Enhanced cellular cholesterol efflux by naringenin is mediated through inhibiting endoplasmic reticulum stress - ATF6 activity in macrophages

Naringenin improves lipoprotein profile and protects against cardiovascular disease. ATF6 is an endoplasmic reticulum (ER) stress sensor with the same activation processes with sterol regulator SREBPs. Clinical data revealed that ATF6 expression was associated with plasma cholesterol level. Here, we investigated whether naringenin was involved in the regulation of cholesterol efflux and tested the role of ER stress-ATF6 in the naringenin function. Results showed that naringenin increased cholesterol efflux to both apoA-I and HDL and gene expressions in ABCA1, ABCG1 and LXRα in RAW264.7 macrophages. Naringenin inhibited the cleaved ATF6 nuclear translocation and its target GRP78 and XBP-1 expressions. Naringenin-induced cholesterol efflux was modulated by treatment with ER stress inhibitor 4-phenylbutyric acid, inducer tunicamycin and ATF6 overexpression in RAW264.7 and/or THP-1 cells, which suggested the naringenin functions were mediated through inhibiting ER stress-ATF6 pathway. Next, we found high-fat diet (HFD) supplemented with naringenin increased by >1.2-fold in cholesterol efflux capacity in primary peritoneal macrophage in apoE−/− mice compared to only HFD-fed mice. The increase was significantly reduced by tunicamycin treatment. Naringenin decreased GRP78, XBP-1 and nuclear ATF6 levels in peritoneal macrophage and aorta and reduced atherosclerotic lesion at aortic root, but reversed by tunicamycin. These confirmed participation of ER stress-ATF6 in naringenin efficacy. Finally, we found naringenin promoted AKT phosphorylation; PI3K inhibitor LY294002 treatment increased nuclear ATF6 and reduced naringenin-enhanced ABCA1 expression and cholesterol efflux. We concluded naringenin as a regulator for cholesterol efflux, and the regulation was mediated by ATF6 branch of ER stress and PI3K/AKT pathway.     

Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells.