Oxidized low-density lipoprotein depletes PKCalpha and attenuates reactive oxygen species formation in monocytes/macrophages.
OBJECTIVE
Preexposure of macrophages to oxidized low-density lipoprotein (oxLDL) attenuates formation of reactive oxygen species (ROS) upon stimulation with phorbol 12-myristate 13-acetate (PMA) or acute exposure to oxLDL. We examined whether attenuation of the oxidative burst is attributed to down-regulation of protein kinase C alpha (PKCalpha).


RESULTS
Acute exposure of a mouse macrophage cell line (RAW 264.7) to both PMA and oxLDL provoked ROS generation that was blocked by the PKCalpha/beta1 inhibitor Go 6967. However, in RAW 264.7 macrophages preincubated with oxLDL, ROS formation in response to stimulation with oxLDL or PMA was reduced. Attenuated ROS production correlated with down-regulation of the amount of PKCalpha protein in a time-dependent manner and was maximal at 8 h and concentrations of 50-100 microg/ml oxLDL. PMA, a well-established PKCalpha activator, inhibited ROS formation as well when preincubated for 8 to 16 h. In cells stably overexpressing PKCalpha-EGFP, we noticed that ROS formation remained intact upon pre-exposure of these cells to oxLDL. Moreover, in these cells endogenous but not overexpressed PKCalpha-EGFP disappeared, further substantiating the importance of PKCalpha in stimulating ROS production. In addition, we noticed a concentration-dependent ability of oxLDL to halt ROS production. Whereas 10 microg/ml oxLDL was insufficient in attenuating ROS formation over an 8-h incubation period in RAW 264.7 cells, 50 microg/ml oxLDL impaired ROS generation.


CONCLUSIONS
These results indicate that attenuation of the oxidative burst in oxLDL-pretreated macrophages is closely associated with down-regulation of PKCalpha, which is elicited in a dose- and time-dependent manner.
Köhl
Roman
R
Institute of Biochemistry I, Faculty of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
Preiss
Stefan
S
von Knethen
Andreas
A
Brüne
Bernhard
B
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D051571:Protein Kinase C-alpha / Q000096:biosynthesis / Q000187:drug effects* / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016897:Respiratory Burst / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D008077:Lipoproteins, LDL; D017382:Reactive Oxygen Species; C099252:oxidized low density lipoprotein; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate