okadaic acid treatment. The different TNF-alpha forms exhibited different sensitivity to dex treatment. Based on these data, we suggest that the effect of dex on TNF-alpha translation is mediated by an okadaic acid-sensitive protein phosphatase. In conclusion, dex inhibits the synthesis of cPLA2. Studies with okadaic acid revealed protein phosphatases to be likely targets for dex both in the inhibition of cPLA2 activation and in inhibition of TNF-alpha translation.}},
  author       = {{Gewert, Karin}},
  keywords     = {{pharmacognosy; Pharmacological sciences; signal transduction; dexamethasone; cytosolic phospholipase A2; glucocorticoids; macrophages; protein kinase C; mitogen-activated protein kinases; TNF-alpha; IL-1beta; protein phosphatases; okadaic acid; pharmacy; toxicology; Farmakologi; farmakognosi; farmaci; toxikologi}},
  language     = {{eng}},
  publisher    = {{Center for Molecular Biomedicine, Lund University}},
  school       = {{Lund University}},
  title        = {{Molecular targets for glucocorticoids in macrophages: cytosolic phospholipase A2 and cytokine formation.}},
  url          = {{https://lup.lub.lu.se/search/files/5922756/1693387.pdf}},
  year         = {{2000}},
}