PKC alpha depletion in RAW264.7 macrophages following microbial/IFNgamma stimulation is PC-PLC-mediated.
Under chronic inflammatory conditions, monocytes/macrophages often exhibit a desensitized phenotype, which is characterized by attenuated reactive oxygen species (ROS) production in close association with depletion of protein kinase C alpha (PKC alpha). This behavior has been observed in monocytes derived from septic blood although the stimulus responsible for initiating these alterations remained obscure. Using RAW264.7 macrophages, we provide evidence that components of neither gram-negative nor gram-positive bacteria deplete PKC alpha, whereas the T(H)1 cytokine interferon-gamma (IFNgamma) does. As shown by western blot analysis, lipopolysaccharide, as well as lipoteichoic acid, did not alter PKC alpha expression, but IFNgamma dose-dependently decreased PKC alpha protein level. Taking into consideration that diacylglycerol and Ca2+ as established PKC alpha activators are released in response to phospholipase C activation, we pretreated cells with the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor tricyclodecan-9-yl potassium xanthate (D609) and the phosphatidylinositol-specific phospholipase C inhibitor 1-(6-(17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). In cells preincubated with D609, IFNgamma-mediated PKC alpha depletion was attenuated, whereas U73122 did not impair this process. Moreover, phorbol 12-myristate 13-acetate-initiated ROS formation, which was attenuated in macrophages pretreated with IFNgamma, was restored in the presence of the PC-PLC inhibitor. These results suggest that IFNgamma causes PC-PLC stimulation, diacylglycerol release, Ca2+ influx, and concomitant PKC alpha activation, which subsequently depletes PKC alpha. Strategies to antagonize IFNgamma might be helpful to prevent monocyte/macrophage desensitization.
von Knethen
Andreas
A
Biochemistry I, University of Frankfurt Medical School, Frankfurt, Germany.
Brüne
Bernhard
B
D000818:Animals; D015153:Blotting, Western; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004963:Estrenes / Q000494:pharmacology; D005434:Flow Cytometry; D005609:Free Radicals; D007371:Interferon-gamma / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009000:Monocytes / Q000166:cytology; D009636:Norbornanes; D010100:Oxygen / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D017382:Reactive Oxygen Species; D013682:Teichoic Acids / Q000494:pharmacology; D013859:Thiocarbamates; D013871:Thiones / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001952:Bridged-Ring Compounds; D004075:Diglycerides; D004963:Estrenes; D005609:Free Radicals; D008070:Lipopolysaccharides; D009636:Norbornanes; D011760:Pyrrolidinones; D017382:Reactive Oxygen Species; D013682:Teichoic Acids; D013859:Thiocarbamates; D013871:Thiones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C009900:lipoteichoic acid; C046498:tricyclodecane-9-yl-xanthogenate; D007371:Interferon-gamma; D051571:Protein Kinase C-alpha; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C; D010100:Oxygen; D002118:Calcium