Stress within the endoplasmic reticulum (ER) can be induced by misfolded proteins accumulating in the lumen of this organelle. Signaling of ER stress to other parts of the cell results in altered gene expression, physiological adaptation, and with sustained stress, apoptosis (cell suicide). ER stress is often studied with highly toxic compounds that create severe ER stress rapidly, and a condition that is likely not physiologically relevant within an organism. In this study, we examine the apoptotic signaling induced by moderate ER stress, and in particular the inositol 1,4,5-trisphosphate receptor (ITPR). The ITPR regulates Ca2+ release from the ER lumen, and can induce apoptosis. We hypothesize that moderate levels of ER stress activate apoptosis via an ITPR-dependent signal. To induce moderate ER stress, we expose cells to 20-30nM concentrations of tunicamycin, an inhibitor of N-linked glycosylation in the ER. In this study, inclusion of an ITPR inhibitor (2-aminoethoxyphenyl borate, 2APB) protected cells from moderate ER stress, but did not protect cells from severe ER stress. A second methodology of assessing ITPR regulation of apoptosis includes overexpression of an ER-localized form of Bcl-2. The B-cell lymphoma 2 protein (Bcl-2has the ability to block the activation of cell suicide (apoptosis) by binding and inhibiting pro-apoptotic proteins (Bax family members). Bcl-2 is a membrane localized protein, found primarily in the mitochondrial outer membrane, and the endoplasmic reticulum (ER) membrane. In recent years, ER localized Bcl-2 has been shown to interact with the ITPR and inhibit pro-apoptotic Ca2+ signaling from the ER. We transfected cells with plasmids bearing forms of a Bcl-2 fusion protein to assess the capability of ER-Bcl-2 to protect cells from moderate apoptosis. The results of initial experiments did not show protection to either moderate or severe ER stress though some replicates of the experiment seemed to indicate protection. As this result is inconsistent with other results in our lab, we propose additional replicates of the experiment and using a drug-based mimic of this interaction to assess moderate ER stress signaling (Akl et al., 2013).
Vargas, Agustin and Brewster, Jay L., "Evaluating ITPR-dependence of Apoptotic Signaling from the Endoplasmic Reticulum" (2013). Pepperdine University, Featured Research. Paper 55.