Assessment of BHK-21 cell viability at low and high concentrations of Tunicamycin after transfection of Bcl-2 ER localized anti-apoptotic protein
Mentor:Jay Brewster, Professor of Biology, Associate Provost, Pepperdine University Seaver College
Accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen induces stress in cells. Signaling of ER stress to other parts of the cell results in altered gene expression; with sustained stress, apoptosis. As a known component of aging; ER stress is often studied by creating severe ER stress, a condition that is not easily compared to cellular aging. In this study, we examine the apoptotic signaling induced by moderate ER stress. We hypothesize that moderate levels of ER stress activate apoptosis via release of ER Ca2+ stores via the inositol trisphosphate receptor(ITPR). 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 protected cells from moderate ER stress, but did not protect cells from severe ER stress; indicating that there may be different pathways by which moderate and high levels of ER stress activate apoptosis. 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-2). Bcl-2 is a membrane localized protein, found primarily in the mitochondrial outer membrane, and the endoplasmic reticulum membrane. 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 we are continuing to evaluate this hypothesis with additional replicates. In the presentation, we will discuss our working model that posits moderate ER stress to activate apoptosis via an ITPR-mediated Ca2+ release.