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Jiahn-Dar Huang, Jung Lee, Ignacio Rodriguez; 7-Ketocholesterol induces cytokines through NF-κB activation and CHOP in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1811.
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© ARVO (1962-2015); The Authors (2016-present)
7-ketocholesterol (7KCh) is a major oxysterol found oxidized lipoprotein deposits in sub-RPE region. 7KCh has been shown to significantly induce cytokines via several inflammatory pathways. The purpose of this study is to determine the relative contribution of ER stress response and other NF-κB-related pathways to 7KCh-mediated cytokine induction.
Cultured ARPE-19 cells were treated with Cholesterol (Ch) and 7KCh in serum-free medium. The signaling pathways were blocked using specific inhibitors and small interfering RNA (siRNAs). NF-κB activity was inhibited by adenovirus overexpressing dominant negative IκBα (dnIκBα). After treatment, the mRNA expression of VEGF, IL-1β, IL-6, IL-8, and ER stress markers were determined by quantitative RT-PCR. The protein expression of Akt, ERK1/2, P38 MAPK, CHOP, and GRP78 were detected by immunoblot. The 7KCh-mediated disruption of intracellular calcium level was evaluated using Fluo-4 assay.
Ch did not significantly induce the expression of cytokines or ER stress markers. However, both Ch and 7KCh significantly induced phosphorylation of Akt and ERK1/2. Ch also induced phosphorylation of P38 MAPK but to a lesser degree than 7KCh. Inhibiting PI3K-Akt, ERK1/2, and P38 MAPK activation did not reduce cytokine induction by 7KCh but further enhanced some of them. By contrast, CHOP and ATF4 knockdown significantly reduced IL-6 induction. ATF4 knockdown also seemed to slightly reduced VEGF induction. Inhibiting NF-κB reduced the induction of all three interleukins to basal level and approximately 50% induction of VEGF, CHOP and GRP78. Both 7KCh and Ch induced a brief and transient intracellular calcium response immediately after treatment. The cell permeable calcium chelator, BAPTA-AM, was able to completely prevent the calcium increase but failed to inhibit the 7KCh-mediated ER stress. However, BAPTA-AM did significantly inhibit IL-6 induction and to a lesser extent of IL-1β and IL-8.
7KCh-mediated cytokine induction seems to occur mainly through NF-κB activation. Nevertheless inhibiting NF-κB does not ablate the 7KCh-mediated induction of VEGF and ER stress markers. Since the ATF4 knockdown reduces VEGF and IL-6 induction, the 7KCh-mediated cytokine induction seems to occur via a synergistic effect between NF-κB and ER stress. This suggests that 7KCh activates kinase pathways that affect both NF-κB and ER, likely via PERK activation.
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