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Regulation of IRE1? by the small molecule inhibitor 4?8c in hepatoma cells.

ABSTRACT: The unfolded protein response (UPR) is activated in response to impairments of the folding environment in the endoplasmic reticulum (ER). The most conserved arm of the UPR, inositol-requiring ER-to-nucleus signaling protein (IRE1?), has been linked to the regulation of a diverse array of cellular processes including ER-associated degradation, inflammatory signaling, cell proliferation and membrane biogenesis. Recent studies have utilized the selective, small molecule inhibitor, 4?8c, to examine the role of IRE1? endoribonuclease (RNase) activity in various cell types including multiple myeloma, mouse embryonic fibroblasts and pancreatic beta cells [1-5]. In the present study we utilized this inhibitor to examine the role of IRE1? RNase activity in hepatoma cells (H4IIE), specifically concentrating on cell proliferation and the identification of potential off target effects under both unstressed and stressed conditions. Experiments were performed in H4IIE hepatoma cells in the absence (control conditions (LG)) or presence (LG + Thapsigargin (Thap)) of ER stress. The presence of 4?8c decreased IRE1? RNase activity, based on reduced splicing of X-box binding protein-1 (XBP1s) and regulated IRE1?-dependent decay of mRNA in both treatments and at concentrations ranging from 10-90 ?M. Cell proliferation was significantly reduced at higher concentrations (> 60 ?M 4?8c) in unstressed cells and displayed a dose-response relationship with 4?8c in both treatments. The presence of 4?8c did not promote cytoxicity in either of the treatment conditions but higher concentrations of the inhibitor (60 ?M) were associated with apparent off-target or compensatory responses that were not observed at 10 ?M. Overall, the small-molecule inhibitor, 4?8c is an effective inhibitor of IRE1? RNase activity in H4IIE cells. Potential off-target effects associated with this inhibitor require the use of multiple inhibitor concentrations in all experiments.


PROVIDER: S-EPMC5661992 | BioStudies | 2017-01-01

REPOSITORIES: biostudies

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