7-Ketocholesterol-Induced Inflammation: Involvement of Multiple Kinase Signaling Pathways via NFκB but Independently of Reactive Oxygen Species Formation

PURPOSE. 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-beta-cyclodextrin.Cytokines were quantified by qRT-PCR (mRNA) and ELISA (protein) using commercially available products. NF kappa B activation was determined by I kappa B alpha mRNA induction RESULTS. Treatment of ARPE-19 cells with 15 mu M 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 I kappa B alpha mRNA expression. By contrast, inhibition of PI3K and PKC zeta significantly decreased the cytokine and I kappa B alpha mRNA expression. Inhibition of the I kappa B kinase complex essentially ablated all cytokine induction CONCLUSIONS. 7KCh induces cytokines via three kinase signaling pathways, AKT-PKC zeta-NF kappa B, p38 MAPK, and ERK. The MAPK/ERK pathways seem to preferentially enhance cytokine induction downstream from NF kappa B activation. The results of this study suggest that 7KCh activates these pathways through interactions in the plasma membrane, but the mechanism(s) remains unknown. (Invest Ophthalmol Vis Sci. 2010;51:4942-4955) DOI:10.1167/iovs.09-4854