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Jiahn-Dar Huang, Jung Wha Lee, Ignacio R. Rodriguez; Sterculic Acid Inhibits 7-ketocholesterol-mediated Unfolded Protein Response In ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4294.
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7-ketocholesterol (7KCh) is a potent inducer of unfolded protein response (UPR) and a major oxysterol in oxidized lipoprotein deposits in sub-RPE region. The prolonged UPR mediated by 7KCh may lead to local chronic inflammation which in turn may contribute to the development of age-related macular degeneration (AMD). We have previously shown that sterculic acid shuts down 7KCh-mediated inflammatory responses. Here we examine the effect of sterculic acid on 7KCh-mediated UPR in ARPE-19 cells.
Cultured ARPE-19 cells were treated with 7KCh in serum-free medium with or without sterculic acid. Stearic acid was used as a structurally similar control. After 24hr treatment, the mRNA expression of the UPR components including PERK, EIF2α, ATF4, CHOP, IRE1, XBP1, GRP78, and P58IPK in ARPE-19 cells were determined by quantitative RT-PCR. The protein levels of phosphorylated-EIF2α, EIF2α, phosphorylated-IRE1α, IRE1α, CHOP, GRP78, and P58IPK were visualized using immunoblot. The 7KCh-mediated disruption of cytoplasmic calcium level was evaluated using Fluo-4 assay with an EnVision Multilabel Reader.
Sterculic acid significantly attenuated 7KCh-mediated mRNA expressions of UPR components in ARPE-19 cells while stearic acid did not show any effect. Immunoblot showed that sterculic acid suppressed the phosphorylation of EIF2α and IRE1α as well as the induction of CHOP and GRP78 by 7KCh treatment. The protein expression of P58IPK did not change by 7KCh treatment either with or without sterculic acid. In ARPE-19 cells, a transient increase of cytoplasmic calcium level was observed immediately after 7KCh treatment. Adding a calcium ion chelator (EGTA) or a calcium channel (i.e. IP3 receptor) inhibitor (2-APB) suppressed the transient calcium increase. However, adding sterculic acid to 7KCh-treated cells did not suppress the transient calcium increase.
Sterculic acid significantly inhibits all three UPR pathways induced by 7KCh. This antagonist effect is not due to the induction of P58IPK, an inhibitor of the PERK pathway. This antagonist effect is also not caused by suppressing the transient calcium flow from extracellular sources or the endoplasmic reticulum. In summary, our results suggest that sterculic acid did not antagonize 7KCh-mediated inflammation by inhibiting specific UPR pathways. Sterculic acid seems to inhibit upstream to UPR pathways and possibly downstream of the calcium response.
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