7-ketocholesterol-induced inflammation: involvement of multiple kinase signaling pathways via NFκB but independently of reactive oxygen species formation.
OBJECTIVE
7-Ketocholesterol (7KCh) accumulates in oxidized lipoprotein deposits and is known to be involved in macrophage foam cell formation and atherosclerosis. 7-KCh is present in the primate retina and is associated with oxidized lipoprotein deposits located in the choriocapillaris, Bruch's membrane, and retinal pigment epithelium (RPE). 7-KCh can also be formed in the retina as a consequence of light-induced iron release. The purpose of this study was to examine the signaling pathways involved in the 7KCh-mediated inflammatory response focusing on three cytokines, VEGF, IL-6, and IL-8.


METHODS
ARPE-19 cells were treated with 7KCh solubilized in hydroxypropyl-β-cyclodextrin. Cytokines were quantified by qRT-PCR (mRNA) and ELISA (protein) using commercially available products. NFκB activation was determined by IκBα mRNA induction.


RESULTS
Treatment of ARPE-19 cells with 15 μM 7KCh markedly induced the expression of VEGF, IL-6, and IL-8. No increase in NOX-4 expression or ROS formation was detected. 7KCh induced the phosphorylation of ERK1/2 and p38MAPK, and inhibitors to these kinases markedly reduced the cytokine expression but did not affect the IκBα mRNA expression. By contrast, inhibition of PI3K and PKCζ significantly decreased the cytokine and IκBα mRNA expression. Inhibition of the IκB kinase complex essentially ablated all cytokine induction.


CONCLUSIONS
7KCh induces cytokines via three kinase signaling pathways, AKT-PKCζ-NFκB, p38 MAPK, and ERK. The MAPK/ERK pathways seem to preferentially enhance cytokine induction downstream from NFκB activation. The results of this study suggest that 7KCh activates these pathways through interactions in the plasma membrane, but the mechanism(s) remains unknown.
Larrayoz
Ignacio M
IM
Mechanisms of Retinal Diseases Section, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.
Huang
Jiahn-Dar
JD
Lee
Jung Wha
JW
Pascual
Iranzu
I
Rodríguez
Ignacio R
IR
D002460:Cell Line; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D007653:Ketocholesterols / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D007653:Ketocholesterols; D016328:NF-kappa B; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D011494:Protein Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C003001:7-ketocholesterol