Abstract

The Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that is located in endoplasmic reticulum (ER) membranes and protects cells from ER stress-induced apoptosis. The ER is associated with generation of reactive oxygen species (ROS) through oxidative protein folding. This study examined the role of BI-1 in the regulation of ER stress-induced accumulation of ROS and expression of unfolded protein response-associated proteins. BI-1 reduced the expression levels of glucose response protein 78, C/EBP homologous protein, phospho-eukaryotic initiation factor 2alpha, IRE1alpha, XBP-1, and phospho-JNK and inhibited the cleavage of ATF-6alpha p-90, leading to the inhibition of ROS. Although ROS scavengers offer some protection against ER stress-induced apoptosis, the expression of pro-apoptotic ER stress proteins was not affected. This study shows that the response of unfolded proteins is followed by ROS accumulation under ER stress, which is regulated in BI-1 cells. The mechanism for these BI-1-associated functions involves the expression of heme oxygenase-1 (HO-1) through nuclear factor erythroid 2-related factor 2. In BI-1 cells, the transfection of HO-1 small interfering RNA completely abolished the BI-1-induced protection. The endogenous expression of HO-1 through ER stress-initiated ROS is believed to be as a protection signal. In conclusion, these observations suggest that BI-1 can inhibit the ER stress proteins as well as the accumulation of ROS, thereby protecting the cells. Moreover, HO-1 plays an important role in the BI-1-associated protection against ER stress.

Highlights

  • The endoplasmic reticulum (ER)3 is the key organelle in cells where the important steps in the folding and modification of proteins as well as the selection for transport to other compartments occur [1, 2]

  • The overall number of propidium iodide-positive stained cells was relatively low compared with the annexin V-stained cells, the protective effect of Bax inhibitor-1 (BI-1) and that of NAC and GSH in Neo cells were similar to the result of annexin V staining

  • These results suggest that BI-1 overexpression protects the cells against ER stress through the regulation of ROS

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Summary

EXPERIMENTAL PROCEDURES

Thapsigargin and tunicamycin were supplied by Calbiochem. DiOC6 (3,3Ј-dihexyloxacarbocyanine iodide) and DCF-DA were obtained from Molecular Probes (Eugene, OR). The antibodies against actin, HO-1, GRP78, JNK, IRE-1, XBP-1, CHOP, and Nrf-2 siRNA were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). ATF-6 antibody was obtained from Imgenex (San Diego, CA). The antibodies against phospho-JNK, eIF-2␣, and phospho-eIF-2␣ were purchased from Cell Signaling Technologies (Beverly, MA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, trypsin, and other tissue culture reagents were supplied by Invitrogen. Bicinchoninic acid protein assay reagents were obtained from Pierce. All other chemicals were at least of analytical grade and were purchased from Sigma

Cell Culture and Viability
Apoptosis Assays
Microsomal Fractionation
Statistical Analysis
Western Blotting
Transfection of siRNA
RESULTS
Previous studies have shown that
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